Stabilized ready-to-drink coffee beverages

Abstract

The present invention relates to the use of a protein deamidase and an enzyme having mannanase activity for obtaining a ready-to-drink coffee beverage with reduced turbidity and / or gelation.

Description

[0001] STABILIZED READY-TO-DRINK COFFEE BEVERAGES

[0002] Reference to sequence listing

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

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the use of a protein deamidase and an enzyme having mannanase activity for obtaining a ready-to-drink coffee beverage with reduced turbidity and / or gelation.

[0006] BACKGROUND OF THE INVENTION

[0007] Ready-to-drink (RTD) coffee beverages are sold worldwide as prepackaged, on-the-go beverages ready for consumption. The RTD coffee beverages may be stored and consumed warm, cold or at ambient temperatures. Examples of common commercially available RTD coffee beverages include, but are not limited to, iced coffees, coffee lattes, and cold brew coffees.

[0008] To ensure their continued appeal to the consumer, the commercially available RTD coffee beverages should meet various requirements in terms of storage stability without phase separation, creaming, gelation, taste and / or sedimentation and the like. In particular, the RTD coffee beverages must meet consumers’ expectations and demands concerning taste and visual appearance, including smooth mouthfeel, creaminess, richness in taste and reduced turbidity. In this regard, a particular challenge, which remains to be sufficiently solved, is the tendency of RTD coffee drinks to form a hazy and turbid appearance, precipitate, gelate, and sediment over time.

[0009] Thus, it is an object of the present invention to provide ready-to-drink coffee beverages with reduced turbidity.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have surprisingly found that by combining the use of a protein deamidase and an enzyme having mannanase activity to obtain a ready-to-drink (RTD) coffee beverage, a RTD coffee beverage is obtained which has reduced turbidity, precipitation, gelling, and sedimentation over time. As a result, a shelf-stable, sensorially superior RTD coffee beverage, comprising a blend of an enzymatically deamidated dairy- or plant-based composition and a mannanase-treated coffee composition, can be obtained which has reduced immediate turbidity and precipitation, reduced gelling and reduced sedimentation over time. Thereby, the ready-to-drink coffee beverage disclosed herein not only meets consumers’ demands in terms of satisfactory organoleptic properties, but also has superior storage stability which in turn ensures its continuous sensory appeal.

[0012] Therefore, the invention relates to method for obtaining a ready-to-drink coffee beverage, comprising the steps of:

[0013] (a) providing a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material, wherein a protein deamidase is used for the enzymatic deamidation;

[0014] (b) providing a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity; and

[0015] (c) mixing the dairy or plant composition of step (a) with the coffee composition of step (b) to obtain the ready-to-drink coffee beverage.

[0016] The invention further relates to a ready-to-drink coffee beverage comprising:

[0017] (a) a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material, wherein a protein deamidase is used for the enzymatic deamidation; and

[0018] (b) a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity, wherein the ready-to-drink coffee beverage has reduced turbidity and / or gelation compared to a ready-to-drink coffee beverage obtained without the use of a protein deamidase and an enzyme having mannanase activity.

[0019] The RTD coffee beverage obtained according to methods of the invention has reduced turbidity, precipitation and gelling. In particular, the inventors have found that the RTD coffee beverage as disclosed herein is resistant to sedimentation, both immediately after its preparation and following weeks and months of storage, both at refrigerated conditions, at ambient temperatures and at heated conditions.

[0020] Thus, a RTD coffee beverage can be obtained which has the sensory properties, including creaminess, taste and visual appearance, demanded and expected by consumers during the entire lifespan envisaged for the beverage. The reduced turbidity of the herein disclosed RTD coffee beverage further avoids the need for adding emulsifiers and / or stabilizers to the final product and, thereby, may also meet consumers’ requirements for clean label food products.

[0021] The invention also relates to the use of a protein deamidase and an enzyme having mannanase activity in the production of a ready-to-drink coffee beverage to reduce turbidity and / or gelation.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 illustrates stability of a RTD coffee drink prepared using a pea-based beverage, with both immediate stability (Day 0) and storage stability (Day 7 and Day 35) shown. Figure 2 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 and Day 35) shown.

[0024] Figure 3 illustrates stability of a RTD coffee drink prepared using cow’s milk, with both immediate stability (Day 0) and storage stability (Day 7 and Day 35) shown.

[0025] Figure 4 illustrates sedimentation after 7 days of storage at 58°C of cold brew samples prepared according to Example 3.

[0026] SEQUENCES

[0027] 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.

[0028] SEQ ID NO: 2: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium viscerum.

[0029] SEQ ID NO: 3: Protein deamidase derived from Chryseobacterium proteolyticum having the mature polypeptide sequence shown as SEQ ID NO: 4.

[0030] SEQ ID NO: 4: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium proteolyticum.

[0031] SEQ ID NO: 5: Protein deamidase derived from Chryseobacterium gambrini having the mature polypeptide sequence shown as SEQ ID NO: 6.

[0032] SEQ ID NO: 6: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium gambrini.

[0033] SEQ ID NO: 7: Protein deamidase derived from Chryseobacterium culicis having the mature polypeptide sequence shown as SEQ ID NO: 8.

[0034] SEQ ID NO: 8: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium culicis.

[0035] SEQ ID NO: 9: Protein deamidase derived from Chryseobacterium defluvii having the mature polypeptide sequence shown as SEQ ID NO: 10.

[0036] SEQ ID NO: 10: Mature polypeptide sequence of protein deamidase derived from Chryseobacterium defluvii.

[0037] SEQ ID NO: 11: Mannanase derived from Talaromyces leycettanus having the mature polypeptide sequence shown as SEQ ID NO: 12.

[0038] SEQ ID NO: 12: Mature polypeptide sequence of mannanase derived from Talaromyces leycettanus.

[0039] SEQ ID NO: 13: Mannanase derived from Chaetomium virescens having the mature polypeptide sequence shown as SEQ ID NO: 14.

[0040] SEQ ID NO: 14: Mature polypeptide sequence of mannanase derived from Chaetomium virescens. SEQ ID NO: 15: Mannanase derived from Sordaria macrospora having the mature polypeptide sequence shown as SEQ ID NO: 16.

[0041] SEQ ID NO: 16: Mature polypeptide sequence of mannanase derived from Sordaria macrospora.

[0042] SEQ ID NO: 17: Mannanase derived from Caldicellulosiruptor saccharolyticus having the mature polypeptide sequence shown as SEQ ID NO: 18.

[0043] SEQ ID NO: 18: Mature polypeptide sequence of mannanase derived from Caldicellulosiruptor saccharolyticus.

[0044] SEQ ID NO: 19: Mannanase derived from Bacillus sp.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] 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.

[0047] As used herein, the terms “drink" and "beverage" are used interchangeably and have the same meaning.

[0048] Unless defined otherwise or clearly indicated by context, all percentages are percentage by weight (percent w / w or “% (w / w)”).

[0049] 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.

[0050] The term “ready-to-drink” beverage, also referred to herein as “ready-to-drink coffee beverage”, “RTD coffee 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 beverage as claimed herein at least comprises a blend of a dairy- or plant-based composition, comprising enzymatically deamidated dairy or plant material, and a coffee composition, comprising a coffee component having been treated with an enzyme having mannanase activity. The ready-to-drink coffee beverage can be ingested by humans or animals, preferably by humans.

[0051] Preferably, the ready-to-drink coffee beverage has a protein content of at least 0.5% (w / w). Preferably, the ready-to-drink coffee beverage has a protein content of at most 20% (w / w). In an embodiment, the ready-to-drink coffee beverage has a protein content in the range of 1-5% (w / w), such as about 1.5% (w / w), about 2%(w / w), about 2.5% (w / w), about 3% (w / w), about 3.5% (w / w) or about 4% (w / w).

[0052] In another embodiment, the ready-to-drink coffee beverage has a protein content in the range 6-15% (w / w). For example, the RTD coffee beverage may be a high-protein RTD coffee drink. Such high-protein RTD coffee drink may be obtained by fortifying the RTD coffee beverage with a protein, such as, e.g., a plant-based protein, a whey protein or a casein protein.

[0053] Preferably, the ready-to-drink coffee beverage has a lipid content of at least 0.1% (w / w).

[0054] Preferably, the ready-to-drink coffee beverage has a lipid content of at most 5% (w / w).

[0055] In an embodiment, the ready-to-drink coffee beverage has a lipid content of about 1.5% (w / w), about 2.5% (w / w), about 3.5% (w / w) or about 4% (w / w).

[0056] In an embodiment, the ready-to-drink coffee beverage contains no added carbohydrates, such as no added sugars. In the context of the invention, “added carbohydrates” or "added sugars" refers to caloric mono- and di-saccharides added during manufacture of the beverage, such as glucose, sucrose, maltose, fructose, which are not naturally found in the ingredients used for producing the RTD beverage. For instance, lactose is naturally found in milk, therefore, for the purpose of this disclosure, lactose is not taken into account in "added sugar".

[0057] In an embodiment, the ready-to-drink coffee beverage has a reduced free monosaccharide content compared to a ready-to-drink coffee beverage prepared without the use of an enzyme having mannanase activity. In an embodiment, the ready-to-drink coffee beverage has an increased di- and / or trisaccharide content compared to a ready-to-drink coffee beverage prepared without the use of an enzyme having mannanase activity. In an embodiment, the ready- to-drink coffee beverage has a reduced free monosaccharide content compared to a ready-to- drink coffee beverage prepared with the use of an alternative enzyme having mannanase activity and exo activity.

[0058] The RTD coffee beverage may or may not be combined with additional food ingredients to produce the RTD coffee beverage. The additional food ingredients which may be added to the ready-to-drink coffee 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. The additional food ingredient may be a solid or liquid ingredient. The additional food ingredient may or may not be plant-based. The selection of additional food ingredients to add to the ready-to-drink coffee beverage may be based on the type of ready-to-drink coffee beverage desired. The additional food ingredients may be any food ingredient deemed useful by a practitioner of skill in the art.

[0059] The ready-to-drink coffee beverage may be fortified with a plant-based dairy alternative powder, such as, e.g., a soymilk powder, or concentrated or isolated protein, such as soy protein isolate, soy protein concentrate, pea protein isolate or pea protein concentrate, or with a dairybased powder, such as, e.g., a milk protein concentration or a whey protein concentrate. In an embodiment, the ready-to-drink coffee beverage is fortified, such as, e.g., an oat-based drink fortified with pea protein or a soy-based drink fortified with soy protein. 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, a SPI or PPI is used for fortification of the RTD coffee beverage when the RTD coffee beverage is plant-based. Also contemplated is the use of SPI or PPI for fortification of a dairy-based RTD coffee beverage.

