WO2025008364A1

WO2025008364A1 - Methods for obtaining plant-based ready-to-drink coffee or tea beverages

Info

Publication number: WO2025008364A1
Application number: PCT/EP2024/068637
Authority: WO
Inventors: Zhen Long; Yuchen Wang; Hanne Vang Hendriksen
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2023-07-05
Filing date: 2024-07-02
Publication date: 2025-01-09
Anticipated expiration: 2026-01-05
Also published as: AU2024290001A1

Abstract

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

Description

METHODS FOR OBTAINING PLANT-BASED REA DY-TO- DRINK COFFEE OR TEA BEVERAGES

Reference to sequence listing

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

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

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

Ready-to-drink (RTD) acidic beverages, such as, e.g., sports drinks, RTD coffee beverages and RTD tea beverages, are sold worldwide as prepackaged, on-the-go beverages ready for consumption. RTD acidic beverages may be stored and consumed at room temperatures and cold. Some of the more common commercially available RTD beverages include, but are not limited to, iced coffees, coffee lattes, cold brew coffees and iced milk teas. While many of the RTD beverages sold today are based on animal milk, a few RTD beverages based on dairy alternative beverages have recently been introduced to the market, including, for example, the coconut milk based iced coffee sold by ALOHA.

Dairy alternative plant-based beverages should meet the same requirements as the conventional dairy-based beverages in terms of storage stability without phase separation, creaming, gelation, taste and/or sedimentation and the like. In particular, the plant-based beverages must meet consumers’ expectations and demands concerning taste and texture, including smooth mouthfeel, creaminess and richness in taste. In this regard, a particular challenge, which remains to be sufficiently solved, is the poor solubility of plant proteins, off- flavors caused by them and tendency of the plant proteins to precipitate in acidic products, such as coffee or tea drinks.

US 2022/0079187 A1 discloses non-dairy analogs, such as pea milk, containing deamidated refined protein component, which when mixed with a heated coffee beverage has reduced immediate feathering. The exemplified non-dairy analogs suitable for mixing with the heated coffee are all prepared with at least a gellan gum and phosphate salts. The disclosure does not relate to ready- to-d rink coffee or tea beverages and fails to demonstrate any improvement in long-term stability of the coffee and plant milk mixed beverages.

US 2022/0151255 A1 discloses nut milks treated with protein deamidase to reduce the immediate aggregation occurring when mixing the nut milks with a weakly acidic to weakly alkaline liquid, such as a coffee. The exemplified nut milks are only tested for immediate aggregation in heated (90°C) coffee and the disclosure is silent in regard to any storage stability of the mixed coffee and nut milk, nor does it relate to ready-to-drink coffee or tea beverages.

US 2022/0142210 A1 discloses protein solutions and methods of preparing such, comprising the use of a protein deamidating enzyme to treat a solution comprising a protein and a stabilizer. Long-term stability is only examined in relation to a pasteurized formulation comprising deamidated pea protein, pectin gum, gellan gum, and a juice concentrate. The disclosure is silent to the use of protein deamidase for improving stability, in particular storage stability, of plant-based ready-to-drink coffee or tea beverages.

US 2023/0240312 A1 discloses protein deamidase-treated plant milks, such as oat milk or pea milk, which when added to heated coffee show improved dispersibility, measured by tendency to coagulation. The disclosure fails to mention the use of protein deamidase to obtain storage-stable ready-to-drink coffee or tea beverages.

Thus, it is an object of the present invention to identify improved processes for obtaining plant-based ready-to-drink coffee or tea beverages with improved stability, in particular plantbased ready-to-drink coffee or tea beverages with improved storage stability.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that by treating a plant material with a protein deamidase, a plant material is obtained which when mixed with an acidic beverage, in particular a coffee or tea drink, has improved dispersibility and reduced risk of flocculation, both at the time of mixing and during long-term storage. As a result, a shelf-stable, organoleptically satisfactory plant-based ready-to-drink acidic beverage comprising a blend of an enzymatically deamidated plant material and an acidic drink, can be obtained which can be stored under cool (refrigerated) conditions or at room temperatures for prolonged time, such us up to several weeks, before consumption, without suffering from any product deterioration. The plant-based ready-to-drink coffee or tea beverage as claimed herein thus not only meets consumers’ demands in terms of satisfactory organoleptic properties, but also ensures storage stability.

The invention therefore provides a method for obtaining a plant-based ready-to-drink coffee or tea beverage, comprising the steps of:

(a) providing an aqueous solution comprising enzymatically deamidated plant material; (b) mixing the aqueous solution of step (a) with a coffee or tea beverage to obtain the plant-based ready-to-drink coffee or tea beverage; and

(c) storing the plant-based ready-to-drink coffee or tea beverage for at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, before consumption.

The methods claimed herein comprise the further benefits that the mixing of the aqueous solution comprising enzymatically deamidated plant material with the coffee or tea beverage can be carried out both under cold conditions and at room temperatures, such as, e.g., at temperatures in the range of 3°C-30°C, such as, e.g., at temperatures in range of 4°C-8°C or alternatively, for example, in the range of 18°C-22°C. This provides producers of plant-based RTD coffee or tea beverages with improved and cost-efficient production methods.

The invention thus further relates to a plant-based ready-to-drink coffee or tea beverage obtainable by any of the methods claimed herein. The plant-based RTD coffee or tea beverage obtained according to methods of the present invention has improved immediate and long-term stability. In particular, the inventors have found that the plant-based RTD coffee or tea beverage as claimed herein is resistant to flocculation, both immediately after its preparation and following weeks of storage, both in refrigerated conditions and at room temperature. Thus, a plant-based RTD coffee or tea beverage can be obtained which has the functional properties, including creaminess, taste and stability, demanded and expected by the consumers. The improved stability of the plant-based RTD coffee or tea beverage prepared according to methods of the present invention, further avoids the need for adding emulsifiers and stabilizers to the final product and, thus, also meets consumers’ requirements for clean label plant-based food products. Thus, the plant-based RTD coffee or tea beverage as claimed herein and prepared using the methods claimed herein, comprises satisfactory organoleptic properties without compromising health, and with that a healthier on-the-go food product, free of additives, can be obtained which is high in protein and low in added fat and sugar.

The invention also provides for the use of a protein deamidase in the production of a plant-based ready-to-drink coffee or tea beverage. In particular, the invention provides the use of a protein deamidase in the production of a storage-stable plant-based ready-to-drink coffee or tea beverage..

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates stability of soy beverage in instant coffee at 4°C.

Figure 2 illustrates stability of pea beverage in coffee brew at 4°C.

Figure 3 illustrates stability of soy beverage in Earl Grey tea at 4°C.

Figure 4 illustrates stability of soy beverage in Lipton black tea at 4°C.

Figure 5 illustrates stability of pea beverage in Earl Grey tea at 4°C.

Figure 6 illustrates stability of pea beverage in Lipton black tea at 4°C. Figure 7 illustrates stability of soy beverage in Lipton green tea at 4°C.

Figure 8 illustrates stability of pea beverage in Lipton green tea at 4°C.

Figure 9 illustrates stability of pea beverage in Lipton Jasmine tea at 4°C.

Figure 10 illustrates stability of a RTD coffee drink prepared using a pea-based beverage, with both immediate stability (Day 0) and storage stability (Day 7) shown.

Figure 11 illustrates stability of a RTD coffee drink prepared using a pea-based beverage formulated with phosphate, with both immediate stability (Day 0) and storage stability (Day 7) shown.

SEQUENCES

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

SEQ ID NO: 2: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium viscerum.

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

SEQ ID NO: 4: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium proteolyticum.

SEQ ID NO: 5: Protein deamidase derived from Chryseobacterium gambrini having the mature polypeptide sequence shown as SEQ ID NO: 6.

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

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

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

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

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

DETAILED DESCRIPTION OF THE INVENTION

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

As used herein, the terms “drink" and "beverage" are used interchangeably and have the same meaning. Unless defined otherwise or clearly indicated by context, all percentages are percentage by weight (percent w/w or “% (w/w)”).

