foaming dairy products

Abstract

The present invention provides a dairy product composition with improved aeration properties, comprising a protein deamidase and, optionally, a second polypeptide derived from the propeptide of the protein deamidase proform. The present invention further provides an improved method for producing an aerated dairy product from the dairy product composition, wherein the protein deamidase and, optionally, a second polypeptide derived from the propeptide of the protein deamidase proform are added to the dairy product composition.

Description

Technical Field

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

[0002] The present invention relates to dairy products that have been treated to enhance the foaming properties of the dairy products. The present invention also provides a method for treating dairy products to enhance their foaming properties.

Background Art

[0003] Typically, cappuccino-style beverages that mix coffee and milk, top it with milk foam, and often apply a design, i.e., "latte art," to the top layer of the foam, are very popular today. Milk provides both a creamy texture to the beverage and smoothness to the foam. Typically, whole milk is used for the beverage and is often preferred. However, many health-conscious consumers prefer low-fat milk, especially milk containing less than 2% milk fat, or skim milk containing a very minimal amount of milk fat. Unfortunately, it is difficult to produce foam of the same quality using low-fat milk or skim milk compared to the foam produced using whole milk. Furthermore, sometimes even whole milk can be difficult to form foam with the desired viscosity and texture.

Summary of the Invention

Means for Solving the Problems

[0004] The present invention provides a dairy composition with improved aeration properties, comprising a first polypeptide which is a mature protein defimidase and, optionally, a second polypeptide which is a propeptide derived from a protein defimidase proform. The present invention further provides an improved method for producing an aerated dairy product from the dairy composition, wherein the protein defimidase and, optionally, a second polypeptide derived from a propeptide of the protein defimidase proform are added to the dairy composition. Unexpectedly, the presence of the propeptide in the dairy composition improves the foaming properties of the dairy composition and produces an improved aerated dairy product compared to a method in which only mature protein defimidase is added.

[0005] The present invention also provides a method for improving the frothing of a dairy composition, the dairy composition comprising a first polypeptide which is a mature protein deamidase and optionally a second polypeptide which is a propeptide derived from a protein deamidase proform. The present invention further provides a method for improving latte art produced from a dairy composition, the dairy composition comprising a first polypeptide which is a mature protein deamidase and optionally a second polypeptide which is a propeptide derived from a protein deamidase proform.

[0006] array The amino acid sequence of the proform polypeptide of deamidase derived from Chryseobacterium viscerum, containing the propeptide variant of SEQ ID NO: 1:F99G. Sequence ID 2: Amino acid sequence of mature deamidase derived from Sequence ID 1. The amino acid sequence of the propeptide derived from Chryseobacterium viscerum, including SEQ ID NO: 3:F99 (wild type). Sequence ID 4: Amino acid sequence of the proform polypeptide of deamidase derived from Chryseobacterium gambrini. SEQ ID NO: 5: The amino acid sequence of mature deamidase derived from SEQ ID NO: 4. SEQ ID NO: 6: The amino acid sequence of the propeptide derived from SEQ ID NO: 4. Sequence ID 7: Amino acid sequence of the proform polypeptide of deamidase derived from Chryseobacterium culicis. Sequence ID 8: Amino acid sequence of mature deamidase derived from Sequence ID 7. SEQ ID NO: 9: The amino acid sequence of the propeptide derived from SEQ ID NO: 7. Sequence ID 10: Amino acid sequence of the proform polypeptide of deamidase derived from Chryseobacterium defluvii. Sequence ID 11: Amino acid sequence of mature deamidase derived from Sequence ID 10. SEQ ID NO: 12: The amino acid sequence of the propeptide derived from SEQ ID NO: 10. Sequence ID 13: Amino acid sequence of the proform polypeptide of deamidase derived from Chryseobacterium proteolyticum. SEQ ID NO: 14: Amino acid sequence of mature deamidase derived from SEQ ID NO: 13. SEQ ID NO: 15: The amino acid sequence of the propeptide derived from SEQ ID NO: 13. SEQ ID NO: 16: Propeptide variant F99G of SEQ ID NO: 3. SEQ ID NO: 17: Propeptide variant F99A of SEQ ID NO: 6. SEQ ID NO: 18: Propeptide variant F99G of SEQ ID NO: 9 SEQ ID NO: 19: L101G, a propeptide variant of SEQ ID NO: 12. SEQ ID NO: 20: L100G, a propeptide variant of SEQ ID NO: 15. SEQ ID NO: 21: A variant of SEQ ID NO: 4 containing the propeptide variant of SEQ ID NO: 17. SEQ ID NO: 22: A variant of SEQ ID NO: 7 containing the propeptide variant of SEQ ID NO: 18. SEQ ID NO: 23: A variant of SEQ ID NO: 10 containing the propeptide variant of SEQ ID NO: 19. SEQ ID NO: 24: A variant of SEQ ID NO: 13 containing the propeptide variant of SEQ ID NO: 20.