[0060] The ready-to-drink coffee beverage obtained according to the methods disclosed herein has reduced turbidity and gelation compared to a ready-to-drink coffee beverage obtained using a similar method but without the use of a protein deamidase and an enzyme having mannanase activity. As used herein, the terms “turbidity” and “haze” are used interchangeably and have the same meaning. In the context of the invention, the terms “turbidity” and “haze” refers to the cloudiness or haziness in a liquid caused by suspended particles that are generally invisible to the naked eye, but forms a hazy appearance in suspension. Haze formation and turbidity can affect the visual appeal of beverages and other liquid foods. The more total suspended solids in a liquid, the cloudier the liquid seems and the higher the turbidity. Turbidity or haze may be quantitatively measured using a nephelometer with units expressed as Nephelometric Turbidity Units (NTU).

[0061] As used herein, the term “gelation” refers to the transition from a liquid state to a gel state. In the context of ready-to-drink coffee drinks, gelation may occur due to the liberation and polymerization of certain saccharides present in the coffee extract. This process can create a change in texture, visual appearance, and mouthfeel of these beverages. The present inventors have found that the solution offered with this invention, in particular the combined use of a protein deamidase and an enzyme having mannanase activity to produce RTD coffee beverages, results in less gelation occurring in the RTD coffee drink. In particular, it has been observed that upon storage of a RTD coffee beverage prepared according to the methods disclosed herein, no gelation occurs, as could be visually confirmed by flipping the RTD drink upside down and examining the bottom of the drink for any sedimentation or gelation.

[0062] The ready-to-drink coffee beverage obtained using the methods as disclosed herein has improved storage stability and shelf-life. In the context of the invention, “storage stability” and “shelf life" are terms used to refer to the period of time after production of the RTD beverage, during which the beverage is transported, and stored in retailers' or consumers' shelves, before consumption. In particular, using the methods as claimed herein, a ready-to-drink coffee beverage is obtained with improved stability, in particular improved storage stability, meaning that the ready- to-drink coffee beverage does not flocculate or precipitate even after long-term storage, such as after several weeks or months of storage.

[0063] The ready-to-drink coffee beverage obtained according to the methods disclosed herein does not require the addition of emulsifiers and / or stabilizers to achieve the properties claimed. In particular, using the methods as claimed herein, a ready-to-drink coffee beverage can be obtained with improved sensorial properties and stability, in particular improved storage stability, meaning that the ready-to-drink coffee beverage does not form a haze, gel or sedimentation, even after long-term storage, such as after several weeks or months of storage. Thus, in one embodiment, the ready-to-drink coffee beverage 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 ready-to- drink coffee 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 term “essentially free of’ is used to describe a composition, a product, or a process feature 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" means 0% (w / w) or 0% (w / v), or 0%. In a preferred embodiment, the ready-to-drink coffee beverage is essentially free of carboxymethylcellulose and / or microcrystalline cellulose.

[0064] The ready-to-drink coffee beverage can be stored under refrigerated conditions, at ambient temperature, or under heated conditions. In the context of the invention, ambient temperature means temperatures in the range of 15-38°C, such as about 15-25°C, such as about 18-22°C, cold and / or refrigerated conditions means temperatures in the range of 1-8°C, such as 2-5°C, such as about 4°C, and heated or warm conditions means temperatures in the range of 55-65°C, such as about 60°C. In some embodiments, the ready-to-drink coffee beverage is meant for storage at 1-70°C, such as at 1-8°C and / or at 15-38°C and / or at 55-65°C. In some embodiments, the ready-to-drink coffee beverage is meant for storage at 3-70°C, such as at 4- 8°C and / or at 15-25°C and / or at 55-65°C. The temperature at which the RTD coffee beverage is stored does not influence the properties of the RTD coffee beverage and the RTD coffee beverage remains free of haze formation, precipitation and sedimentation over time.

[0065] In some embodiments, the ready-to-drink coffee beverage is stored for at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, before being consumed. The prolonged storage does not influence the properties of the RTD coffee beverage and the RTD coffee beverage remains free of haze formation, precipitation and sedimentation over time. Without wishing to be bound by any theory, it is believed that the use an enzyme having mannanase activity to treat the coffee component, results in the cleavage of mannans having the effect of reducing viscosity and preventing haze to form in the coffee composition comprising the coffee component. Over time the haze particles get bigger and turn into precipitate, which will be seen as a sediment formed in the RTD coffee beverage comprising the coffee composition. In the context of the present invention, the term “stability” or “stable” when used to describe a ready-to- drink coffee beverage means the resistance of the beverage to haze formation or sedimentation, both immediately after its production and following long-term storage at storage conditions typical for consumer beverages and beverage additive products. The stability may be determined by any method known in the art for evaluation of such, including by visual evaluation and taste testing. Further, the RTD coffee drink remains stable over time, i.e., the RTD coffee drink is stable against precipitation of the proteins and other components contained therein under refrigerated, heated and ambient temperature conditions for extended periods of time, such as periods 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. In a preferred embodiment, the ready-to-drink coffee beverage is stored for at least one month before being consumed.

[0066] The inventors have observed a reduction in viscosity of coffee components with high dry matter contents when treating with an enzyme having mannanase activity. The benefit of reducing viscosity in such coffee components has multiple implications in coffee processing and manufacturing setups. In particular, the use of an enzyme having mannanase activity to treat a coffee extract in a process to produce a soluble coffee, such as an instant coffee, enables obtaining a coffee extract having a high dry matter content, without the thereto linked high increase in viscosity, whereby the manufacturing process is greatly improved in terms of time spent and energy needed. This has been confirmed at least on Example 6 and 7 disclosed herein.

[0067] For example, the point of addition of the enzyme having mannanase activity in a process of obtaining a soluble coffee product may preferably be before the evaporator, such as in a holding tank before the evaporator. In a process wherein a coffee component, such as a coffee extract, enters the evaporator at a dry matter of for example 5% and water is evaporated up to approx. 50% dry matter, reaching a certain viscosity at the end of the step, the addition of an enzymes having mannanase activity during the process has been shown to decrease the viscosity of the resulting evaporated coffee extract, allowing to reach higher dry matter at the end of evaporator step at the same viscosity. A more efficient evaporator performance by going to higher dry matter in the evaporator will save energy and time in the following drying step. Also, the lesser amount of water to be evaporated in the drying step, e.g., the freeze drying or spray drying step, enables for a more efficient overall performance in the soluble coffee manufacturing, in terms of saving time and energy. Thus, the use of the enzyme having mannanase activity as disclosed herein in processes wherein freeze- or spray-drying processes are used for obtaining a soluble coffee product based on a coffee extract, provides a highly desirable benefit in terms of increase in production capacity in plants where freeze- or spray-drying forms a capacity bottleneck. An additional benefit of adding an enzyme having mannanase activity during the evaporation step, is that a more efficient and robust coffee processing process can be established, requiring fewer cleaning stops in the process, due to, e.g., less film and precipitate formation in the system. In standard processes for obtaining soluble coffee, high viscosity and formation of precipitates in the system is known to affect quality due to the risk of burning of the coffee concentrate pushed to high dry matter and viscosity in the evaporator, with a risk of off-flavor generation. The mannanase according to the present invention solves this issue with its capacity to reduce viscosity of the coffee extract. Also, the addition of the enzyme after the evaporator will result in quality improvement of the soluble coffee by, for example, keeping mannan oligosaccharides in solution.

[0068] In an embodiment, the ready- to-d rink coffee beverage is a canned beverage or a packaged beverage. The RTD coffee beverage may be contained and stored in any type of can or packaging material deemed suitable by the person skilled in the art.

[0069] In some embodiments, the ratio of dairy or plant composition to coffee composition in the ready-to-drink coffee 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 an embodiment, the ratio of dairy or plant composition to coffee composition is 1 :1. In another embodiment, the ratio of dairy or plant composition to coffee composition is 3:1.

[0070] The ready-to-drink coffee beverage may be heat treated. Thus, in some embodiments, a step of heat treatment, such as sterilization, is performed before or after mixing the dairy or plant composition with the coffee composition to obtain the ready-to-drink coffee beverage. In a preferred embodiment, the heat treatment is performed after mixing the dairy or plant composition with the coffee composition to obtain the ready-to-drink coffee beverage.

[0071] The heat treatment may be a Low Temperature Long Time (LTLT), High Temperature Short Time (HTST), Higher Heat Shorter Time (HHST), Ultra-High Temperature (UHT), Ultra Pasteurized (UP) treatment, or any other form of heat treatment known in the art. In some embodiments, the heat treatment of the ready-to-drink coffee beverage further inactivates the enzymes used its production, i.e., the protein deamidase and the enzyme having mannanase activity. In some embodiments, the ready-to-drink coffee beverage is subjected to 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,

[0072] 7, 8, 9, or 10 seconds. In further embodiments, the UHT treatment is 140-145°C for 3, 4, 5, 6, 7,

[0073] 8, 9, or 10 seconds. In some embodiments, the UHT treatment is 143°C for 4, 5, 6, 7, or 8 seconds. In some embodiment, the ready-to-drink coffee beverage is UHT or Extended Shelf-Life (ESL) treated and aseptically packed.

[0074] In the methods of the invention, a protein deamidase is used to obtain a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material. Without wishing to be bound by any theory, it is believed that the use of a protein deamidase to prepare the dairy or plant composition results in a composition having both more total protein and more soluble protein. Thereby, a dairy or plant composition is obtained which has improved solubility, which can enhance the overall texture and mouthfeel of the beverage, and increased emulsification properties, which can lead to a more stable product with a better shelf life. In the context of the invention, this provides for an improved RTD coffee beverage, having improved visual and textural appearance due to no precipitation, haze, or sedimentation in the beverage. The dairy or plant material is subjected to enzymatic treatment with a protein deamidase to obtain the dairy or plant composition comprising enzymatically deamidated dairy or plant material. Thus, in the context of the present invention, the terms “enzymatically deamidated dairy material” and “enzymatically deamidated plant material” means a dairy or 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 dairy or plant material. One such method is exemplified in Example 1 by the measurement of free ammonium content (NH4).

[0075] As used herein, a “dairy composition” refers to a composition that contains one or more components derived from milk. This can include products such as raw milk, pasteurized milk, homogenized milk, pasteurized and homogenized milk, whole milk, skim milk, low fat milk, cream, whitener, creamer, concentrated milk, condensed milk, evaporated milk, reconstituted milk powder, or milk proteins such as casein and whey.

[0076] The term “dairy milk” generally refers to the milk from a cow, however, it may also refer to milk from a goat, sheep, buffalo, or camel. Milk is typically available in three versions which differ by their fat content. Whole milk has on average 3.8% milk fat, with a minimum of 3.2% milk fat. Low-fat milk, also referred to as lite milk or reduced-fat milk, generally contains 2% milk fat or less. In some embodiments, low-fat milk contains about 2% milk fat. In some embodiments, low- fat milk contains about 1% milk fat. Skim milk, also referred to as skimmed milk, nonfat milk or fat-free milk, generally has 0.5% milk fat or less. In some embodiments, skim milk has 0.1% milk fat or less. In some embodiments, skim milk might have extra milk solids, such as lactose and / or protein, to optimize the taste and texture due to its very low fat content.