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

The term “ready-to-drink” beverage, also referred to herein as “RTD acidic beverage”, “RTD coffee or tea beverage”, “RTD beverage” and “RTD drink”, refers to a liquid food product that is ready to be consumed directly at purchase, without the need for any additional preparation steps, such as, e.g., addition of water, heating, cooling or cooking. The ready-to- drink coffee or tea beverage as claimed herein at least comprises a mixture of an enzymatically deamidated plant material and a coffee or tea drink, and may or may not be combined with additional food ingredients to produce the ready-to-drink coffee or tea beverage. The ready-to- drink coffee or tea beverage can be ingested by humans or animals, preferably by humans. The ready-to-drink coffee or tea beverage claimed herein is plant-based.

The plant material may be in the form of an aqueous solution or suspension of a plantbased dairy alternative powder. Or the plant material may be any other suitable preparation obtained from a plant, such as, e.g., an aqueous suspension of a flour or the like obtained from a plant, such as from a part of a plant. The plant material may be a combination of any of the above.

The plant material used to obtain the plant-based ready-to-drink beverage is subjected to enzymatic treatment with a protein deamidase to obtain the aqueous solution comprising enzymatically deamidated plant material. Thus, in the context of the present invention, the term “enzymatically deamidated plant material” means a plant material treated with a protein deamidase for deamidation. A person skilled in the art will know of suitable analytical methods to determine enzymatic deamidation of a plant material. One such method is exemplified in Example 1 disclosed herein by the measurement of free ammonium content (NH4) (step 2 of the assay procedure).

In some embodiments, additional food ingredients are added to the plant-based ready- to-drink beverage. The additional food ingredients may be any food ingredient deemed useful by a practitioner of skill in the art. The additional food ingredient may be a solid or liquid ingredient. The additional food ingredient may or may not be plant-based. In some embodiments, the additional food ingredient is water.

The plant-based ready-to-drink coffee or tea beverage may be fortified with a plantbased dairy alternative powder, such as, e.g., a soymilk powder, or concentrated or isolated protein, such as soy protein isolate, soy protein concentrate, pea protein isolate or pea protein concentrate. In an embodiment, the plant-based ready-to-drink coffee or tea beverage is fortified, such as, e.g., an oat-based drink fortified with pea protein or a soy-based drink fortified with soy protein. In an embodiment, the plant-based ready-to-drink coffee or tea beverage is an oat-based drink 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 drink fortified with soy or pea protein to a protein level of 6-8% (w/w).

Soy and pea belong to the family of legumes or Fabaceae. Based on protein contents, soy and pea protein products may be classified into three main categories: soy/pea flour, soy/pea protein concentrate (SPC or PPG), and soy/pea protein isolate (SPI or PPI), with the highest protein contents being in the isolates followed by concentrates and lastly flours.

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

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

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

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

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

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

The additional food ingredients which may be added to the plant-based ready-to-drink coffee or tea beverage, include, but are not limited to, e.g., lipids, such as oils, in particular plant oils, sugars, such as sucrose, proteins, various forms of synthetic amino acids, dietary fibres, salts, minerals, flavoring agents, vitamins, and any combinations thereof.

In an embodiment, lipid is added to the plant-based ready-to-drink coffee or tea beverage and/or to the aqueous solution comprising enzymatically deamidated plant material. The lipid may be a plant oil or a mixture of plant oils. The lipid may be selected from rapeseed oil, flaxseed oil, safflower oil, flaxseed oil, soybean oil, olive oil, sunflower oil, palm oil and combinations thereof. In one embodiment the lipid is a soybean oil. The selection of suitable lipid may be based on the type of plant-based ready-to-drink coffee or tea beverage desired.

In a further embodiment, sugar is added, optionally together with a lipid, to the plantbased ready-to-drink coffee or tea beverage and/or to the aqueous solution comprising enzymatically deamidated plant material. In one embodiment the sugar is sucrose. In a preferred embodiment, sucrose and soybean oil is added to the aqueous solution comprising enzymatically deamidated plant material.

In an embodiment, salt is added to the plant-based ready-to-drink coffee or tea beverage and/or to the aqueous solution comprising enzymatically deamidated plant material. The salt may be sodium chloride, dicalcium carbonate, dicalcium phosphate, tricalcium phosphate, calcium carbonate and any combinations thereof. In an embodiment, vitamins and/or minerals are added to the plant-based ready-to-drink coffee or tea beverage and/or to the aqueous solution comprising enzymatically deamidated plant material. The vitamins may be vitamin A, vitamin C, vitamin D, vitamin E, vitamin B12, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6, vitamin K, folic acid (vitamin B9, and mixtures thereof. The mineral may be calcium, phosphorous, magnesium, sodium, potassium, chloride, iron, zinc, iodine, selenium, copper and mixtures thereof.

The plant-based ready-to-drink coffee or tea beverage obtained according to the methods claimed herein does not require the addition of emulsifiers and/or stabilizers to achieve the properties claimed herein. In particular, using the methods as claimed herein, a plant-based ready-to-drink coffee or tea beverage is obtained with improved stability, in particular improved storage stability, meaning that the plant-based ready-to-drink coffee or tea beverage does not flocculate or precipitate even after long-term storage, such as after several weeks or months of storage. Thus, in one embodiment, the plant-based ready-to-drink coffee or tea beverage is substantially free from added emulsifiers and/or stabilizers. In another embodiment, the aqueous solution comprising enzymatically deamidated plant material is substantially free from added emulsifiers and/or stabilizers. For example, the aqueous solution comprising enzymatically deamidated plant material may be a plant-based dairy alternative drink, such as a pea-, soy- or oat-based drink, which is essentially free of added emulsifiers and/or stabilizers.

As used herein, the terms “emulsifier” and “stabilizer” are meant as added emulsifiers and stabilizers, i.e. ingredients not naturally found in the material used for preparing the plantbased ready-to-drink coffee or tea beverage. Examples of such emulsifiers and stabilizers include, but are not limited to, thickening agents, such as, e.g., carboxymethylcellulose, gellan gum, hydroxypropyl starch and agar, and emulsifiers, such as, e.g., monoglyceride and diglyceride.

In the context of the invention, the terms “substantially free from” and “essentially free of” are used interchangeably to describe a composition, a product, or a process that contains only trace amounts or negligible quantities of a particular substance, component, or process feature. It indicates that the presence of the specified substance or process feature is minimal and does not impact the overall characteristics or functionality of the invention. In an embodiment, "essentially free of' or “substantially free from” means 0% (w/w) or 0% (w/v).

Using the methods of the invention, a plant-based ready-to-drink coffee or tea beverage can be obtained which is stable, in particular storage stable, without the need for the addition of a buffering salt, such as a citrate salt or a phosphate salt, neither during production of the plantbased ready-to-drink coffee or tea beverage, nor when formulating the obtained plant-based ready-to-drink coffee or tea beverage. This has been confirmed at least in the data shown in Example 6 herein.

In an embodiments, the aqueous solution comprising enzymatically deamidated plant material is substantially free from added buffering salts. In a further embodiment, the aqueous solution comprising enzymatically deamidated plant material is obtained using a method substantially free from added buffering salts.

In another embodiment, the plant-based ready-to-drink coffee or tea beverage is substantially free from added buffering salts.

In another embodiment, the method of obtaining the plant-based ready-to-drink coffee or tea beverage is carried out in the absence of added buffering salts.

Examples of buffering salts include chloride salts, such as sodium chloride and potassium chloride, citrate salts, and phosphate salts, such as tricalcium phosphate, potassium phosphate, dipotassium phosphate, sodium phosphate, disodium phosphate.

In some embodiments, the added buffering salt is a phosphate salt.

In further embodiments, the aqueous solution comprising enzymatically deamidated plant material is substantially free from added phosphate salts.