[0007] definition Deamidase: The term "deamidase" refers to the protein-glutaminase (also known as glutaminylpeptide glutaminase) activity described in EC 3.5.1.44 that catalyzes the hydrolysis of glutamine gamma-amides (e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine) substituted at the carboxyl position or at both the alpha-amino and carboxyl positions. Thus, deamidase can deamidate glutamine residues in proteins to glutamic acid residues and is also called protein glutamine deamidase. Deamidase contains three catalytic residues (Cys-156, His-197, and Asp-217, as shown in, for example, Hashizume et al. "Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex," Journal of Biological Chemistry, vol.286, no.44, pp.38691-38702) and belongs to InterPro entry IPR041325.

[0008] Deamidase activity: Deamidase (protein glutaminase) activity was measured using the assay described in the examples. The activity assay consists of two separate parts: (1) an enzymatic step in which ammonia is formed by the catalytic action of protein deamidase, and (2) a non-enzymatic detection step in which the ammonia formed in step (1) is derivatized into a blue indophenol compound having an absorption maximum at 630 nm. The amount of enzyme that produces 1 μmol of ammonia per minute at 37°C is defined as 1 unit (indophenol assay unit: denoted as IPA(U)). Activity can be determined against a nominal strength standard.

[0009] Deamidase inhibitory domain: The term "deamidase inhibitory domain" refers to an amino acid sequence that interacts with amino acid residues in the deamidase active site to inhibit or reduce deamidase activity. For example, deamidase activity can be reduced to less than 50%, preferably less than 40%, in the presence of a deamidase inhibitory domain (compared to deamidase activity without the presence of deamidase inhibitory activity).

[0010] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from spliced ​​mature mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed through a series of steps, including splicing, before it becomes spliced ​​mature mRNA.

[0011] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by an open reading frame that begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. A coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0012] Regulatory Sequences: The term “regulatory sequence” refers to a nucleic acid sequence that is involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each regulatory sequence may be native (i.e., derived from the same gene) or heterologous (i.e., derived from a different gene) to the polynucleotide encoding the polypeptide, and may be native or heterologous to each other. Such regulatory sequences include, but are not limited to, leaders, polyadenylations, prepropeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, a regulatory sequence includes a promoter and transcription and translation termination signals. A regulatory sequence may have linkers for the purpose of introducing specific restriction sites that facilitate ligation of the regulatory sequence with the coding region of the polynucleotide encoding the polypeptide.

[0013] Expression: The term "expression" means all processes involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression vector: An expression vector refers to a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, the encoding sequence being operably linked to appropriate regulatory sequences that can result in DNA expression in a suitable host. Such regulatory sequences may include promoters that induce transcription, optional operator sequences that control transcription, sequences encoding appropriate ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0014] Elongation: The term "elongation" refers to the addition of one or more amino acids to the amino and / or carboxyl terminals of a polypeptide, and an "elongated" polypeptide has deamidase activity.

[0015] Fragment: The term "fragment" refers to a polypeptide having one or more amino acids that are not present at the amino and / or carboxyl terminus of a mature polypeptide, and this fragment has deamidase activity.

[0016] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide of the present invention is fused at the N-terminus and / or C-terminus of another polypeptide of the present invention. A fusion polypeptide is produced by fusion of two or more polynucleotides that together encode the polypeptides of the present invention. Techniques for producing fusion polypeptides are known in the art and involve ligating the coding sequences encoding the polypeptides so that they are in-frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein techniques in which the fusion polypeptide is produced post-translation (Cooper et al., 1993, EMBO J.12:2575-2583; Dawson et al., 1994, Science 266:776-779). A fusion polypeptide may further include cleavage sites between the two polypeptides. Thus, a fusion polypeptide may include, for example, cleavage sites for site-specific endopeptidase within 20 amino acids, preferably 10 amino acids, of the C-terminus of the first polypeptide. Well-known examples of site-specific endopeptidases include glutamyl endopeptidases (e.g., EC3.4.21.19 or EC3.4.21.82), trypsin- and chymotrypsin-like endopeptidases (including enteropeptidases).Many other examples of cleavage sites and corresponding endopeptidases are disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48, but are not limited thereto.