[0077] In some embodiments, the dairy composition comprises cream, milk, concentrated milk, condensed milk, evaporated milk, reconstituted milk, milk protein concentrate, whey protein concentrate, micellar casein, and any combination thereof. In some embodiments, the milk, concentrated milk, condensed milk, evaporated milk, reconstituted milk, milk protein concentrate, whey protein concentrate and / or micellar casein is or is prepared from low-fat or skim milk. In some embodiments, the methods and compositions of the present invention include dairy compositions which have a lower amount of milk fat, such as dairy compositions derived from low-fat or skim milk. In some embodiments, the milk, concentrated milk, condensed milk, evaporated milk, reconstituted milk, milk protein concentrate, whey protein concentrate and / or micellar casein comprises 3.8% or less, 3.25% or less, 3% or less, 2.5% or less, 2% or less, 1 .5% or less, 1% or less, 0.5% or less, 0.25% or less, or 0.15% or less fat.

[0078] The dairy composition may be heat treated. Heat treatments may be a Low Temperature Long Time (LTLT), High Temperature Short Time (HTST), Higher Heat Shorter Time (HHST), Ultra-High Temperature (UHT), Ultra Pasteurized (UP) treatment, or any other form of heat treatment known in the art. In some embodiments, the protein deamidase is added to the dairy composition prior to heat treatment. In other embodiments, the protein deamidase is added to the dairy composition after heat treatment. In some embodiments, the dairy composition may be heat treated both before the deamidase is added and after, as part of the process of producing the resulting dairy composition.

[0079] Those skilled in the art will readily recognize the suitable dairy composition and processes for preparing such for a ready-to-drink coffee beverage having desired balance of flavor, creaminess, and stability.

[0080] As used herein, a “plant composition” refers to plant-based composition which may be obtained by suspending plant material in an aqueous suspension. 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.

[0081] 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, 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.

[0082] In an embodiment, the plant material is derived or obtained from almond, cashew, chickpea, coconut, fava bean, hazelnut, lentil, lupin, macadamia, mung bean, peanut, pecan, pistachio, corn, quinoa, flax, hemp, oat, spelt, pea, rice, sesame, sunflower, soy, walnut, or any combination thereof. In a preferred embodiment, the plant material is derived or obtained from almond, pea, soy, oat or any combination. In an embodiment, the plant material is derived or obtained from almond. In an embodiment, the plant material is derived or obtained from pea, soy, or a combination thereof. In an embodiment, the plant material is derived or obtained from oat.

[0083] 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, a concentrate, an isolate 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.

[0084] The plant composition 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 an embodiment, the plant composition comprising enzymatically deamidated plant material is standardized and / or homogenized. In another embodiment, the plant composition comprising enzymatically deamidated plant material has been further treated with one or more further hydrolyzing enzymes selected from the group of pectinases, hemicellulases, xylanases, beta-glucanases, mannanases, glucanases, glucoamylases, iso-amylases, alphaamylases, beta-amylases, and mixtures thereof. The enzymes used in the methods of the invention may be added to 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.

[0085] In some embodiments, the plant composition is a plant-based dairy alternative drink. Examples of plant-based dairy alternative drinks include almond drinks, cashew drinks, chickpea drinks, coconut drinks, fava bean drinks, hazelnut drinks, lentil drinks, lupin drinks, macadamia drinks, mung bean drinks, peanut drinks, pecan drinks, pistachio drinks, walnut drinks, corn drinks, quinoa drinks, flax drinks, hemp drinks, oat drinks, spelt drinks, pea drinks, rice drinks, sesame drinks, sunflower drinks, soy drinks and drinks comprising any combination thereof. In an embodiment, the plant composition is a soy drink, a pea drink, or any combinations thereof. In an embodiments, the plant composition is formulated with one or more further ingredients. Examples of such further ingredients include concentrated or isolated proteins, lipids, sugars, a calcium source, such as CaCOs, and emulsifiers, such as, e.g., a gellan gum.

[0086] Preferably, the plant composition has a protein content of at least 0.3% (w / w).

[0087] Preferably, the plant composition has a protein content of at most 20% (w / w).

[0088] In some preferred embodiments, the plant composition has a protein content in the range of 1-10% (w / w), such as about 1 .5% (w / w), about 2% (w / w), about 2.5% (w / w), about 3.5% (w / w), or about 5%. In some embodiments, the plant composition is diluted with water after the treatment with protein deamidase and before mixing with the coffee composition to obtain the RTD coffee beverage.

[0089] In an embodiment, the plant composition has a lipid content of about 1.5%, about 2% or about 3.5% (w / w).

[0090] Those skilled in the art will readily recognize the suitable plant composition and processes for preparing such for obtaining a ready- to-d rink coffee beverage having desired balance of flavor, creaminess, nutritional goals, stability, allergen considerations, and sustainability concerns.

[0091] To obtain the dairy or plant composition, the treatment with the protein deamidase is performed at temperatures in the range of 20-80°C. In some embodiments, the temperature used for the enzymatic deamidation of the dairy or plant material is between 25-40°C, 30-45°C, 35- 50°C, 40-55°C, 50-60°C, or 50-65°C. In further embodiments, the temperature used for enzymatic deamidation of the dairy or plant material is 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. Preferably, the temperature used for enzymatic deamidation is in the range of 55°C-65°C, more preferably around 60°C.

[0092] In an embodiment, the treatment with the protein deamidase is carried out at 20-80°C for at least 10 minutes. In some embodiments, the treatment with the protein deamidase is carried out for about 10, about 15, about 20, about 25, about 30, about 60, about 120, about 180, or about 240 minutes. In some embodiments, the treatment with the protein deamidase is carried out for about 10, 30, or 60 minutes. In preferred embodiments, the treatment with the protein deamidase is carried out for 30-60 minutes.

[0093] After the enzymatic deamidation of the dairy or plant material, the protein deamidase may be inactivated. The enzyme may be inactivated at any step after hydrolysis. In some embodiments, the protein deamidase 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 90°C for 5, 10, 15, 20, 25, or 30 minutes.

[0094] In some embodiments, the protein deamidase 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.

[0095] The process used, including temperature ranges, pH and the length of enzymatic treatment, will vary depending on the dairy or plant material and optionally further added enzymes. The skilled person will know how to determine the best process parameters based on the enzyme(s) and the dairy or plant material used.

[0096] In the methods of the invention, a coffee component is treated with an enzyme having mannanase activity to obtain a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity. Without wishing to be bound by any theory, it is believed that the use of an enzyme having mannanase activity to treat a coffee component results in the polysaccharides, such as the mannans, present in the coffee being broken down resulting in a reduction of turbidity and improvement of clarity and stability of the final ready-to- drink coffee beverage. The reduction in turbidity of the coffee composition has the added benefit of reducing further haze formation and sedimentation forming over time, e.g., during long-term storage of the ready-to-drink coffee beverage comprising the coffee composition. These observations are supported at least by the findings reported in Example 3 herein, wherein it was shown that mannanase treatment of a cold brew sample had the effect of significantly reducing particle size, immediate turbidity formation and turbidity and sedimentation forming after longterm storage of the cold brew. Additionally, and as shown in Example 2 herein, the combination of a mannanase-treated coffee with a deamidase-treated plant- or dairy-based beverage to obtain the RTD coffee beverage as claimed herein displayed a further improvement in storage stability in that no precipitation or sedimentation was seen in the samples treated with both enzymes. This synergistic effect of the two enzymes in reducing turbidity and sedimentation in RTD coffee beverages is not previously described in prior art and thus constitutes a surprising finding. As used herein, the term “coffee component” refers to the part of the coffee composition that is treated with an enzyme having mannanase activity. For example, the coffee component may be an aqueous suspension obtained from brewing roasted coffee beans, a coffee extract, a coffee concentrate or a soluble coffee, such as an instant coffee powder. In an embodiments, the coffee component has a high yield of dry matter. In an embodiment, the coffee component is or is derived from instant coffee solids, a coffee extract, roasted ground coffee beans, or a coffee concentrate. In an embodiment, the coffee component is or is derived from instant coffee solids, roasted ground coffee beans, or a coffee concentrate.

[0097] In an embodiments, the coffee component is or is derived from a coffee extract. As used herein, a “coffee extract” is meant as a concentrated substance that is produced by brewing coffee beans in water. Such coffee extract contains the flavors, oils, and other chemical components of the coffee beans, and can be used to add coffee flavor to food and drink products. It can come in various forms such as liquid, powder, or oil. The coffee extract can be dehydrated, such as a soluble coffee or dry mix composition, or it can be a liquid mix composition, a frozen composition or a liquid concentrate composition.

[0098] The methods of the invention can be applied to any coffee component obtained by conventional coffee processing. The coffee component may be obtained directly out of an industrial process. In an embodiment, the coffee component is a cold coffee brew, a warm coffee brew, or a coffee brew prepared from soluble coffee.

[0099] In an embodiment, the coffee component is obtained using an enzymatically aided coffee extraction process.

[0100] In an embodiment, the coffee component is produced using a method comprising the steps of: i. providing roast and ground coffee beans; ii. optionally performing one or more first extractions of said coffee beans; iii. adding to said coffee beans, which have optionally been subjected to one or more first extractions, water and an enzyme having mannanase activity; iv. incubating to make an aqueous coffee component; and v. separating the coffee component from the extracted coffee beans.

[0101] The coffee component may be separated from extracted coffee beans by any means known in the art.

[0102] In an embodiment, the coffee component is essentially free of initial insoluble solids.

[0103] In an embodiment, the coffee component has been subjected to a step of centrifugation or filtration prior to the treatment with the enzyme having mannanase activity. In another preferred embodiment, the coffee component has been subjected to a step of centrifugation or filtration after the treatment with the enzyme having mannanase activity and before mixing the coffee composition with the dairy or plant composition. For example, the filtration of the coffee component may be a belt filtration, which removes most of the insoluble solids, prior to the treatment with the enzyme having mannanase activity. Further, the separated coffee component, still containing, e.g., fine particulates and oil, may be recirculated through a cross-flow membrane device, which removes all initial insolubles.