The plant-based ready-to-drink coffee or tea beverage obtained in the methods of the present invention has improved stability, in particular improved storage stability. Thus, the plantbased ready-to-drink coffee or tea beverage is stored for at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, before being consumed. This prolonged storage does not influence the properties of the plant-based RTD beverage and the plant-based RTD beverage remains free from precipitation and flocculation over time. In the context of the present invention, the term “stability” or “stable” when used to describe a plant-based ready-to-drink beverage means the resistance of the beverage to flocculation or precipitation, both immediately after its production and following long-term storage at storage conditions typical for consumer beverages and beverage additive products. In the context of the invention, stability and storage stability of the plant-based ready-to-drink coffee or tea beverage is to be understood as not only the aqueous solution comprising enzymatically deamidated plant material being resistant to feathering when added to a coffee or tea beverage, but also resistance of the RTD beverage to precipitation, flocculation and sedimentation, which may occur over time when storing the RTD coffee or tea beverage. The stability may be determined by any method known in the art for evaluation of such, including by visual evaluation. The plant-based ready-to-drink coffee or tea beverage as claimed herein is largely unaffected by the acidic food matrix, such as the coffee drink or tea drink, used to prepare the plant-based ready-to-drink beverage. Further, the plantbased RTD coffee or tea drink is stable over time, i.e., the plant-based RTD coffee or tea drink is stable against precipitation of the proteins and other components contained therein under refrigerated and room temperature conditions for extended periods of time, such as a period of time of 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, and 12 months, such as 4 months or 8 months.

The plant-based ready-to-drink coffee or tea beverage as claimed herein is shelf-stable and can be stored under refrigerated conditions or at room temperature conditions. In the context of the present invention, room temperature conditions means temperatures at about 15- 25°C, such as about 18-22°C, and cold and/or refrigerated conditions means temperatures at about 2-5°C, such as about 4°C. In one embodiment, the plant-based ready- to-d rink coffee or tea beverage is meant for storage at 3-30°C, such as at 3-5°C and/or at 15-25°C. In one embodiment the plant-based ready-to-drink coffee or tea beverage is meant for storage at about 4°C and/or at about 18-22°C.

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

In some embodiments, the aqueous solution comprising enzymatically deamidated plant material and the coffee or tea beverage have a temperature in the range of 3°C-30°C prior to and/or during the mixing. In some embodiments, the aqueous solution comprising enzymatically deamidated plant material and the coffee or tea beverage have a temperature in the range of 3°C-30°C prior to the mixing. In some embodiments, the aqueous solution comprising enzymatically deamidated plant material and the coffee or tea beverage have essentially the same temperature when mixed. The ability to mix the coffee or tea beverage and the enzymatically deamidated plant material at cool and/or room temperatures provides manufacturers of plant-based ready-to-drink coffee or tea beverages with improved production methods, which are cost and energy efficient and more reliable, i.e. avoiding the risk for flocculation upon and/or after preparation of the plant-based RTD coffee or tea drink.

In some embodiments, the ratio of the aqueous solution comprising enzymatically deamidated plant material to coffee or tea beverage is from 1 :10 to 10:1, such as 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, based on weight/weight. In one embodiment, the ratio of the aqueous solution comprising enzymatically deamidated plant material to coffee or tea beverage is 1 :1. In another embodiment, the ratio of the aqueous solution comprising enzymatically deamidated plant material to coffee or tea beverage is 1:4. In another embodiment, the ratio of the aqueous solution comprising enzymatically deamidated plant material to coffee or tea beverage is 3:1.

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

Preferably, the aqueous solution comprising enzymatically deamidated plant material has a protein content of at most 5% (w/w).

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

In another preferred embodiment, the aqueous solution comprising enzymatically deamidated plant material has a protein content of about 2% (w/w).

Preferably, the aqueous solution comprising enzymatically deamidated plant material has a lipid content of at least 0.5% (w/w). Preferably, the aqueous solution comprising enzymatically deamidated plant material has a lipid content of at most 4% (w/w).

In a preferred embodiment, the aqueous solution comprising enzymatically deamidated plant material has a lipid content of about 1.5%, about 2% or about 3.5% (w/w).

In the context of the present invention, the terms “acid/acidic food matrix”, “acidic beverage” and “acidic drink” mean a coffee or tea drink having a low pH, i.e. pH of at most 7. Thus, in one embodiment the coffee or tea beverage has a pH of 3-7, such as a pH of 4.5-6.5. In a preferred embodiment, the coffee or tea beverage has a pH of 5.0-5.5. In the context of the present invention, the coffee drink or tea drink may have a pH in the range of 3-7, such as a pH in the range of about 4-6, such as a pH in a range of about 4.5-5.5. For example, the coffee drink may be a highly acidic coffee having pH below 5.0. Examples of highly acidic coffees include, but are not limited to, espresso and ristretto. The coffee drink may also be a coffee drink prepared from instant coffee, such as, e.g., Nescafe Gold Instant coffee, or a coffee brew. The acidic beverage may also be a tea beverage, such as tea beverage based on Chai tea blends, spiced tea blends, black teas, such as, e.g., Assam and Darjeeling black teas, green teas, earl grey, oolong tea, and rooibos tea. Such tea beverages may be used to prepare chai lattes or milk tea beverages.

In one embodiment, the plant-based ready-to-drink beverage prepared by mixing a coffee or tea beverage with an aqueous solution comprising enzymatically deamidated plant material has a pH of 5-7, such as a pH of 5.2-6.5.

In some embodiments, the aqueous solution comprising enzymatically deamidated plant material is obtained using a method comprising the steps of: i. obtaining a slurry of a plant material in water; ii. treating the slurry of step (i) with a protein deamidase to obtain an aqueous solution comprising deamidated plant material in water; and iii. optionally inactivating the protein deamidase.

The protein deamidase used to treat the slurry of plant material in water is held at a temperature in the range of 20-80°C, so that the plant material is enzymatically deamidated by the protein deamidase to produce an enzymatically deamidated plant material. In some embodiments, the slurry is held at a temperature between 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 held at a temperature of about 20°C, 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C.

In some embodiments, the slurry with the added protein deamidase is held at 20-80°C for at least 10 minutes to allow for enzymatic deamidation of the plant material. In some embodiments, the slurry is held for about 10, about 15, about 20, about 25, about 30, about 60, about 120, about 180, or about 240 minutes to allow for enzymatic deamidation of the plant material. In some embodiments, the slurry is held for at least about 10, 30, or 60 minutes. In some embodiments, the slurry is held for 30 minutes. In some embodiments, the slurry is held for 60 minutes.

In preferred embodiments, the slurry with the added protein deamidase is held at a temperature in the range of 50-60°C for about 30-60 minutes.

In some embodiments, the method of obtaining the aqueous solution comprising enzymatically deamidated plant material is substantially free from added emulsifiers and/or stabilizers and/or added buffering salts. In some embodiments, the slurry of a plant material in water is treated with a protein deamidase in the absence of added buffering salts.

In some embodiments, a lipid is added to the slurry. The lipid may be added before, during or after the slurry is treated with protein deamidase. In one embodiment, the lipid is added before the slurry is treated with protein deamidase. In one embodiment, the lipid is added after the slurry has been treated with protein deamidase. The lipid may be selected from rapeseed oil, flaxseed oil, safflower oil, flaxseed oil, soybean oil, olive oil, sunflower oil, palm oil and combinations thereof. In one embodiment, the lipid is a soybean oil.

The process used, including temperature ranges, pH and the length of enzymatic treatment, will vary depending on the plant material and the enzymes added to the slurry. The skilled person will know how to determine the best process parameters based on the plant material and enzymes used.