[0017] Heterologous: The term "heterologous" with respect to a host cell means that a polypeptide or nucleic acid is not naturally present in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means that a control sequence of the polypeptide or nucleic acid, such as a promoter, does not naturally associate with the polypeptide or nucleic acid, i.e., the control sequence is derived from a gene other than the gene encoding the mature polypeptide.

[0018] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) that can express a polypeptide of interest and / or ferment saccharides. The term "host cell" includes protoplasts generated from the cell.

[0019] Introduced: In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection", "transformation", or "transduction" as known in the art.

[0020] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other specific material or component separated from at least one other material or component to which it is naturally associated as found in nature (e.g., other proteins, nucleic acids, cells, etc., but not limited thereto). Isolated polypeptides include, but are not limited to, culture broths containing secreted polypeptides.

[0021] Mature polypeptide: The term "mature polypeptide" or "mature protein" means the polypeptide in its mature form after N-terminal processing (e.g., removal of the signal peptide and / or propeptide).

[0022] Signal peptide: A "signal peptide" is a sequence of amino acids that binds to the N-terminal portion of a protein and promotes the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0023] Native: The term "native" means a nucleic acid or polypeptide that is naturally present within a host cell.

[0024] Nucleic acid: The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules that can encode polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since the genetic code is degenerate, two or more codons can be used to encode a particular amino acid, and the present compositions and methods include nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are shown in the 5' to 3' direction.

[0025] Nucleic acid construct: The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, modified to contain a nucleic acid segment in a manner not normally found in nature, or synthesized, and contains one or more regulatory sequences operably linked to the nucleic acid sequence.

[0026] Operablely coupled: The term "operably coupled" means that certain components are in a relationship (including, but not limited to, juxtaposition) that allows them to function in the intended way. For example, a regulatory sequence is operably coupled to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

[0027] Purified: The term "purified" means a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified polypeptides or nucleic acids may form distinct bands in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation). Purified nucleic acids or polypeptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, or about 99.8% or more pure (e.g., by weight percentage on a molar basis). In a related sense, a composition is enriched with respect to molecules if there is a substantial increase in the concentration of molecules after the application of purification or enrichment techniques. The term "enriched" refers to compounds, polypeptides, cells, nucleic acids, amino acids, or other specific materials or components present in the composition at a higher relative or absolute concentration than that of the starting composition.

[0028] In one embodiment, the term “purified” as used herein refers to a polypeptide or cell substantially free of components from the producing organism (in particular, insoluble components). In other embodiments, the term “purified” refers to a polypeptide substantially free of insoluble components from the natural organism from which it is obtained (in particular, insoluble components). In one embodiment, the polypeptide is separated from a portion of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified (i.e., separated) by one or more unit operations of filtration, precipitation, or chromatography.

[0029] Therefore, polypeptides can be purified such that only trace amounts of other proteins (in particular, other polypeptides) are present. As used herein, the term “purified” may mean the removal of other components present in the cell from which the polypeptide originated, in particular other proteins, and most particularly other enzymes. Polypeptides can be “substantially pure,” that is, they may not contain other components from the organism from which they were produced (e.g., the host organism in the case of recombinant polypeptides). In one embodiment, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one embodiment, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, “substantially pure polypeptide” may mean a polypeptide preparation containing up to 10% by weight, preferably up to 8% by weight, more preferably up to 6% by weight, more preferably up to 5% by weight, more preferably up to 4% by weight, more preferably up to 3% by weight, even more preferably up to 2% by weight, most preferably up to 1% by weight, and most preferably up to 0.5% by weight, other polypeptide materials to which the polypeptide is naturally or recombinantly associated.

[0030] Therefore, a substantially pure polypeptide is preferably at least 92% pure by weight of the total polypeptide material present in the preparation, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure. The polypeptide of the present invention is preferably in a substantially pure form (i.e., the preparation substantially contains no other polypeptide material that is naturally or recombinantly associated). This can be achieved, for example, by preparing the polypeptide by a well-known recombinant method or a classical purification method.