[0104] In an embodiment, the enzyme having mannanase activity has an enzyme activity profile resulting in a low concentration of free monosaccharides in the coffee component. Preferably, the free monosaccharides are selected from mannose, galactose and a combination thereof. The low concentration of the free monosaccharides in the enzyme-treated coffee component may be determined by any method deemed suitable by a person of skill in the art. For example, the content of free monosaccharides in the coffee component may be determined by High pH anion- exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and capillary electrophoresis (CE) or high performance liquid chromatography (HPLC) separation with UV detection. An aspect of the invention therefore relates to a coffee component having been treated with an enzyme having mannanase activity which coffee component has a lower concentration of free monosaccharides compared to a coffee component having been treated with an enzyme having an enzyme activity profile different from the enzyme having mannanase activity as disclosed and claimed herein. In an embodiment, the lower concentration of free monosaccharides in the coffee component is obtained using an enzyme having a sequence identity to the polypeptide of SEQ ID NO: 12 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, a higher concentration of free di- or trisaccharides in the coffee component is obtained using an enzyme having a sequence identity to the polypeptide of SEQ ID NO: 12 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, a higher concentration of free trisaccharides in the coffee component is obtained using an enzyme having a sequence identity to the polypeptide of SEQ ID NO: 12 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. A further aspect of the invention relates to a coffee component having been treated with an enzyme having mannanase activity, which coffee component has a mannose and galactose concentration below a certain threshold. In the context of the invention, the use of an enzyme having mannanase activity enables obtaining of a ready-to-drink coffee beverage having a lower amount of free monosaccharides, such as, e.g., galactose and mannose, compared to a ready-to-drink coffee beverage prepared without the use of the enzyme having mannanase activity according to this invention. Thereby, a ready-to-drink coffee beverage can be obtained having a reduced or low sugar content. The treatment with the enzyme having mannanase activity is to be performed at a temperature where the enzymes are active and for sufficiently long time to permit enzyme reaction. The incubation is to be performed at a temperature where the enzyme having mannanase activity is active, typically in the range of about 25°C to about 100°C.

[0105] Preferably, the enzyme having mannanase activity is a thermostable enzyme. In the aspects of the invention where a thermostable enzyme is used, incubation may be performed at a temperature typically in the range of about 50°C to about 100°C, such as about 60°C to about 100°C, about 70°C to about 100°C, or about 80°C to about 100°C. In an embodiment, incubation with the enzyme having mannanase activity is performed at a temperature in the range of 3-30°C, such as at a temperature of about 4-25°C.

[0106] The incubation with the enzyme having mannanase activity may be performed for about 30 minutes to about 48 hours, such as about 1 hour to about 24 hours or about 2 to about 24 hours to permit enzyme reaction. In preferred embodiments, the incubation with the enzyme having the mannanase activity is at 60-80°C for 30-120 minutes, such as at 60°C for about 60 minutes.

[0107] In some embodiment, the enzyme having mannanase activity is inactivated. The enzyme may be inactivated at any step in the process. For example, the inactivation of the enzyme having mannanase activity may be before or after the coffee composition is mixed with the dairy or plant composition to obtain the ready-to-drink coffee beverage. In an embodiment, the enzyme having mannanase activity is inactivated by heat treatment of the ready-to-drink coffee beverage. In some embodiments, the enzyme having mannanase activity 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 other embodiments, the enzyme having mannanase activity is not inactivated.

[0108] In some embodiments, the enzyme having mannanase activity 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.

[0109] Suitable processes to make a coffee composition from a coffee component are known to a person skilled in the art. As used herein, the term “coffee composition” refers to a suspension comprising at least a coffee component having been subjected to treatment with an enzyme having mannanase activity. The coffee composition may comprise one or more additional components or ingredients. In some embodiments, the coffee composition is an aqueous suspension comprising a coffee component having been subjected to treatment with an enzyme having mannanase. The specific composition and concentration of the coffee component in the coffee composition can vary depending on the desired flavor profile, caffeine content, acidity, and other attributes of the final RTD coffee beverage. In some embodiments, the coffee composition has a pH of 3-7, such as a pH of about 3.5-6.5, 4.5-6.0, or 5-5.5. In some embodiments, the coffee composition has a pH of 5-5.5. In other embodiments, the coffee composition is an acidic coffee composition having pH below 5.0.

[0110] In some embodiments, the dairy or plant composition and the coffee composition have a temperature in the range of 3-30°C prior to the mixing. The ability to mix the coffee composition and the dairy or plant composition at cool and / or room temperatures provides manufacturers of ready-to-drink coffee beverages with improved production methods, which are cost and energy efficient and more reliable, i.e. avoiding the risk of flocculation upon and / or after preparation of the RTD drink.

[0111] Protein deamidase

[0112] In the methods of the invention, a dairy or plant material is treated with a protein deamidase to obtain a dairy or plant composition comprising enzymatically deamidated dairy or plant material.

[0113] A further aspect of the invention relates to the use of protein deamidase to obtain a coffee component. Thus, some embodiments of the invention further relates to a method of obtaining an enzymatically deamidated coffee component. The coffee component treated with protein deamidase may be or be derived from, e.g., instant coffee solids, a coffee extract, roasted ground coffee beans, spent coffee grounds or a coffee concentrate. In a preferred embodiment, protein deamidase is used to treat spent coffee grounds to produce a coffee component. For example, such coffee component obtained by treatment with at least a protein deamidase may find use as a protein ingredient for use in food and beverages applications. Without wishing to be bound by any particular theory, the inventors believe the use of protein deamidase to treat spent coffee grounds in the production of a coffee component leads to increased yield as more dry matter is extracted, increased solubility, reduced sedimentation and haze formation, and more flavor in the resulting in the coffee composition, such as a more full body of the coffee composition.

[0114] In an embodiment, both a protein deamidase and an enzyme having mannanase activity is used in the production of the coffee component.

[0115] 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 without crosslinking the protein.

[0116] 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 alphaamino and carboxyl positions, e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine. Thus, deamidases can deamidate glutamine residues in proteins to glutamate residues and are also referred to as protein glutamine deamidase. Deamidases comprise a Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al. “Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex”, Journal of Biological Chemistry, vol. 286, no. 44, pp. 38691-38702) and belong to the InterPro entry IPR041325.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] The protein deamidase may be purified. The term "purified" as used herein covers protein deamidase enzyme protein essentially free from insoluble components from the production organism. The term "purified" also covers protein deamidase enzyme protein essentially free from insoluble com-ponents from the native organism from which it is obtained. Preferably, it is also separated from some of the soluble components of the organism and culture medium from which it is derived. More preferably, it is separated by one or more of the unit operations: filtration, precipitation, or chromatography. 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.

[0121] 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.

[0122] In some embodiments, the protein deamidase may be derived from Chryseobacterium genus, such as Chryseobacterium viscerum, 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.

[0123] 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.).

[0124] For example, protein deamidases can be obtained from a culture broth of the abovedescribed microorganisms.

[0125] 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.

[0126] 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.

[0127] The methods and compositions of the invention include a mature deamidase and optionally a second polypeptide which is derived from the propeptide of a deamidase. The second polypeptides described herein are mutated variants of the naturally occurring propeptides. These variant propeptide sequences have been found to bind less strongly to their corresponding deamidase, so that they are more easily enzymatically cleaved off after recombinant expression and secretion from of the host cell. The polypeptides of SEQ ID NOs: 1 to 10 are derived from Chryseobacterium spp. and are described in PCT application PCT / EP2023 / 055936; filed March 8, 2023, herein incorporated by reference.

[0128] 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.

[0129] According to a preferred embodiment the protein deamidase applied in the process of the invention is derived from or obtained from a Chryseobacterium species, e.g., Chryseobacterium proteolyticum or Chryseobacterium viscerum.

[0130] In the context of the present invention, the term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one embodiment, the mature polypeptide is obtained 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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:

[0141] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0142] 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.

[0143] The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0144] 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.

[0145] 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.

[0146] 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 example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

[0147] 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).

[0148] 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.

[0149] 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 0.1-15 IPA(U) / g substrate protein, 0.5-10 IPA(U) / g substrate protein.

[0150] 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.

[0151] 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.

[0152] Enzyme having mannanase activity

[0153] In the methods of the invention, a coffee component is treated with an enzyme having mannanase activity to obtain a coffee composition comprising the coffee component.

[0154] In an embodiment, the coffee component is treated with an enzyme having mannanase activity and a protein deamidase to obtain a coffee composition. For example, the coffee component may be treated simultaneously or sequentially with a mannanase and protein deamidase to obtain the coffee composition.

[0155] In the context of the present invention a “mannanase” is a beta-mannanase. It may be an enzyme defined according to the art as a mannan endo-1 , 4-p-mannosidase (EC 3.2.1.78) which catalyzes the hydrolysis of 1 ,4-p-D-mannosidic linkages in mannans, galactomannans and glucomannans, which enzyme has the alternative names: mannan endo-1 ,4-p-mannosidase; 1 ,4- P-D-mannan mannanohydrolase; endo-1 , 4-p-mannanase; p-mannanase B; p-1 ,4-mannan 4- mannanohydrolase; endo-p-mannanase; and p-D-mannanase. For purposes of the present invention, mannanase activity may be determined using the activity assay described by Staalbrand etal. (1993), Purification and characterization of two p-mannanases from Trichoderma reesei, J. Biotechnol., 29:229-42.

[0156] In an embodiment, the enzyme having mannanase activity is an endo-beta-1 ,4- mannanase, preferably a GH5 endo-beta-1 , 4-mannanase, more preferably a GH5_7 endo-beta- 1 ,4-mannanase or a GH5_8 endo-beta-1 , 4-mannanase. In a preferred embodiment, the enzyme having mannanase activity is a GH5_7 endo-beta-1 , 4-mannanase. In another preferred embodiment, the enzyme having mannanase activity is a GH5_8 endo-beta-1 , 4-mannanase.

[0157] In an embodiment, the enzyme having mannanase activity is thermostable. In some embodiment, the enzyme having mannanase activity has a melting temperature (Tm) determined by Differential Scanning Calorimetry (DSC) of at least 80°C, preferably at least 85°C, more preferably at least 90°C. The Tmmay be determined as described, e.g., in Example 4 of WO 2016 / 207384 A1.

[0158] In an embodiment, the enzyme having mannanase activity is essentially free of exo activity. Using such enzyme having mannanase activity and being essentially free of exo activity may result in obtaining a coffee component having a low concentration of free monosaccharides.

[0159] An enzyme having mannanase activity to be used in the 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.

[0160] Mannanases have been identified, e.g., in several Bacillus organisms. For example, Talbot et al. (1990), Appl. Environ. Microbiol., Vol. 56, No. 11 , pp. 3505-3510, describes a beta- mannanase derived from Bacillus stearothermophilus having an optimum pH of 5.5-7.5. Mendoza et al. (1994), World J. Microbiol. Biotech., Vol. 10, No. 5, pp. 551-555, describes a p-mannanase derived from Bacillus subtilis having an optimum activity at pH 5.0 and 55°C. JP-03047076 discloses a p-mannanase derived from Bacillus sp., having an optimum pH of 8-10. JP-63056289 describes the production of an alkaline, thermostable p-mannanase. JP-08051975 discloses alkaline p-mannanases from alkalophilic Bacillus sp. AM-001. A purified mannanase from Bacillus amyloliquefaciens is disclosed in WO 97 / 11164. WO 94 / 25576 discloses an enzyme from Aspergillus aculeatus, CBS 101.43, exhibiting mannanase activity and WO 93 / 24622 discloses a mannanase isolated from Trichoderma reesei. A suitable commercial mannanase preparation is Mannaway® produced by Novozymes A / S.