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

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

After the optional enzyme inactivation, the aqueous solution comprising enzymatically deamidated plant material may be cooled. The hydrolyzed plant material may be separated into a solid and a liquid stream, for example by centrifugation. Centrifugation may occur in a decanter centrifuge. Following centrifugation, the liquid stream may be harvested or collected and used as the aqueous solution comprising enzymatically deamidated plant material. The liquid stream may still comprise some solid matter. In some embodiments, the liquid stream comprises 1-80% solids. In further embodiments, the liquid stream comprises 1-10%, 5-20%, 10-25%, 20-35%, 25-40%, 30-45%, 35-50%, 40-55%, 45-60%, 50-65%, 55-70%, 60-75%, or 65-80% solids. In some embodiments, the liquid stream comprises 10-15% solids.

In some embodiments, the liquid stream is further processed to remove water, also referred to as concentrated. Concentration also increases the relative amounts of solids in the concentrated liquid stream. In some embodiments, water removal will concentrate the products of hydrolysis, namely the released sugars. Concentration may occur by evaporation of the water in the liquid stream. In some embodiments, the concentrated liquid stream comprises 10- 100% solids. In further embodiments, the concentrated liquid stream comprises 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 comprising enzymatically deamidated plant material.

In some embodiments, the liquid stream is used directly as the aqueous solution comprising enzymatically deamidated plant material. Additional food ingredients may be added to the liquid stream to produce the aqueous solution comprising enzymatically deamidated plant material. In some embodiments, the liquid stream is derived from a soy material or a pea material. For example, this soy or pea derived liquid stream may be formulated using for instance sodium chloride (NaCI), oil, sugar and flavoring agents. It may be homogenized. It may be UHT or ESL treated and aseptically packed.

In some embodiments, the aqueous solution comprising enzymatically deamidated plant material is a plant-based dairy alternative drink. Examples of plant-based dairy alternative drinks include soy drinks, cashew drinks, macadamia drinks, coconut drinks, pea drinks, rice drinks, quinoa drinks, flax drinks, hemp drinks, oat drinks and drinks comprising any combination thereof. In an embodiment, the plant-based beverage is a soy drink, a pea drink, or any combinations thereof. In some embodiments, the plant-based beverage is prepared from soy protein isolate or pea protein isolate.

The plant material is or is derived from the edible portions of a plant. In some embodiments, the plant material is derived from edible portions of a plant which are high in starch. In some embodiments, the edible portion of the plant may be tubers, roots, stems, cobs, legumes, fruits, or seeds. In some embodiments, the plant is a cereal and the plant material is or is derived from the cereal grain, also referred to as the whole grain. In further embodiments, the cereal grain may be from corn, rice, millet, milo, quinoa, or oat. In yet further embodiments, the cereal grain may be from barley, wheat, buckwheat, or rye. In some embodiments, the plant material is or is derived from a tuber or root (including rhizomes), such as a potato, sweet potato, cassava, tiger nut (chufa nut), canna, or tapioca. In some embodiments, the plant material is or is derived from a fruit, such as banana, jack fruit, or bread fruit. In some embodiments, the plant material is or is derived from a hemp, fava, sago, pea, or soy bean plant.

In some embodiments, the plant material is heat treated. In some embodiments, the plant material is dehydrated. In some embodiments, the plant material is de-hulled, ground, wet- milled, and/or dry milled. 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, pea flour, soy flour, bean flour, de-hulled oats, de-hulled barley, de-hulled wheat, de-hulled peas, de-hulled beans, or a combination or any thereof. In preferred embodiments, the plant material is an oat material, a pea material, a soy material or any combinations thereof.

In some embodiments, the plant material is suspended in water to produce an aqueous solution comprising plant material, where the ratio of plant material to water is 1:3 to 1:10 (w/w), such as, e.g., 1:3 to 1:8 (w/w) or 1:6 to 1:10 (w/w).

The aqueous solution comprising enzymatically deamidated plant material may be subjected to further processing, such as, e.g., treatment with further enzymes, including, e.g., further hydrolyzing enzymes. In some embodiment, the aqueous solution comprising enzymatically deamidated plant material may be standardized and/or homogenized.

The aqueous solution comprising enzymatically deamidated plant material may be further treated with one or more hydrolyzing enzymes selected from the group of pectinases, hemicellulases, xylanases, beta-glucanases, mannanases, glucanases, glucoamylases, isoamylases, alpha-amylases, beta-amylases, and mixtures thereof. The enzymes used in the methods of the invention may be added to the slurry comprising the plant material in any suitable form, such as in the form of a liquid, in particular a stabilized liquid, or it may be added as a substantially dry powder or granulate. Granulates may be produced, e.g., as disclosed in US Patent No. 4,106,991 and US Patent No. 4,661 ,452. Liquid enzyme preparations may, for instance, be stabilized by adding a sugar or sugar alcohol or lactic acid according to established procedures. Other enzyme stabilizers are well-known in the art.

The inventors have found that the protein deamidase enables obtaining a sterilized plant-based ready-to-drink coffee or tea beverage, which is stable to increased temperatures and acidic conditions. Thus, an embodiment relates to a method of obtaining a sterilized plantbased ready-to-drink coffee or tea beverage, comprising the steps of: obtaining a slurry of a plant material in water;

- treating the slurry of plant material in water with a protein deamidase to obtain an aqueous solution comprising deamidated plant material in water; optionally inactivating the protein deamidase, optionally by subjecting the aqueous solution comprising deamidated plant material in water to a heat treatment; mixing the aqueous solution comprising enzymatically deamidated plant material with a coffee or tea beverage to obtain a plant-based ready-to-drink coffee or tea beverage; heat treating, preferably UHT treating, the plant-based ready-to-drink coffee or tea beverage to obtain the sterilized plant-based ready-to-drink coffee or tea beverage, optionally wherein the plant-based ready-to-drink coffee or tea beverage is a canned beverage or a packaged beverage; and storing the sterilized plant-based ready-to-drink coffee or tea beverage for at least 4 hours before consumption, preferably wherein the method is substantially free from added emulsifiers, stabilizers and/or added buffering salts.

Protein deamidase

In the methods of the invention, a plant material is treated with a protein deamidase to obtain an enzymatically deamidated plant material.

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

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

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

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

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

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

The types or origins of the protein deamidase used in the present invention are not particularly limited. Examples of the protein deamidase includes protein deamidases derived from Chryseobacterium genus, Flavobacterium genus, Empedobacter genus, Sphingobacterium genus, Aureobacterium genus, or Myroides genus.

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

In some embodiments, the protein deamidase may be derived from Chryseobacterium genus, such as Chryseobacterium viscerum (the strain has formerly been referred to as Chryseobacterium sp-62563), C. gambrini, C. culicis, C. defluvii, or C. proteolyticum. In some embodiments, the deamidase in the methods of the invention is derived from or obtained from Chryseobacterium viscerum.

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

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

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

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

The methods and compositions of the invention include a mature deamidase and optionally a second polypeptide which is derived from the propeptide of a deamidase. The second polypeptides described herein are mutated variants of the naturally occurring propeptides. These variant propeptide sequences have been found to bind less strongly to their corresponding deamidase, so that they are more easily enzymatically cleaved off after recombinant expression and secretion from of the host cell. The polypeptides of SEQ ID NOs: 1 to 10 are derived from Chryseo bacterium spp. and are described in PCT application PCT/EP2023/055936; filed March 8, 2023, herein incorporated by reference.

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

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

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

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 to SEQ ID NO: 2.

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 to SEQ ID NO: 4.

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 to SEQ ID NO: 6.

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 to SEQ ID NO: 8.

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 to SEQ ID NO: 10.

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 to a mature polypeptide of SEQ ID NO: 1.

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 to a mature polypeptide of SEQ ID NO: 3. 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 to a mature polypeptide of SEQ ID NO: 5.

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 to a mature polypeptide of SEQ ID NO: 7.

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 to a mature polypeptide of SEQ ID NO: 9.

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

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

In the context of the present invention, the term “variant” means a polypeptide having enzymatic activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding one or more (e.g., several) amino acids, e.g., 1-5 amino acids, adjacent to and immediately following the amino acid occupying a position.

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

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.