[0031] Recombinant: The term "recombinant," in its traditional sense, is used to refer to the manipulation of nucleic acid sequences, such as cleavage and recombination, to form constellations different from those found in nature. The term "recombinant" refers to cells, nucleic acids, polypeptides, or vectors that have been modified from their natural state. Thus, for example, a recombinant cell may express genes not found in the natural (non-recombinant) form of the cell, or it may express natural genes at different levels or under different conditions than those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0032] To recover: The term “recover” or “recover” means the recovery of polypeptides from total fermentation broth or cell-free fermentation broth by means of separating polypeptides from at least one fermentation broth component selected from a list of cells, nucleic acids, or other designated materials, e.g., by polypeptide crystal collection, by filtration, e.g., deep filtration (using filter aids or packed filter media, cloth filtration in a chamber filter, rotary drum filtration, drum filtration, rotary vacuum drum filter, candle filter, horizontal leaf filter or similar, using seed or pad filtration in a flame or modular setting), or by membrane filtration (using sheet filtration, modular filtration, candle filtration, microfiltration, ultrafiltration in any cross-flow, dynamic cross-flow, or dead-end operation), or by centrifugation (using a decanter centrifuge, plate centrifuge, liquid cyclone or similar), or by precipitation of polypeptides and collection of polypeptides from the broth medium by particle size classification using the relevant solid-liquid separation method. Recovery includes the isolation and / or purification of polypeptides.

[0033] Sequence Identity: The relationship between two amino acid sequences, or between two nucleotide sequences, is described by a parameter called "sequence identity." For the purposes of this invention, sequence identity between two amino acid sequences is determined as the "longest identity" output using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48;443-453), which is implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), 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 an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The nobrief option must be specified on the command line for the Needle program to report the longest identity. The output of Needle labeled "Longest Identity" is calculated as follows: (Number of identical residues × 100) / (Length of alignment - Total number of gaps in the alignment)

[0034] Variant: The term "variant" means a polypeptide having deamidase activity that contains artificial mutations (i.e., substitutions, insertions (including extensions), and / or deletions (e.g., truncations)) at one or more positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding 1 to 5 amino acids (e.g., 1 to 3 amino acids, in particular 1 amino acid) adjacent to or immediately following an amino acid occupying a position.

[0035] Wild-type: The term "wild-type" in relation to amino acid sequences or nucleic acid sequences means that the amino acid sequence or nucleic acid sequence is natural, i.e., naturally occurring. As used herein, the term "naturally occurring" refers to something found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "not naturally occurring" refers to something not found in nature (e.g., a recombinant nucleic acid and protein sequence produced in the laboratory, or a modification of a wild-type sequence).

[0036] Milk: The term "milk," also called "dairy milk," generally refers to milk from cows, but can also refer to milk from goats, sheep, buffaloes, or camels. Milk is typically available in three versions with different fat content. Whole milk contains an average of 3.8% milk fat and a minimum of 3.2% milk fat. Low-fat milk, also called light milk or reduced-fat milk, generally contains 2% or less milk fat. In some embodiments, low-fat milk contains about 2% milk fat. In some embodiments, low-fat milk contains about 1% milk fat. Skimmed milk, also called nonfat milk or fat-free milk, generally contains 0.5% or less milk fat. In some embodiments, skimmed milk has 0.1% or less milk fat. In some embodiments, skim milk may contain additional milk solids, such as lactose and / or protein, to optimize its taste and texture due to its very low fat content.

[0037] Barista Milk: The term "barista milk" refers to milk that may contain additional ingredients to produce a better foam that is more cohesive, pours more evenly, and can be used to create latte art. Barista milk may contain more fat and / or more protein compared to standard whole milk.

[0038] Dairy product composition: The term "dairy product composition" refers to a milk-derived composition that includes cream, milk, butter, concentrated milk, evaporated milk, condensed milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof.

[0039] Dairy desserts: The term "dairy desserts" refers to ice cream, frozen custard, frozen yogurt, gelato, and ice milk.

[0040] Aeration: The term "aeration" refers to the introduction of air into a material such as a dairy composition. In relation to the present invention, aeration is typically achieved by whipping or beating the dairy composition, and sometimes using steam. Aeration produces a lighter, fluffier dairy composition. Aerationd dairy compositions may be called foams. Examples of aerated dairy products include ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped butter, and whipped butter spread.

[0041] Milk Foam: The term “milk foam” is also known as “foamed milk” or “foam,” and is typically milk or cream that has been heated and aerated. Foam can be produced with steam, such as by using the steam wand of an espresso machine. It can also be produced by frothing, shaking, or whipping. Electric milk frothers can also be used to produce milk foam. Milk foam can be further characterized as “microfoam.” Microfoam has fine, uniform bubbles, can be glossy, and slightly thick. Microfoam has a viscosity similar to wet paint. Microfoam is preferred for latte art because it provides clarity and stability to patterns and / or etchings.