[0161] The enzyme having mannanase activity may be a fungal enzyme. For example, the enzyme may be obtained from yeast such as from Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia', or from a filamentous fungus such as from Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.

[0162] In another embodiment, the enzyme is obtained from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.

[0163] In another embodiment, the enzyme is obtained from Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0164] In an embodiment, the enzyme is obtained from Talaromyces, e.g., from Talaromyces leycettanus.

[0165] In another embodiment, the enzyme is obtained from Chaetomium, e.g., from Chaetomium virescens.

[0166] In another embodiment, the enzyme is obtained from Sordaria, e.g., from Sordaria macrospora.

[0167] In another embodiment, the enzyme is obtained from Caldicellulosiruptor, e.g., from Caldicellulosiruptor saccharolyticus.

[0168] It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0169] The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sam brook et a / ., 1989, supra).

[0170] In one embodiment, the enzyme having mannanase activity is not obtained from Aspergillus niger.

[0171] In an embodiment, the enzyme having mannanase activity has a sequence identity to the polypeptide of SEQ ID NO: 12 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, the enzyme differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 12. In one embodiment, such enzyme is thermostable.

[0172] In an embodiment, the enzyme having mannanase activity has a sequence identity to the polypeptide of SEQ ID NO: 14 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, the enzyme differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 14.

[0173] In an embodiment, the enzyme having mannanase activity has a sequence identity to the polypeptide of SEQ ID NO: 16 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, the enzyme differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 16.

[0174] In an embodiment, the enzyme having mannanase activity has a sequence identity to the polypeptide of SEQ ID NO: 18 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, the enzyme differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 18. In one embodiment, such enzyme is thermostable.

[0175] In an embodiment, the enzyme having mannanase activity has a sequence identity to the polypeptide of SEQ ID NO: 19 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an embodiment, the enzyme differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 19.

[0176] The enzyme having mannanase activity may be added at a concentration of at least 0.001 g enzyme protein / kg dry matter, preferably at least 0.005 g enzyme protein / kg dry matter, such as at a concentration of 0.001-1 g enzyme protein / kg dry matter, preferably 0.005-0.5 g enzyme protein / kg dry matter.

[0177] The enzyme having mannanase activity may be added at a concentration of at least 0.001 g enzyme protein / kg coffee beans, preferably at least 0.005 g enzyme protein / kg coffee beans, such as at a concentration of 0.001-0.5 g enzyme protein / kg coffee beans, preferably 0.005-0.2 g enzyme protein / kg coffee beans.

[0178] The enzyme having mannanase activity is preferably added as an enzymatic preparation characterized in that at least 5%, preferably at least 10% or at least 20%, of the total protein in the preparation is an enzyme having mannanase activity as its predominant enzymatic activity.

[0179] The enzyme having mannanase activity may be added as a mixture with other enzymes such as, e.g., cellulase and / or galactanase enzyme(s). In such enzymatic composition, the enzyme having mannanase activity may be the major enzymatic component, e.g., a monocomponent composition. Alternatively, the compositions may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, beta- xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. In an embodiment, the enzyme having mannanase activity is added as a composition comprising the enzyme having mannanase activity and an enzyme having amylase activity, such as an enzyme having glucoamylase activity.

[0180] The compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art. The invention is further defined by the following numbered embodiment:

[0181] Embodiment 1 . A method for obtaining a ready-to-drink coffee beverage, comprising the steps of:

[0182] (a) providing a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material, wherein a protein deamidase is used for the enzymatic deamidation;

[0183] (b) providing a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity; and

[0184] (c) mixing the dairy or plant composition of step (a) with the coffee composition of step (b) to obtain the ready-to-drink coffee beverage.

[0185] Embodiment 2. Method of embodiment 1 , wherein the ready-to-drink coffee beverage has reduced turbidity and / or gelation compared to a ready-to-drink coffee beverage obtained using a similar method but without the use of a protein deamidase and an enzyme having mannanase activity.

[0186] Embodiment 3. Method of embodiment 2, wherein the reduced turbidity and / or gelation is immediately upon obtaining the ready-to-drink coffee beverage and / or following storage of the ready-to-drink coffee beverage.

[0187] Embodiment 4. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage has reduced sedimentation compared to a ready-to-drink coffee beverage obtained using a similar method but without the use of a protein deamidase and an enzyme having mannanase activity.

[0188] Embodiment 5. Method of embodiment 4, wherein the reduced sedimentation is immediately upon obtaining the ready-to-drink coffee beverage and / or following storage of the ready-to-drink coffee beverage.

[0189] Embodiment 6. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is meant for storage at 3-70°C, such as storage at 4-8°C and / or at 15-25°C and / or at 55-65°C.

[0190] Embodiment 7. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is meant for storage at 4-8°C or at 15-25°C.

[0191] Embodiment 8. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is stored for at least 4 hours before being consumed.

[0192] Embodiment 9. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is stored for at least 8 hours before being consumed, preferably at least 12 hours before being consumed.

[0193] Embodiment 10. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is stored for at least 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 before being consumed. Embodiment 11 . Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is stored for at least 7 days, 14 days, 21 days, or 1 month before being consumed.

[0194] Embodiment 12. Method of any of the preceding embodiments, wherein the ratio of dairy or plant composition to coffee composition in the ready-to-drink coffee beverage is from 1 :10 to 10:1 based on w / w.

[0195] Embodiment 13. Method of any of the preceding embodiments, wherein the ratio of dairy or plant composition to coffee composition in the ready-to-drink coffee beverage is 1 :1 , 2:1 or 3:1 based on w / w.

[0196] Embodiment 14. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is essentially free of added emulsifiers and / or stabilizers.

[0197] Embodiment 15. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is essentially free of carboxymethylcellulose and / or microcrystalline cellulose.

[0198] Embodiment 16. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is essentially free of carboxymethylcellulose.

[0199] Embodiment 17. Method of any of the preceding embodiments, wherein the dairy composition or plant composition comprises one or more additional food ingredients.

[0200] Embodiment 18. Method of any of the preceding embodiments, wherein the dairy composition or plant composition 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.

[0201] Embodiment 19. Method of embodiment 18, wherein the additional food ingredient is a lipid, such as an oil, preferably a plant oil, and / or a sugar, such as a sucrose, and / or calcium carbonate.

[0202] Embodiment 20. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is subjected to heat treatment after mixing the dairy or plant composition of step (a) with the coffee composition of step (b).

[0203] Embodiment 21. Method of embodiment 20, wherein the heat treatment is an Extended Shelf-Life (ESL), Low Temperature Long Time (LTLT), High Temperature Short Time (HTST), Higher Heat Shorter Time (HHST), Ultra-High Temperature (UHT), or Ultra Pasteurized (UP) treatment, preferably a UHT or ESL treatment.

[0204] Embodiment 22. Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage is a canned beverage or a packaged beverage.

[0205] Embodiment 23. Method of any of the preceding embodiments, wherein the plant material is derived or obtained from almond, cashew, chickpea, coconut, fava bean, hazelnut, lentil, lupin, macadamia, mung bean, peanut, pecan, pistachio, corn, quinoa, flax, hemp, oat, spelt, pea, rice, sesame, sunflower, soy, walnut, or any combination thereof. Embodiment 24. Method of any of the preceding embodiments, wherein the plant material is derived or obtained from almond, pea, soy, oat or any combination thereof.

[0206] Embodiment 25. Method of any of the preceding embodiments, wherein the plant material is derived or obtained from pea or soy.

[0207] Embodiment 26. Method of any of the preceding embodiments, wherein the dairy composition comprises cream, milk, concentrated milk, condensed milk, evaporated milk, reconstituted milk, milk protein concentrate, whey protein concentrate, micellar casein, or any combination thereof.

[0208] Embodiment 27. Method of any of the preceding embodiments, wherein the dairy composition comprises 3.8% or less, 3.25% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.25% or less, or 0.15% or less fat.

[0209] Embodiment 28. Method of any of the preceding embodiments, wherein the dairy composition is whole milk, low fat milk, or skim milk.

[0210] Embodiment 29. The method of any of embodiments, wherein the dairy composition is heat treated prior to treatment with the protein deamidase.

[0211] Embodiment 30. The method of embodiment 29, wherein the heat treatment is performed at 60-140°C or wherein the heat treatment is a Low Temperature Long Time (LTLT), High Temperature Short Time (HTST), Higher Heat Shorter Time (HHST), Ultra-High Temperature (UHT), or Ultra Pasteurized (UP) treatment.

[0212] Embodiment 31. Method of any of embodiments 1-28, wherein the dairy composition is not heat treated prior to treatment with the deamidase.

[0213] Embodiment 32. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase is at a temperature in the range of 50-70°C, preferably in the range of 55- 65°C.

[0214] Embodiment 33. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase is at a temperature of about 60°C.

[0215] Embodiment 34. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase is carried out for at least 10 minutes, preferably for 15-90 minutes, such as for 30-60 minutes.

[0216] Embodiment 35. Method of any of the preceding embodiments, wherein the treatment with the protein deamidase is in the presence of a chloride salt, preferably in the presence of potassium chloride or a sodium chloride.

[0217] Embodiment 36. Method of any of the preeceding embodiments, wherein the protein deamidase is inactivated by a heat treatment, such as an Ultra-High Temperature (UHT) treatment.

[0218] Embodiment 37. Method of any of the preceding embodiments, wherein the protein deamidase is derived from or obtained from a Chryseobacterium species, such as from Chryseobacterium proteolyticum or Chryseobacterium viscerum. Embodiment 38. Method of any of the preceding embodiments, wherein the protein deamidase comprises a polynucleotide sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NOs: 2, 4, 6, 8, or 10.

[0219] Embodiment 39. Method of any of the preceding embodiments, wherein the protein deamidase comprises the polynucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, or 10.

[0220] Embodiment 40. Method of any of the preceding embodiments, wherein the coffee composition of step (b) has a pH of 3-7, such as a pH of 3.5-6.5, 4.5-6.5, or 5.0-5.5.

[0221] Embodiment 41. Method of any of the preceding embodiments, wherein the coffee composition of step (b) has a pH below 5.0.

[0222] Embodiment 42. Method of any of the preceding embodiments, wherein the coffee component is obtained using an enzymatically aided coffee extraction process.