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

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are intrOoduced at every residue in the molecule, and the resultant mutant molecules are tested for enzymatic activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for ex-ample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

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

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

A protein deamidase to be used in the methods of the invention may be added at a concentration of 0.01-20 IPA(U)/g substrate protein, such as 0.1-12 IPA(U)/g substrate protein, 0.5-7 IPA(U)/g substrate protein. In some embodiments, the protein deamidase to be used in the methods of the invention is added at a concentration in the range of 0.3-2 IPA(U)/g substrate protein, such as 0.5-1.5 IPA(U)/g substrate protein. Deamidase (protein glutaminase) activity was measured using the assay described in Example 1. The activity assay consists of two separate de-coupled parts: (1) an enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and (2) a non- enzymatic detection step, wherein the ammonia formed in step (1) is derivatized to a blue indophenol compound with an absorption maximum at 630 nm. The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (given in Indophenol Assay Unit: IPA(U)). The activity may be determined relative to a standard of declared strength.

The enzymes dosage will depend on parameters such as the temperature, the incubation time and the dairy alternative recipe. The skilled person will know how to determine the optimal enzyme dosage.

Without wishing to be bound by any particular theory, the inventors believe that use of protein deamidase to yield an enzymatically deamidated plant material for use in the production of plant-based ready- to-d rink coffee or tea beverages contribute to the superior benefits reported herein, including, but not limited to, the improved stability, including storage stability, of the plant-based ready-to-drink acidic or tea beverage.

The invention is further defined by the following numbered embodiments:

Embodiment 1. Method of obtaining a plant-based ready-to-drink acidic beverage, comprising the steps of:

(a) providing an aqueous solution comprising enzymatically deamidated plant material;

(b) mixing the aqueous solution of step (a) with an acidic beverage to obtain the plantbased ready-to-drink acidic beverage, wherein the plant-based ready-to-drink acidic beverage is stored for at least 4 hours before being consumed.

Embodiment 2. Method of obtaining a plant-based ready-to-drink coffee or tea beverage, comprising the steps of:

(b) mixing the aqueous solution of step (a) with a coffee or tea beverage to obtain the plant-based ready-to-drink coffee or tea beverage; and

(c) storing the plant-based ready-to-drink coffee or tea beverage for at least 4 hours before consumption.

Embodiment 3. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is stored for at least 8 hours before being consumed.

Embodiment 4. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is stored for at least 12 hours before being consumed.

Embodiment 5. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have a temperature in the range of 3°C-30°C prior to and/or during the mixing in step (b). Embodiment 6. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have a temperature in the range of 3°C-30°C prior to the mixing in step (b).

Embodiment 7. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have a temperature in the range of 3°C-12°C, such as in the range of 3°C-10°C, 4°C-9°C or 5°C-8°C, prior to and/or during the mixing in step (b).

Embodiment 8. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have a temperature in the range of 3°C-12°C, such as in the range of 3°C-10°C, 4°C-9°C or 5°C-8°C, prior to the mixing in step (b).

Embodiment 9. Method of any of embodiments 1-6, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have a temperature in the range of 15°C- 25°C, such as in the range of 16°C-24°C, 17°C-22°C or 18°C-21°C, prior to and/or during the mixing in step (b).

Embodiment 10. Method of any of embodiments 1-6, wherein the aqueous solution of step (a) and the coffee or tea beverage of step (b) have a temperature in the range of 15°C- 25°C, such as in the range of 16°C-24°C, 17°C-22°C or 18°C-21°C, prior to the mixing in step (b).

Embodiment 11. Method of any of the preceding embodiments, wherein the aqueous solution comprising enzymatically deamidated plant material and the coffee or tea beverage have essentially the same temperature prior to being mixed.

Embodiment 12. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) has a protein content in the range of 0.3 to 5% (w/w), such as 1 to 3% (w/w).

Embodiment 13. Method of any of the preceding embodiments, wherein the aqueous solution of step (a) has a lipid content in the range of 0.5 to 4% (w/w), such as 1 to 2% (w/w), such as about 1.5% (w/w).

Embodiment 14. Method of any of the preceding embodiments, wherein the aqueous solution is a plant-based dairy alternative drink.

Embodiment 15. Method of any of the preceding embodiments, wherein the aqueous solution is a soy drink, a cashew drink, a macadamia drink, a coconut drink, a pea drink, a rice drink, a quinoa drink, a flax drink, a hemp drink, an oat drink or any combinations thereof.

Embodiment 16. Method of any of the preceding embodiments, wherein the aqueous solution is a pea drink, a soy drink or a combination thereof.

Embodiment 17. Method of embodiment 16, wherein the soy drink is prepared from soy bean flour, soy protein isolate, soy protein concentrate, or any combination thereof, preferably from soy protein isolate. Embodiment 18. Method of embodiment 16, wherein the pea drink is prepared from pea flour, pea protein isolate, pea protein concentrate, or any combination thereof, preferably from pea protein isolate.

Embodiment 19. Method of embodiment 15, wherein the aqueous solution is an oatbased drink.

Embodiment 20. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage and/or the aqueous solution comprising enzymatically deamidated plant material is substantially free from added emulsifiers and/or stabilizers.

Embodiment 21. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is substantially free from added emulsifiers and/or stabilizers.

Embodiment 22. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage and/or the aqueous solution comprising enzymatically deamidated plant material is substantially free from gums, such as gellan gum and pectin gums.

Embodiment 23. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage and/or the aqueous solution comprising enzymatically deamidated plant material is substantially free from added buffering salts.

Embodiment 24. Method of any of the preceding embodiments, wherein the plant-based the aqueous solution comprising enzymatically deamidated plant material is substantially free from added buffering salts.

Embodiment 25. Method of any of embodiments 23-24, wherein the buffering salt is a chloride salt, a citrate salt, a phosphate salt, and any combination thereof.

Embodiment 26. Method of any of embodiments 23-25, wherein the buffering salt is a phosphate salt.

Embodiment 27. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage has a pH of 3-7, such as a pH of 3.5-6.5, 4.5-6.5, or 5.0- 5.5.

Embodiment 28. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is a canned beverage or a packaged beverage.

Embodiment 29. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is meant for storage at a temperature of 3°C-30°C.

Embodiment 30. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is meant for storage at a temperature of 3°C-5°C, such as at about 4°C, or at a temperature of 16°C-24°C, such as about 20°C.

Embodiment 31. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage further comprises one or more additional food ingredients selected from the group of lipids, sugars, proteins, vitamins, minerals, amino acids, flavoring agents, dietary fibres, salts and any combinations thereof. Embodiment 32. Method of embodiment 31, wherein the additional food ingredient is a lipid, such as an oil, preferably a plant oil, more preferably a soybean oil, and/or a sugar, such as a sucrose.

Embodiment 33. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is heat treated.

Embodiment 34. Method of any of the preceding embodiments, wherein the plant-based ready-to-drink coffee or tea beverage is heat treated and the heat treatment is an Ultra-High Temperature (UHT) treatment, an Extended Shelf Life (ESL) treatment or a pasteurization, preferably a UHT treatment.

Embodiment 35. Method of any of embodiments 33-34, wherein the heat treatment is carried out after step (b) and prior to step (c).

Embodiment 36. Method of any of the preceding embodiments, wherein the coffee or tea beverage is a coffee drink.

Embodiment 37. Method of any of embodiments 1-35, wherein the coffee or tea beverage is a tea drink.

Embodiment 38. Method of any of the preceding embodiments, wherein the plant material is derived from soy, cashew, macadamia, coconut, pea, bean, rice, quinoa, flax, hemp or oat, preferably from oat, pea or soy.

Embodiment 39. Method of any of the preceding embodiments, wherein the plant material is derived from pea or soy.

Embodiment 40. Method of any of the preceding embodiments, wherein the ratio of aqueous solution to coffee or tea beverage is from 1 : 10 to 10:1 based on w/w.

Embodiment 41. Method of any of the preceding embodiments, wherein the ratio of aqueous solution to coffee or tea beverage is 1:1 , 1 :4 or 3:1 based on w/w.

Embodiment 42. Method of any of the preceding embodiments, wherein the aqueous solution comprising enzymatically deamidated plant material is obtained using a method comprising the steps of i. obtaining a slurry of a plant material in water; ii. treating the slurry of step (i) with a protein deamidase to obtain an aqueous solution comprising deamidated plant material in water; and iii. optionally inactivating the protein deamidase.

Embodiment 43. Method of embodiment 42, wherein the treatment with the protein deamidase is carried out at 50-60°C for 30-60 minutes.

Embodiment 44. Method of any of embodiments 42-43, wherein the treatment of step ii. further comprises treating with one or more hydrolyzing enzymes selected from the group of pectinases, hemicellulases, xylanases, beta-glucanases, mannanases, glucanases, glucoamylases, iso-amylases, alpha-amylases, beta-amylases, and mixtures thereof. Embodiment 45. Method of any of embodiments 42-44, wherein the protein deamidase and, when dependent on embodiment 44, the one or more hydrolyzing enzymes is inactivated by a heat treatment, such as an Ultra-High Temperature (UHT) treatment.

Embodiment 46. Method of any of embodiments 42-45, wherein step (ii) is substantially free from added emulsifiers, stabilizers and/or buffering salts, preferably free from added buffering salts.

Embodiment 47. Method of any of the preceding embodiments, wherein the aqueous solution comprising enzymatically deamidated plant material is obtained using a protein deamidase derived from or obtained from a Chryseobacterium species, such as from Chryseobacterium proteolyticus or Chryseobacterium viscerum.

Embodiment 48. Method of any of the preceding embodiments, wherein the aqueous solution comprising enzymatically deamidated plant material is obtained using a protein deamidase comprising a polynucleotide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

Embodiment 49. Method of any of the preceding embodiments, wherein the aqueous solution comprising enzymatically deamidated plant material is obtained using a protein deamidase comprising the polynucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, or 10.

Embodiment 50. Plant-based ready-to-drink acidic beverage obtainable by the method of any of the preceding embodiments.

Embodiment 51. Plant-based ready-to-drink acidic beverage according to embodiment 50, further comprising a protein deamidase.

Embodiment 52. Plant-based ready-to-drink acidic beverage according to any of embodiments 50-51, wherein the protein deamidase comprises a polypeptide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

Embodiment 53. Plant-based ready-to-drink acidic beverage according to any of embodiments 50-52, wherein the plant-based ready-to-drink acidic beverage is a plant-based ready-to-drink coffee or tea beverage.

Embodiment 54. Plant-based ready-to-drink acidic beverage according to any of embodiments 50-53, characterized by being substantially free from added emulsifiers, stabilizers and/or buffering salts.

Embodiment 55. Plant-based ready-to-drink acidic beverage according to any of embodiments 50-54, characterized by being substantially free from added buffering salts.

Embodiment 56. Plant-based ready-to-drink acidic beverage according to any of embodiments 50-55, characterized by being substantially free from added phosphate salts.

Embodiment 57. Plant-based ready-to-drink acidic beverage according to any of embodiments 50-56, characterized by being heat treated. Embodiment 58. Plant-based ready-to-drink acidic beverage according to any of embodiments 50-57, characterized by being UHT treated, ESL treated, or pasteurized.

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

Embodiment 60. Plant-based ready-to-drink coffee or tea beverage of embodiment 59, characterized by being essentially free of added buffering salts, emulsifiers and/or stabilizers.

Embodiment 61. Plant-based ready-to-drink coffee or tea beverage of any of embodiments 59-60, characterized by being storage stable.

Embodiment 62. Pea- and/or soy-based ready-to-drink coffee or tea beverage obtainable by the method as embodied in any of the embodiments 1-49.

Embodiment 63. Pea- and/or soy-based ready-to-drink coffee or tea beverage of embodiment 62, characterized by being essentially free of added buffering salts, emulsifiers and/or stabilizers.

Embodiment 64. Use of a protein deamidase in the production of a plant-based ready-to- drink acidic beverage to improve storage stability.

Embodiment 65. 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.

Embodiment 66. Use according to any of embodiments 64-65, wherein the protein deamidase comprises a polypeptide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

Embodiment 67. Use according to any of embodiments 64-66, wherein the protein deamidase is used in the absence of added buffering salts, emulsifiers and/or stabilizers.

Embodiment 68. Use according to any of embodiments 64-67, wherein the plant-based ready-to-drink acidic beverage is a pea- and/or soy-based ready-to-drink acidic beverage.

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

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

Materials

The following enzymes are used throughout the examples:

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

Example 1 : Protein deamidase activity assay

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

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

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

In step (1), the ammonia is developed by the deamidating action of the protein deamidase. In step (2), the generated ammonia reacts with phenol to form dioxyphenylamine under alkaline conditions. The reaction is catalyzed by sodium pentacyanonitrosylferrate(lll) (sodium nitroprusside). “Color Reagent solution A” contains phenol and sodium nitroprusside. “Color Reagent Solution B” provides alkaline reaction conditions. The intermediate is then oxidized by addition of sodium hypochlorite (“Color Reagent Solution C”) to form indophenol blue. This compound absorbs visible light at 630 nm. The enzyme activity is then calculated using a standard curve.

Assay Procedure:

Step (1) Enzymatic step with ammonia formation

Reagents:

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

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

Substrate solution: 30 mM Z-GIn-Gly (Merck C6154-1G) in assay dilution solution (check pH after dissolution). Stop solution: 0.4M TCA.

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

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

Incubation:

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

2. Add 100 pL of substrate solution to each well.

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

4. Seal the plate using transparent plate sealer.

5. Incubate the plate for 10 minutes at 37°C, 500 rpm, on a thermomixer equipped with a lid heating function.

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

Total reaction volume: 210 pL.

Step (2) Ammonia detection step

Reagents:

Color reagent A: 4% (w/v) Phenol, 0.015% (w/v) sodium pentacyanonitrosylferrate(lll) dihydrate (sodium nitroprusside) (Na2[Fe(CN)sNO]-2H2O).

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

Color reagent C: 28% (w/v) Potassium carbonate, 6% (v/v) sodium hypochlorite (Sigma- Aldrich 239305-25ml, < 5% available CI2).

Incubation:

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

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

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

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

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

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

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

Total reaction volume: 210 pL Standard curve:

Standard stock solution: 1000 mg NH₄ ⁺/L.

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

The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (Indophenol Assay Unit; IPA(U)):

which can be shortened to 2¹ _ P^mo1 - * Or -

180.04 mL * min. where

• C_NH4+ is the ammonia concentration in the reaction solution derived from the ammonium standard curve (i.e., taking into account the dilution of the prediluted ammonium standard solution in the ammonia derivatization step).

• 18.04 is the molecular mass of ammonium used for the standard solution.

• Vreadion is the reaction volume in the well when ammonia is generated (210 pL).

• Venzyme is the volume of enzyme solution added to the well when ammonia is generated

(10 pL).

• V_NH3 detection is the reaction volume in the well when ammonia is detected (210 pL).

Example 2: Preparation of legume-based beverages

Soy beverage preparation

100 mL commercially available soy beverage (DouBenDou WeiZhen soy beverage manufactured by DaLi group; protein content: 2.5 g, fat: 1.7 g, carbohydrate 7.2 g per 100 mL product; Ingredients: water, soy (non-genetically modified (non-GM)), sucrose) was treated with protein deamidase in amount 2 IPA(U)/g protein in soy beverage. The enzyme treatment was conducted at 60°C for 1 hour. Then the enzyme was deactivated at 85°C for 10 minutes. The resulting soy beverage was cooled to room temperature (between 20 to 25°C). The soy beverage control sample was prepared using the same procedure but without protein deamidase. Pea beverage preparation

Pea beverage was laboratory prepared using the following procedure. The ShuangTa pea protein isolate (protein content 82.2% based on dry solid) was mixed with deionized H₂O to prepare a 10% solution (m/v). The 10% pea beverage solution was then treated with protein deamidase at the dosage of 0.6 IPA(U)/g protein and 2 IPA(U)/g protein. The reaction was carried out at 60°C for 1 hour followed by homogenization (8000 rpm for 10 minutes) of the pea beverage solution with soy oil (Nissin) and sucrose and H₂O according to the recipe; protein: 3.4 g; soy 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 under stirring. Next, the sample was cooled to 5°C. The pea beverage control sample was prepared with the same procedure without protein deamidase. The pH of the pea beverage was around 7.0 for both enzymatic sample and control sample.