[0223] Embodiment 43. Method of any of the preceding embodiments, wherein the coffee component is an enzymatically deamidated coffee component.

[0224] Embodiment 44. Method of any of the preceding embodiments, wherein the coffee component is an enzymatically deamidated coffee component obtained by treating a coffee component with a protein deamidase.

[0225] Embodiment 45. Method of any of the preceding embodiments, wherein the coffee component is or is derived from instant coffee solids, a coffee extract, roasted ground coffee beans, or a coffee concentrate.

[0226] Embodiment 46. Method of any of the preceding embodiments, wherein the coffee component is or is derived from instant coffee solids, roasted ground coffee beans, or a coffee concentrate.

[0227] Embodiment 47. Method of any of the preceding embodiments, wherein the coffee component is essentially free of initial insoluble solids.

[0228] Embodiment 48. Method of any of the preceding embodiments, wherein the coffee component has been subjected to a step centrifugation or filtration, prior to the treatment with the enzyme having mannanase activity.

[0229] Embodiment 49. Method of any of the preceding embodiments, wherein the coffee component has been subjected to a step of filtration after the treatment with the enzyme having mannanase activity and before mixing the coffee composition with the dairy or plant composition.

[0230] Embodiment 50. Method of any of the preceding embodiments, wherein the coffee component is obtained by treatment with the enzyme having mannanase activity at a temperature of at least 60°C, such as at least 65°C, preferably at least 70°C such as at least 75°C or at least 80°C.

[0231] Embodiment 51. Method of any of the preceding embodiments, wherein the coffee component is obtained by treatment with the enzyme having mannanase activity for at least 30 minutes, preferably for at least 1 hour or at least 2 hours. Embodiment 52. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity has at least 60% sequence identity, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 19.

[0232] Embodiment 53. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity has at least 60% sequence identity, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to SEQ ID NO: 12 or SEQ ID NO: 18.

[0233] Embodiment 54. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity has at least 60% sequence identity, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to SEQ ID NO: 12.

[0234] Embodiment 55. Method of any embodiments 1-53, wherein the enzyme having mannanase activity has at least 60% sequence identity, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to SEQ ID NO: 18.

[0235] Embodiment 56. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity is thermostable.

[0236] Embodiment 57. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity has a melting temperature (Tm) determined by Differential Scanning Calorimetry (DSC) of at least 80°C, preferably at least 85°C or at least 90°C.

[0237] Embodiment 58. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity is an endo-beta-1 , 4-mannanase.

[0238] Embodiment 59. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity is a GH5 endo-beta-1 , 4-mannanase, preferably a GH5_7 endo-beta- 1 , 4-mannanase or a GH5_8 endo-beta-1, 4-mannanase.

[0239] Embodiment 60. Method of any of the preceding embodiments, wherein the enzyme having mannanase activity is essentially free of exo activity.

[0240] Embodiment 61 . Method of any of the preceding embodiments, wherein the ready-to-drink coffee beverage has an increased di- and / or trisaccharide content compared to a ready-to-drink coffee beverage prepared without the use of an enzyme having mannanase activity.

[0241] Embodiment 62. A ready-to-drink coffee beverage obtainable by a method of any of the preceding embodiments.

[0242] Embodiment 63. A ready-to-drink coffee beverage comprising: (a) a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material, wherein a protein deamidase is used for the enzymatic deamidation; and

[0243] (b) a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity, wherein the ready-to-drink coffee beverage has reduced turbidity and / or gelation compared to a ready-to-drink coffee beverage obtained without the use of a protein deamidase and an enzyme having mannanase activity.

[0244] Embodiment 64. Ready-to-drink coffee beverage of embodiment 63, wherein the coffee component is essentially free of insoluble solids.

[0245] Embodiment 65. Ready-to-drink coffee beverage of embodiments 63-64, wherein the coffee component has been subjected to a step of centrifugation or filtration before and / or after the treatment with the enzyme having mannanase activity.

[0246] Embodiment 66. Ready-to-drink coffee beverage of embodiments 63-65, wherein the coffee component has been subjected to a step of centrifugation or filtration before the treatment with the enzyme having mannanase activity.

[0247] Embodiment 67. Ready-to-drink coffee beverage of embodiments 63-66, wherein the ready-to-drink coffee beverage has an increased di- and / or trisaccharide content compared to a ready-to-drink coffee beverage prepared without the use of an enzyme having mannanase activity.

[0248] Embodiment 68. Use of a protein deamidase and an enzyme having mannanase activity in the production of a ready-to-drink coffee beverage to reduce turbidity and / or gelation.

[0249] Embodiment 69. Use of embodiment 68, wherein the ready-to-drink coffee beverage comprises a blend of a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material; and a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity.

[0250] Embodiment 70. Use of embodiment 68-69, wherein the protein deamidase is used to treat the dairy material or plant material and the enzyme having mannanase activity is used to treat the coffee component.

[0251] Embodiment 71. Use of embodiments 68-70, wherein the ready-to-drink coffee beverage has reduced sedimentation compared to a ready-to-drink coffee beverage obtained without the use of a protein deamidase and an enzyme having mannanase activity.

[0252] Embodiment 72. Use of a protein deamidase in the production of a coffee composition to increase dry matter content, reduce sedimentation, reduce haze formation, and / or improve flavor.

[0253] Embodiment 73. Use according to embodiment 72, wherein the coffee composition is intended for use as a cold brew coffee or in a ready-to-drink coffee beverage. Embodiment 74. Use according to any of embodiments 72-73, wherein the coffee composition is a cold brew coffee or a ready-to-drink coffee beverage.

[0254] Embodiment 75. Use according to any of embodiments 72-74, wherein the coffee composition is or is derived from instant coffee solids, roasted ground coffee beans, spent coffee grounds, a coffee concentrate, or a coffee extract.

[0255] Embodiment 76. Use according to any of embodiments 72-75, wherein the coffee composition is derived from spent coffee grounds.

[0256] Embodiment 77. Use of an enzyme having mannanase activity in the production of a coffee composition to improve shelf-stability.

[0257] Embodiment 78. Use according to embodiment 77, wherein the coffee composition has reduced turbidity and / or haze formation compared to a coffee composition obtained without the use of an enzyme having mannanase activity.

[0258] Embodiment 79. Use according to any of embodiments 77-78, wherein the coffee composition has reduced initial turbidity and / or haze formation compared to a coffee composition obtained without the use of an enzyme having mannanase activity.

[0259] Embodiment 80. Use according to any of embodiments 77-79, wherein the coffee composition has reduced turbidity and / or haze formation after storage for at least 24 hours compared to a coffee composition obtained without the use of an enzyme having mannanase activity.

[0260] Embodiment 81. Use according to any of embodiments 77-80, wherein the enzyme with mannanase activity is a GH5 endo-beta-1 , 4-mannanase, preferably a GH5_7 endo-beta-1 ,4- mannanase or a GH5_8 endo-beta-1 , 4-mannanase.

[0261] Embodiment 82. Use according to any of embodiments 77-81 , wherein the enzyme having mannanase activity is essentially free of exo activity.

[0262] Embodiment 83. Use according to any of embodiments 77-82, wherein the coffee composition is a cold brew.

[0263] Embodiment 84. Use according to any of embodiments 77-83, wherein the coffee composition has an increased di- and / or trisaccharide content compared to a coffee composition prepared without the use of an enzyme having mannanase activity.

[0264] Embodiment 85. Use of a protein deamidase and an enzyme having mannanase activity in the production of a coffee composition.

[0265] Embodiment 86. Use according to embodiment 85, wherein the coffee composition is or is derived from instant coffee solids, roasted ground coffee beans, a coffee concentrate, a coffee extract or spent coffee grounds.

[0266] Embodiment 87. Use according to any of embodiments 85-86, wherein the coffee composition is derived from spent coffee grounds.

[0267] Embodiment 88. Use according to any of embodiments 85-87, wherein the coffee composition has an increased yield of dry matter and / or increased coffee flavor compared to a coffee composition obtained without the use of protein deamidase and an enzyme having mannanase activity.

[0268] Embodiment 89. Use of an enzyme having mannanase activity in the production of a coffee composition to increase dry matter content and / or reduce viscosity.

[0269] Embodiment 90. Use according to embodiment 89, wherein the coffee composition is obtained by treating a coffee component with the enzyme having mannanase activity, wherein the coffee component is roasted ground coffee beans, a coffee concentrate, a coffee extract or spent coffee grounds.

[0270] Embodiment 91. Use according to any of embodiments 89-90, wherein the coffee component is a coffee extract.

[0271] Embodiment 92. Use according to any of embodiments 89-91 , wherein the coffee component is subjected to freeze- and or spray-drying after treatment with the enzymes having mannanase activity.

[0272] Embodiment 93. Use according to any of embodiment 89-92, wherein the coffee composition is a soluble coffee, such as an instant coffee powder.

[0273] Embodiment 94. A method for producing a soluble coffee, comprising the steps:

[0274] - providing an aqueous coffee extract;

[0275] - treating the aqueous coffee extract with an enzyme having mannanase activity to obtain an enzymatically treated aqueous coffee extract;

[0276] - concentrating the enzymatically treated aqueous coffee extract to obtain a concentrated coffee extract;

[0277] - optionally separating the concentrated coffee extract from insoluble solids, for example by a step of centrifugation; and

[0278] - drying the concentrated coffee extract to obtain the soluble coffee.

[0279] Embodiment 95. Method of embodiment 94, wherein the step of drying the concentrated coffee extract is a step of freeze-drying or spray-drying.

[0280] Embodiment 96. Method of any of embodiments 94-95, wherein the step of concentrating the enzymatically treated aqueous coffee is by evaporation, preferably using an evaporator.

[0281] Embodiment 97. Method of any of embodiments 94-96, wherein the aqueous coffee extract is obtained by a method comprising the steps of:

[0282] - providing roast and ground coffee beans;

[0283] - optionally performing one or more first extractions of said coffee beans;

[0284] - adding to said coffee beans, which have optionally been subjected to one or more first extractions, water and optionally an enzyme having mannanase activity;

[0285] - incubating to make the aqueous coffee extract; and

[0286] - separating the aqueous coffee extract from the extracted coffee beans. Embodiment 98. Method of any of embodiments 94-97, wherein the enzyme having mannanase activity is a GH5 endo-beta-1, 4-mannanase, preferably a GH5_7 endo-beta-1,4- mannanase or a GH5_8 endo-beta-1 , 4-mannanase.

[0287] Embodiment 99. Method of any of embodiments 94-98, wherein the enzyme having mannanase activity is essentially free of exo activity.

[0288] Embodiment 100. Method of any of embodiments 94-99, wherein the enzyme having mannanase activity has at least 70% sequence identity to SEQ ID NO: 12.