Example 3: Improved stability of protein deamidase treated pea-based and soy-based beverages mixed with coffee

Stability of soy beverage in instant coffee drink

2 g Nescafe Gold instant coffee (Nestle) was dissolved in 150 g 85°C hot water to make the instant coffee beverage. Then 2 g soy beverage prepared according to Example 2 was added to 20 g of the instant coffee beverage. For the enzymatically deamidated sample (Treatment with 2 IPA(U)/g protein) no curdling was observed upon mixing, while for the non- enzymatic treated sample a clear aggregation was observed, settling instantly at the bottom of the test tube. The samples were then stored in a refrigerator (~4°C) for the storage stability tests. The results from the storage stability test are shown in Table 1 below (Y: yes, N: no) and in Figure 1. The pH of the soy-based coffee drink was around 5.2.

Table 1 Soy beverage stability in Nescafe Gold instant coffee at 4°C

As can be seen from Table 1 and Figure 1 , the use of protein deamidase to prepare the enzymatically deamidated plant-based beverage resulted in a soy-based ready-to-drink coffee beverage with no visible flocculation after 2, 9 and 16 days of storage.

Pea beverage stability in coffee The coffee was prepared with commercially available Milan Gold coffee bean (Grand Espresso No. 2) which was milled and brewed using a De’Longhi® coffee machine (type: ECAM23.420.SW) under the extra strong and extra-long coffee taste setup.

2 g and 8 g of pea beverage made according to Example 2 was each mixed with 8 g brewed coffee under room temperature. The results are shown in Table 2 and Figure 2. By mixing 2 g pea beverage with 8 g coffee brew, the pH changed from 5.0 to around 5.5, while 8 g pea beverage mixed with 8 g coffee brew resulted in a final pH around 6.0.

Table 2 Pea beverage stability in Milan Gold coffee brew at 4°C

As can be seen from Table 2 and Figure 2, the use of protein deamidase to prepare the enzymatically deamidated plant-based beverage resulted in a pea-based ready-to-drink coffee beverage with no visible flocculation immediately after mixing (t=0) or after 28 and 35 days of storage.

Example 4: Improved stability of protein deamidase treated pea-based and soy-based beverages mixed with tea

Soy beverage stability in black tea

Two commercial black teas were tested for the application. In the first test, the black tea brew was made with commercially available Earl Grey tea bags (2 grams of tea per tea bag). Two tea bags were mixed with 100 mL 85°C hot water and incubated for 30 minutes. The tea bags were then removed from the liquid and discarded. Next, 2 g soy beverage prepared according to Example 2 was mixed with 20 g Earl Grey black tea drink. The sample was stored in a refrigerator (at 4°C) for the storage stability tests. Results from the stability testing are shown in Table 3 and in Figure 3. The pH of the Earl Grey tea and soy beverage mixture was around 5.3. Table 3 Soy beverage stability in Earl Grey tea at 4°C

As can be seen from Table 3 and Figure 3, the use of protein deamidase to prepare the enzymatically deamidated soy-based beverage resulted in a soy-based ready-to-drink tea beverage with no visible flocculation after 2, 9 and 16 days of storage.

In the second test, the black tea brew was made with commercially available Lipton black tea bag. Four tea bags were mixed with 100 mL 85°C hot water and incubated for 30 minutes. Then the tea bags were removed from the liquid and discarded. Next, 3 g soy beverage prepared according to Example 2 was mixed with 9 g Lipton black tea brew. The sample was stored in a refrigerator (at 4°C) for the storage stability tests. Results from the stability testing are shown in Table 4 and in Figure 4. The pH of the Lipton black tea and soy beverage mixture was around 5.4.

Table 4 Soy beverage stability in Lipton black tea at 4°C

As can be seen from Table 4 and Figure 4, the use of protein deamidase to prepare the enzymatically deamidated plant-based beverage resulted in a soy-based ready-to-drink tea beverage with no visible flocculation after 2 days, 9 days and 16 days of storage.

Pea beverage stability in black tea

Two commercial black teas were tested for the application. In the first test, the black tea brew was made with commercially available Earl Grey black tea bag. Two tea bags were mixed with 100 mL 85°C hot water and incubated for 30 minutes. Then the tea bags were removed from the liquid and discarded. Next, 3 g pea beverage prepared according to Example 2 was mixed with 9 g Earl Grey black tea brew. The sample was stored in a refrigerator (at 4°C) for the storage stability tests. Results from the stability testing are shown in Table 5 and Figure 5. The pH of the Earl Grey black tea and pea beverage mixture was around 5.7. Table 5 Pea beverage stability in Earl Grey tea at 4°C

As can be seen from Table 5 and Figure 5, the use of protein deamidase to prepare the enzymatically deamidated plant-based beverage resulted in a pea-based ready-to-drink acidic beverage with no visible flocculation after 2, 9 and 16 days of storage.

In the second test, the black tea brew was made with commercially available Lipton black tea bag. Two tea bags were mixed with 100 mL 85°C hot water and incubated for 30 minutes. Then the tea bags were removed from the liquid and discarded. Next, 3 g pea beverage prepared according to Example 2 was mixed with 9 g Lipton black tea brew. The sample was stored in a refrigerator (at 4°C) for the storage stability tests. Results from the stability testing are shown in Table 6 and in Figure 6. The pH of the Lipton black tea and pea beverage mixture was around 5.7.

Table 6 Pea beverage stability in Lipton black tea at 4°C. Ctrl, control; PD, protein deamidase; prot, protein.

As can be seen from Table 6 and Figure 6, the use of protein deamidase to prepare the enzymatically deamidated plant-based beverage resulted in a pea-based ready-to-drink tea beverage with no visible flocculation after 2, 9 and 16 days of storage. The slight flocculation observed for the samples treated with a lower amount of protein deamidase, i.e. 0.6 IPA(U)/g protein, confirms an optimum range of protein deamidase.

Soy beverage stability in green tea Green tea brew was made with commercially available Lipton green tea bag. Four tea bags were mixed with 100 mL 85°C hot water incubated for 30 minutes. Then the tea bags were removed from the liquid and discarded. Next, 3 g soy beverage made according to Example 2 was mixed with 9 g green tea brew. The sample was stored in a refrigerator (at 4°C) for the storage stability tests. Results from the stability testing are shown in Table 7 and in Figure 7. The pH of the Lipton green tea and soy beverage mixture was around 5.9.

Table 7 Soy beverage stability in Lipton green tea at 4°C.

As can be seen from Table 7 and Figure 7, the use of protein deamidase to prepare the enzymatically deamidated plant-based beverage resulted in a soy-based ready- to-d rink tea beverage with no visible flocculation after 2, 9 and 16 days of storage.

Pea beverage stability in green tea

The green tea brew was made with commercially available Lipton green tea bag. Two tea bags were mixed with 100 mL 85°C hot water and incubated for 30 minutes. Then the tea bags were removed from the liquid and discarded. Next, 3 g pea beverage made according to Example 2 was mixed with 9 g green tea brew. The sample was stored in a refrigerator (at 4°C) for the storage stability tests. Results from the stability testing are shown in Table 8 and in Figure 8. The pH of the Lipton green tea and pea beverage mixture was around 6.0.