[0289] Embodiment 101. Method of any of embodiments 94-100, wherein the concentrated coffee extract has reduced viscosity and / or increased dry matter content compared to a concentrated coffee extract obtained without the use of an enzyme having mannanase activity.

[0290] Embodiment 102. Method of any of embodiments 94-101, wherein the concentrated coffee extract has increased amounts of mannan oligosaccharides in solution compared to a concentrated coffee extract obtained without the use of an enzyme having mannanase activity.

[0291] Embodiment 103. Method of the preceding embodiment, wherein the mannan oligosaccharides are di- and / or trisaccharides.

[0292] Embodiment 104. Method of any of embodiments 94-103, wherein the soluble coffee has an increased di- and / or trisaccharide content compared to a soluble coffee prepared without the use of an enzyme having mannanase activity.

[0293] Embodiment 105. Method of any of embodiments 94-104, wherein the soluble coffee is an instant coffee powder.

[0294] Embodiment 106. A soluble coffee obtainable by a method as embodied in any of embodiments 94-105.

[0295] 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.

[0296] 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.

[0297] EXAMPLES

[0298] Materials

[0299] Enzymes

[0300] The following enzymes are used throughout the examples:

[0301] Protein deamidase: Protein glutaminase derived from Chryseobacterium viscerum 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 sitespecific endopeptidase used is a glutamyl endopeptidase from Bacillus licheniformis. The resulting active deamidase after maturation was the polypeptide shown in SEQ ID NO: 2.

[0302] Enzyme having mannanase activity: Mannanase derived from Talaromyces leycettanus having the mature polypeptide sequence shown as SEQ ID NO: 12.

[0303] Example 1 : Protein deamidase activity assay

[0304] The activity assay consists of two separate de-coupled parts:

[0305] 1) An enzymatic step wherein ammonia is formed by the catalytic action of the protein deamidase; and

[0306] 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.

[0307] 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.

[0308] Assay Procedure:

[0309] Step (1) Enzymatic step with ammonia formation

[0310] Reagents:

[0311] Assay dilution solution: 0.2 M Na-phosphate buffer, 0.01% Triton X-100, pH 6.5.

[0312] Assay buffer: Same as above. Used to prepare stock solution and diluted sample of protein deamidase (referred to in the following as “enzyme”).

[0313] Substrate solution: 30 mM Z-GIn-Gly (Merck C6154-1G) in assay dilution solution (check pH after dissolution).

[0314] Stop solution: 0.4M TCA.

[0315] 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).

[0316] Dissolve / dilute enzyme product in assay buffer and prepare suitable dilution resulting in a linear assay response.

[0317] Incubation: 1. Add 10 pL of diluted enzyme samples in triplicates to the wells of a 96-well microtiter plate (MTP).

[0318] 2. Add 100 pL of substrate solution to each well.

[0319] 3. For blank samples add 100 pL 0.4M TCA solution.

[0320] 4. Seal the plate using transparent plate sealer.

[0321] 5. Incubate the plate for 10 minutes at 37°C, 500 rpm, on a thermomixer equipped with a lid heating function.

[0322] 6. To stop the reaction, carefully add 100 pL 0.4M TCA solution (except for the blank samples, which already contain TCA).

[0323] Total reaction volume: 210 pL.

[0324] Step (2) Ammonia detection step

[0325] Reagents:

[0326] Color reagent A: 4% (w / v) Phenol, 0.015% (w / v) sodium pentacyanonitrosylferrate(lll) dihydrate (sodium nitroprusside) (Na2[Fe(CN)sNO]-2H2O).

[0327] Color reagent B: 5% (w / v) Potassium hydroxide.

[0328] Color reagent C: 28% (w / v) Potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma- Aldrich 239305-25ml, < 5% available C ).

[0329] Incubation:

[0330] 1. Transfer 15 pL from each well from step (1) into a new 96-well MTP.

[0331] 2. Transfer 45 pL Milli-Q water to each well.

[0332] 3. To each well, add 30pL of color reagent B (on lab table, shake gently by hand to mix).

[0333] 4. To each well, add 60pL of color reagent A (on lab table, shake gently by hand to mix).

[0334] 5. To each well, add 60pL of color reagent C (on lab table, shake gently by hand to mix).

[0335] 6. Color development: Carefully seal the plate and leave it on lab table for 30 minutes.

[0336] 7. Carefully transfer the MTP to a plate reader and measure absorbance at 630 nm.

[0337] Total reaction volume: 210 pL

[0338] Standard curve:

[0339] Standard stock solution: 1000 mg NH4+ / L.

[0340] 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.

[0341] The amount of enzyme producing 1 pmol ammonia per minute at 37°C is defined as 1 unit (Indophenol Assay Unit; IPA(U)): IPA(U) _r fmg_ I mol (i»i) 1 dertertton (M) n * * * * which can be shortened to in. where

[0342] • CNH4+ is the ammonia concentration in the reaction solution derived from the ammonium standard curve (i.e., taking into account the dilution of the prediluted ammonium standard solution in the ammonia derivatization step).

[0343] • 18.04 is the molecular mass of ammonium used for the standard solution.

[0344] • V reaction is the reaction volume in the well when ammonia is generated (210 pL).

[0345] • V enzyme is the volume of enzyme solution added to the well when ammonia is generated (10 pL).

[0346] • VNH3 detection is the reaction volume in the well when ammonia is detected (210 pL).

[0347] Example 2: Using mannanase and protein deamidase for obtaining RTD coffee beverage

[0348] Enzyme reactions

[0349] Pea protein samples: 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.

[0350] Dairy protein samples: Fresh pasteurized cow’s milk (protein content: 3.2%) from YILI group was incubated with protein deamidase at a concentration of 0 (control sample) or 1.1 IPA(U) / g protein and held at 60°C for 1 hour with agitation.

[0351] Coffee samples: 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. The mannanase was added to the brewed coffee at 0 and 0.5% g enzyme / g coffee dry basis. The coffee mixture was incubated at 60°C for 1 hour with agitation.

[0352] Pea milk preparation

[0353] 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 1 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.

[0354] Table 1 Pea milk recipe and ingredients

[0355] 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.

[0356] For some of the pea milk samples, 500mg / 100g pea milk K2HPO4 3H2O was added to the pea milk before mixing with coffee.

[0357] RTD coffee beverage

[0358] The milk samples (pea milk and cow’s 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.

[0359] 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.

[0360] 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.

[0361] From the results shown in Table 2-4 (and corresponding Figures 1-3), it was confirmed that protein deamidase alone could avoid curdling of the RTD coffee samples, both with and without phosphate added. Furthermore, the combined use of mannanase and protein deamidase (treatment 3) could significantly reduce the sediment generation during storage. The extended shelf stability was shown both after 7 days incubation at 60°C and after five weeks (35 days) incubation at 60°C.

[0362] Table 2 pea milk and coffee blend without phosphate, ratio 3:1 (milk to coffee, w / w), '+’ indicate degree of sedimentation observed, indicate no sedimentation observed.

[0363] Table 3 pea milk and coffee blend with 500mg K2HPO4 3H2O Pea milk, ratio 1.5:1 (milk to coffee, w / w), '+’ indicate degree of sedimentation observed, indicate no sedimentation observed

[0364] Table 4 Cow’s milk and coffee blend, ratio 3:1 (milk to coffee, w / w), '+’ indicate degree of sedimentation observed, indicate no sedimentation observed Example 3: Using mannanase in preparation of cold brew, impact on shelf-life

[0365] Experimental setup Raw Material: Commercial ground coffee (Brand: BKI Extra, sourced from a supermarket). Moisture content 3,941%.

[0366] Cold Brew preparation:

[0367] 200 g of de-ionized water was distributed to mashing beakers (corresponding to a watergrist ratio of 4:1) and placed in a LP Electronic Mashing Device (Lochner GmbH, Germany) and subsequently pre-warmed to temperature of 25°C. Fifty (50) grams of ground coffee were added to each beaker. The water and coffee mixtures were homogenized by stirring for 5 minutes at 150 rpm. Stirring speed was subsequently reduced to 100 ppm and mannanase was added according to Table 5 below.

[0368] Table 5 Dosages of Mannanase applied in Cold Brew samples.

[0369] After 120 minutes at 25°C under continuous stirring, all samples were adjusted for evaporation losses to the original weight of 250 g at start and filtered at room temperature using filter-funnels equipped with MN614 0 240mm (Macherey-Nagel, Germany) filter-paper. When filtration was completed all the cold brew samples were collected for analyses.

[0370] Cold Brew samples analyses:

[0371] Moisture of ground coffee was determined using a HR73 Halogen Moisture Analyzer (Mettler Toledo, Switzerland) set to drying temperature of 105°C.

[0372] Density, Brix and dynamic viscosity was analyzed using a DMA™ 4500M density analyzer (Anton Paar, Austria). Turbidity was measured by Hach TI2350 (Hach, Germany), Table 6.

[0373] Table 6 Analyses of cold brew samples.

[0374] Cold Brew shelf-life:

[0375] Cold brew samples were boiled for 10 minutes at 98°C for enzyme inactivation, cooled down and stored at 58°C for up to 20 days. At designated time points, samples were equilibrated to room temperature, manually inverted and turbidity was measured by Hach TI2350 (Hach, Germany). Results are shown in Table 7 and Figure 4.

[0376] Table 7 Turbidity in NTU, measured during 58°C storage.

[0377] As seen from the data shown in at least Table 7, it was confirmed that the use of mannanase for preparing a cold brew coffee resulted in a cold brew having increase shelf-life stability, as seen from the little to no increase in turbidity during storage. For comparison, the control sample (1.), which was not treated with a mannanase, displayed a significant increase in turbidity already on day 3 of storage.

[0378] After 7 days of storage at 58°C, the samples were also visually examined for the formation of sediment and pictures were taken to show the presence of mannan precipitate in the samples. The results from visual examination of the cold brew samples are shown in Figure 4. In Figure 4A+B, the sample depicted “1” is the reference sample, wherein no mannanase was added. Remaining samples “2”, “3”, “4” and “5” are cold brew samples prepared with increasing dosage of mannanase, 50 - 100 - 500 - 1000 ppm, respectively. Picture 4B are the same samples as shown in Figure 4A, only difference being that the glasses have been inverted to display the existence of mannan sediment forming at the bottom of the glasses. Particle size analysis of cold brew samples:

[0379] Size of particles formed after 20 days of storage at 58°C was analyzed by laser diffraction using a Mastersizer 3000 (Malvern Ltd. Instruments, United Kingdom). The following parameters were applied for the analyses: laser Obscuration 4%, stirrer speed 1400 rpm, Particles Refractive Index 1.5, Particle Absorption Index 0.010.

[0380] The formation of high size particles was clearly observed in the sample not treated with mannanase, while all the samples treated with increasing dosages of mannanase did not show large size particles.