Table 8 Pea beverage stability in Lipton green tea at 4°C. Ctrl, control; PD, protein deamidase; prot, protein.

As can be seen from Table 8 and Figure 8, the use of protein deamidase at an enzyme dosage of 2 IPA(U)/g protein to prepare the enzymatically deamidated plant-based beverage resulted in a ready-to-drink tea beverage with no visible flocculation after 2, 9 and 16 days of storage. The slight flocculation observed for the samples treated with a lower amount of protein deamidase, i.e. 0.6 IPA(U)/g protein, confirms an optimum range of protein deamidase.

Pea beverage stability in Jasmine herb tea

The Jasmine herb tea brew was made with commercially available Lipton Jasmine tea bag. Two tea bags were mixed with 100 mL 85°C hot water and incubated for 30 minutes. Then the tea bags were removed from the liquid and discarded. Next, 3 g pea beverage made according to Example 2 was mixed with 9 g Jasmine herb tea brew. The sample was stored in a refrigerator (at 4°C) for the storage stability tests. Results from the stability testing are shown in Table 9 and in Figure 9. The pH of the Lipton Jasmine tea and pea drink mixture was around 6.1.

Table 9 Pea drink stability in Lipton Jasmine tea at 4°C. Ctrl, control; PD, protein deamidase; prot, protein.

As can be seen from Table 9 and Figure 9, the use of protein deamidase at PD dosage of 2 IPA(U)/g protein to prepare the enzymatically deamidated plant-based beverage resulted in a ready-to-drink tea beverage with no visible flocculation after 2, 9 and 16 days of storage. The slight flocculation observed for the samples treated with a lower amount of protein deamidase, i.e. 0.6 IPA(U)/g protein, confirms an optimum range of protein deamidase.

Example 5: The role of pH and tannic acid on stability of protein deamidase treated pea and soy beverages

Tannic acids (tannins) are a class of polyphenol compounds present in tea and coffee, which tend to have bitter and astringency taste. In this study, the role of tannic acid on curdling of plant-based beverages was investigated. Tannic acid (CAS 1401-55-4) was purchased from Sigma Aldrich. Soy and pea beverages made according to Example 2 and control samples (no protein deamidase added) were included in the testing. The soy beverage and pea beverage was each mixed with 1 % (m/v) tannic acid solution, respectively, and when flocculation was observed, the pH of the beverage was recorded. Next, 0.02 mol/L HCI was used to adjust pH for another portion of soy beverage or pea beverage to match the recorded pH of tannic acid treated plant based beverages. The HCI treated soy and pea beverages were then visually examined to investigate if any flocculation could be observed. The results from the visual examinations are shown in Table 10. Based on these observations, it was concluded that it is not only pH influencing flocculation of plant based beverages but also tannic acid influences flocculation. It was thus concluded that treatment of plant-based beverages with protein deamidase not only makes the plant-based beverage more stable to acidic pH, but also improves resistance of the plant-based beverages to polyphenol-induced curdling effects such as that resulting from the tannic acid found in coffee and tea.

Table 10 Pea and soy beverage stability with tannic acid or HCI in acidic condition. PD, protein deamidase; prot, protein.

Example 6: Testing protein deamidase with and without phosphate salt for pea-based ready-to-drink coffee beverage

Enzyme reactions

Pea protein sample: Pea protein isolate (Roquette Nutralys S85F 2.0, protein content: 82%) was suspended in tap water to a final protein content of 10% and mixed with magnetic stirrer. Protein deamidase was tested in concentrations of 0 (control sample), 6 and 12 IPA(U)/g protein and the three samples incubated at 60°C for 1 hour in a FINEPCR combi-D24 Rotisserie with rotation speed set at 7.

Coffee sample: The coffee was brewed in a Juracoffee machine using Milan gold coffee bean (Arabica and Robusta mixture, medium roast), with a water to bean ratio of 8 to 1. Pea milk preparation

After incubation of the pea protein samples with the protein deamidase, the pea milk samples were prepared by formulation according to the recipe in Table 11 below. The ingredients were mixed well in a VORWERK Thermomix with rotation speed set at 2.5, and temperature set at 60°C. Then the pea milk samples were homogenized at rotate speed 8 for 3 min under 60°C.

Table 11 Pea milk recipe and ingredients

After formulation, the pea milk samples were pasteurized at 90°C for 10 minutes under agitation. Then the samples were transferred to ice water to cool down to room temperature.

For some of the pea milk samples, 500mg/100g pea milk dipotassium phosphate (K2HPO4 3H2O) was added to the pea milk before mixing with coffee.

RTD coffee beverage

The pea milk samples were mixed with the coffee samples at different ratios and put to autoclave at 121 °C for 5 minutes. Then the RTD coffee samples were transferred to ice water to cool down to room temperature.

The RTD coffee samples were then placed in sealed bottles and placed in a 60°C oven for an acceleration experiment to mimic the stability during shelf life.

Curdling in the RTD coffee samples was evaluated by visual examination and sedimentation observed by turning the sealed bottles upside down and visually examining sediment content forming at the bottom of the flask.

From the results shown in Tables 12 and 13 (and corresponding Figures 10 and 11), it was confirmed that protein deamidase alone could avoid curdling of the RTD coffee samples, both with and without phosphate added.

Table 12 Pea milk and coffee blend without phosphate salt, ratio 3:1 (milk to coffee, w/w), '+’ indicate degree of sedimentation observed, indicate no sedimentation observed.

Table 13 pea milk and coffee blend with 500mg dipotassium phosphate, ratio 1.5:1 (milk to coffee, w/w), '+’ indicate degree of sedimentation observed, indicate no sedimentation observed

Claims

1. Method of obtaining a plant-based ready-to-drink coffee or tea beverage, comprising the steps of:

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

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

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

5. Method of any of the preceding claims, wherein the aqueous solution of step (a) is a plantbased dairy alternative drink.

6. Method of any of the preceding claims, wherein the aqueous solution comprising enzymatically deamidated plant material and/or the plant-based ready-to-drink coffee or tea beverage is substantially free from added emulsifiers and/or stabilizers.

7. Method of any of the preceding claims, wherein the plant-based ready-to-drink coffee or tea beverage is a canned or packaged beverage.

8. Method of any of the preceding claims, wherein the plant-based ready-to-drink coffee or tea beverage is meant for storage at a temperature of 3-30°C.

9. Method of any of the preceding claims, wherein the plant-based ready-to-drink coffee or tea beverage further comprises one or more additional food ingredients selected from the group of lipids, sugars, proteins, vitamins, minerals, amino acids, flavoring agents, dietary fibres, salts and any combination thereof.

10. Method of any of the preceding claims, wherein the plant material is derived from oat, pea or soy, preferably from pea or soy.

11. Method of any of the preceding claims, wherein the ratio of aqueous solution to coffee or tea beverage is from 1 :10 to 10:1 based on w/w.

12. Method of any of the preceding claims, wherein the aqueous solution comprising enzymatically deamidated plant material is obtained using a method comprising the steps of i. obtaining a slurry of a plant material in water; ii. treating the slurry of step (i) with a protein deamidase to obtain an aqueous solution comprising deamidated plant material in water; and iii. optionally inactivating the protein deamidase.

13. Method of any of the preceding claims, wherein the plant-based ready-to-drink coffee or tea beverage is heat treated, optionally wherein the heat treatment is an Ultra-High Temperature (UHT) treatment, an Extended Shelf Life (ESL) treatment or a pasteurization.

14. Method of any of the preceding claims, wherein the aqueous solution comprising enzymatically deamidated plant material and/or the plant-based ready-to-drink coffee or tea beverage is substantially free from added buffering salts, preferably substantially free from added phosphate salts.

15. Method of any of the preceding claims, wherein the aqueous solution comprising enzymatically deamidated plant material is obtained using a protein deamidase derived from or obtained from a Chryseobacterium species.

16. Plant-based ready-to-drink coffee or tea beverage obtainable by the method of any of claims 1-15.

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