[0381] From the data reported in this example, it was concluded that the use of even small dosages of mannanase for production of a cold brew coffee gave rise to smaller size particles, no precipitates forming during storage of the cold brew samples and the turbidity remaining almost unchanged during storage.

[0382] Saccharides distribution in cold brew samples:

[0383] The content of monosaccharides and oligosaccharides (DP1-DP4) in cold brew samples after 1 month storage at 58°C was analyzed with a Dionex HPLC system ICS-5000 using a Rl- 101 Differential Refractive Index Detector (Thermo Scientific, USA), based on Analytica-EBC method 8.7.

[0384] The samples treated with mannanase showed an increase in disaccharides (DP2) and trisaccharides (DP3), compared to the reference untreated sample (no mannanase treatment). After mannanase treatment and after storage for 30 days at 58°C, the amount of free monosaccharides (DP1) remained unchanged compared to the reference untreated samples. The results are shown in Table 8.

[0385] Table 8 Mono- and oligosaccharides content in the cold brew samples after 30 days of storage at 58°C, measured by HPLC. DP1 , free monosaccharides; DP2, disaccharides; DP3, trisaccharides; DP4+, saccharides longer than tri-saccharides. As shown at least in Table 8, using the mannanase of the present invention provides for a solution that gives a pure coffee extract with low amounts of free sugar. The mannanase of the present invention thus provides for a valuable alternative to the mannanases available for purposes of producing higher amounts of monosaccharides from coffee grounds, such as, e.g., the CoffeeMax™ sold by Amano Enzyme and having a high exo activity giving rise to release of monosaccharides. For example the present mannanase provides a better solution for manufacturers of sugar-free / low-sugar coffee beverages.

[0386] Also, the herein disclosed enzyme having mannanase activity gives rise to coffee extracts with increased amounts of di- and trisaccharides, which are healthier alternative to monosaccharides. Mannose oligosaccharides (MOS) are non-digestible oligosaccharides derived via partial hydrolysis of the mannans polysaccharide and are considered functional oligosaccharides, acting as prebiotics. Mannobiose (a disaccharide) and mannotriose (a trisaccharide) are examples of such MOS and increasing the concentration of these, by the action of the herein disclosed mannanase, can give rise to obtainment of a coffee composition, such as a cold brew of a RTD coffee beverage, having prebiotic health claims. Furthermore, using the enzyme having mannanase activity according to the invention enable obtaining a coffee composition, such as the herein exemplified cold brew coffee, which has a low content of the monosaccharide beta-mannose, which is perceived bitter.

[0387] Example 4: Haze reduction in RTDs made from instant coffee

[0388] An instant coffee composition was produced by mixing 1 g of instant coffee powder (Nescafe Gold) with 50 g of boiling water in 100 mL flasks. Enzyme incubation (using the mannanase derived from Talaromyces leycettanus having the mature polypeptide sequence shown as SEQ ID NO: 12) was done for two hours at 60°C and inactivated by incubating the flasks in a water bath at 95°C for 10 minutes. Enzyme treatments were done with a dose response of mannanase at 0, 100, 300, 1000 and 2500 ppm weight by weight instant coffee. After inactivation, samples were transferred to glass tubes used and stored at room temperature. At designated time points, samples were equilibrated to room temperature, manually inverted and turbidity was measured by Hach TI2350 (Hach, Germany), see Table 9.

[0389] Table 9 Turbidity formation over time in a coffee beverage produced from instant coffee As seen from the data shown in at least Table 9, it was confirmed that the use of mannanase for preparing an instant coffee resulted in a RTD beverage having increased shelflife stability, as seen from the little to no increase in turbidity during storage of the mannanase- treated RTD samples. For comparison, the control sample, which was not treated with a mannanase, displayed a significant increase in turbidity already after 1 day of storage.

[0390] Example 5: Haze reduction in RTDs made from brewed drip coffee

[0391] Drip coffee was prepared from a medium roast Arabica coffee. 50 g of coffee grounds were mixed with 750 mL of hot Dl-water, boiled in an electric kettle, and left for 30 minutes to cool down. The coffee extract here prepared was filtered using filter-funnels equipped with MN614 0 240mm (Macherey-Nagel, Germany) filter-paper. The pH (5.07), turbidity (60 NTU) and Brix (2,17°Brix) of the brewed drip coffee were measured. Mannanase dosing was based on the total dissolved solids (TDS) (where TDS=0.85* °Brix) measured in the filtered coffee (mannanase dosed at 3, 15, 30, 150, 500 ppm). Mannanase and filtered coffee was incubated for 20 minutes at 60°C and enzyme inactivated at 95°C for 10 minutes.

[0392] All coffee samples were then cooled down, adjusted to pH 6.8 with 1 M sodium carbonate and stored at 58°C for up to 30 days. At designated time points, samples were equilibrated to room temperature, manually inverted and turbidity was measured by Hach TI2350 (Hach, Germany), see Table 10.

[0393] Table 10 Turbidity development (values given in NTU) in coffee drinks prepared by drip coffee method, measured during 58°C storage.

[0394] As seen from the data shown in at least Table 10, it was confirmed that the use of mannanase for preparing a brewed drip coffee resulted in a RTD beverage having increased shelf-life stability, as seen from the little to no increase in turbidity during storage of the mannanase-treated RTD samples. The results were most pronounced for mannanase dosing of 15ppm and above. For comparison, the control sample, which was not treated with a mannanase (No enzyme, Reference), displayed a significant increase in turbidity already after 6 days of storage.

[0395] Example 6: Viscosity reduction in coffee concentrates at increasing dry matter

[0396] Coffee concentrates at variable dry matter (DM) content were prepared mixing instant coffee powder (Nescafe Gold) with the relative amount of Dl-water and, for the enzyme-treated sample, 1000 ppm Mannanase dosed based on the dry matter. Samples were incubated at 60°C for 1 hour, followed by 10 minutes inactivation at 95°C. Viscosity was measured at 20°C. Measurements are average of twelve readings, viscosity is calculated (MPa*s) by interpolating a calibration curve of standards of known viscosities.

[0397] As seen from at least the data presented in Table 11 , the mannanase of the invention is effective in reducing viscosity of coffee concentrates prepared at various dry matter contents. In general, reduced viscosity was observed for all the mannanase-treated coffee concentrates compared to the reference coffee concentrates (no mannanase treatment). Even at the higher dry matter contents, including DM 30%, 40% and 50%, the mannanase-treated coffee concentrates displayed a significantly reduced viscosity compared to the reference coffee concentrates (not treated with mannanase).

[0398] Table 11 Viscosity (mPa*s) measured in coffee concentrates prepared from soluble coffee at variable dry matter. DM, dry matter.

[0399] Example 7: Mannanase dose response for viscosity reduction of coffee concentrate

[0400] 200 grams of coffee concentrate at 50% dry matter was prepared by mixing 104 g of instant coffee powder (Nescafe Gold, 96% dry matter) with 96 g of Dl-water. 25 g of the mix was transferred to each single-use aluminium cup for the Rapid Visco Analyzer RVA 4500 and mannanase was added at a dosages of 0, 10, 30, 100, 300, 1000 ppm based on dry matter, immediately before the start of the run. Viscosity (cP) was recorded after 20 minutes at 40°C, 160 rpm in RVA 4500 (Perkin Elmer). Table 12 Viscosity (cP) in 50% dry matter coffee concentrates treated with increasing dosages of mannanase. As seen from the data shown at least in Table 12, it was confirmed that even low dosages of the mannanase is effective in lowering the viscosity in the treated coffee concentrates.

Claims

CLAIMS1. A method for obtaining a ready-to-drink coffee beverage, comprising the steps of:(a) providing a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material, wherein a protein deamidase is used for the enzymatic deamidation;(b) providing a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity; and(c) mixing the dairy or plant composition of step (a) with the coffee composition of step (b) to obtain the ready-to-drink coffee beverage.

2. Method of claim 1 , wherein the ready-to-drink coffee beverage has reduced turbidity and / or gelation compared to a ready-to-drink coffee beverage obtained using a similar method but without the use of a protein deamidase and an enzyme having mannanase activity.

3. Method of any of the preceding claims, wherein the ready-to-drink coffee beverage is meant for storage at 1-70°C, such as storage at 4-8°C and / or at 15-25°C and / or at 55-65°C.

4. Method of any of the preceding claims, wherein the ready-to-drink coffee beverage is stored for at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, before being consumed.

5. Method of any of the preceding claims, wherein the ratio of dairy or plant composition to coffee composition in the ready-to-drink coffee beverage is from 1 :10 to 10:1 based on w / w.

6. Method of any of the preceding claims, wherein the plant material is derived or obtained from almond, cashew, chickpea, coconut, fava bean, hazelnut, lentil, lupin, macadamia, mung bean, peanut, pecan, pistachio, corn, quinoa, flax, hemp, oat, spelt, pea, rice, sesame, sunflower, soy, walnut, or any combination thereof, preferably from almond, pea, soy, oat or any combination thereof.

7. Method of any of claims 1-5, wherein the dairy composition comprises cream, milk, concentrated milk, condensed milk, evaporated milk, reconstituted milk, milk protein concentrate, whey protein concentrate, micellar casein, or any combination thereof.

8. Method of any of the preceding claims, wherein the coffee component is or is derived from instant coffee solids, a coffee extract, roasted ground coffee beans, or a coffee concentrate.

9. Method of any of the preceding claims, wherein the coffee component is essentially free of initial insoluble solids, optionally wherein the coffee component has been subjected to a step centrifugation or filtration, prior to the treatment with the enzyme having mannanase activity.

10. Method of any of the preceding claims, wherein the protein deamidase is derived from or obtained from a Chryseobacterium species.

11. Method of any of the preceding claims, wherein the enzyme having mannanase activity is an endo-beta-1 , 4-mannanase, optionally wherein the enzyme having mannanase activity is thermostable.

12. Method of any of the preceding claims, wherein the ready-to-drink coffee beverage is essentially free of added emulsifiers and / or stabilizers.

13. Method of any of the preceding claims, wherein the ready-to-drink coffee beverage has an increased di- and / or trisaccharide content compared to a ready-to-drink coffee beverage prepared without the use of an enzyme having mannanase activity.

14. A ready-to-drink coffee beverage comprising:(a) a dairy composition comprising enzymatically deamidated dairy material or a plant composition comprising enzymatically deamidated plant material, wherein a protein deamidase is used for the enzymatic deamidation; and(b) a coffee composition comprising a coffee component having been treated with an enzyme having mannanase activity, wherein the ready-to-drink coffee beverage has reduced turbidity and / or gelation compared to a ready-to-drink coffee beverage obtained without the use of a protein deamidase and an enzyme having mannanase activity.

15. Use of a protein deamidase and an enzyme having mannanase activity in the production of a ready-to-drink coffee beverage to reduce turbidity and / or gelation.

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