[0042] A key quality of milk foam is its "cohesion." Foam cohesion is determined by the foam-liquid interaction that causes the foam to adhere to the surface of the liquid milk. If the foam lacks cohesion or is insufficient, when the frothed milk is poured, the foam lags behind the liquid milk, resulting in clumps of foam remaining in the frother and coming out later. Such clumps of foam are considered undesirable when preparing beverages containing frothed milk and can hinder, ruin, or destroy latte art. Foam with very good cohesion pours smoothly from the frother, resulting in the foam and liquid pouring simultaneously or almost simultaneously, with little to no foam remaining at the end of the pour.

[0043] Latte Art: Also known as coffee art, barista art, or coffee design, the term “latte art” refers to a specific pattern or design intentionally created on the top layer of milk or cream foam, typically on the top layer of milk foam in hot beverages such as lattes, cappuccinos, or hot chocolates. Latte art can also be made with other hot liquids containing milk or cream, such as cream-based soups like pumpkin soup or tomato soup. Latte art is created by pouring microfoam into a shot, such as an espresso, in a specific way to create patterns and designs. Common latte art patterns include tulips, rosettes, and hearts. Latte art can also use etching, in which a thin rod, such as a toothpick, is used to draw an image on the top layer of milk foam in a hot beverage. Using etching, latte art can depict any image, including faces and animals. Stencils may be used to assist etching. Latte art is particularly difficult to create consistently, partly due to the conditions required for the foamed milk. Variations in milk can make it difficult to predict whether the resulting milk foam will have the high density of microform and high cohesiveness required to create latte art.

[0044] Rules for naming variants: For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 5 is used to determine the corresponding amino acid residue position in another propeptide and / or deamidase. The amino acid sequence of the other propeptide and / or deamidase is aligned with the polypeptide disclosed in SEQ ID NO: 5, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 5 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16;276-277) version 5.0.0 or later. The parameters used are a gap-open penalty of 10, a gap-extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0045] To facilitate reference when describing variants, the following nomenclature should be applied: Approved IUPAC one- or three-letter amino acid abbreviations should be used. Substitutions. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of threonine with alanine at position 226 is indicated as "Thr226Ala" or "T226A". Multiple mutations are separated by an additional mark ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) with arginine I and serine (S) with phenylalanine (F) at positions 205 and 411, respectively. Deletion. For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Therefore, a deletion of glycine at position 195 is indicated as "Gly195*" or "G195*". Multiple deletions are separated by an additional mark ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*". Insertion. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, the insertion of lysine after glycine at position 195 is indicated as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are indicated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as "Gly195GlyLysAla" or "G195GKA".

[0046] In such cases, the inserted amino acid residue is numbered by adding a lowercase letter to the position number of the amino acid residue preceding it. In the example above, the sequence would therefore be as follows:

[0047] [Table 1]

[0048] Multiple modifications. Variants containing multiple modifications are separated by an additional mark ("+"). For example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine with tyrosine and glutamic acid at positions 170 and 195, respectively. Different Modifications. When different modifications can be introduced at a certain position, the different modifications are separated by commas. For example, "Arg170Tyr,Glu" or "R170Y,E" represents the substitution of arginine at position 170 with tyrosine or glutamate. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" or "Y167G,A+R170G,A" represent the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0049] In relation to the present invention, the term “variant” means a polypeptide comprising modifications, i.e., substitutions, insertions, and / or deletions at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids, e.g., 1 to 5 amino acids, adjacent to or immediately following an amino acid occupying a position.

[0050] Amino acid changes may be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1 to 30 amino acids; small amino or carboxyl terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to 20 to 25 residues; or alterations of net charge or other functions, such as small extensions that facilitate purification by polyhistidine tracts, antigen epitopes, or binding domains.

[0051] Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, in H. Neurath and RLHill, 1979, *The Proteins*, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0052] Alternatively, changes in amino acids can alter the physicochemical properties of a polypeptide. For example, amino acid changes can affect the thermal stability of a polypeptide, alter substrate specificity, or change the optimal pH.

[0053] Essential amino acids in polypeptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced into any residue within the molecule to identify amino acid residues critical to the molecule's activity, and the resulting mutant molecules are tested for endopeptidase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interactions can also be determined by physically analyzing the structure, which is determined by techniques such as nuclear magnetic conjugate, crystallography, electron diffraction, or photoaffinity labeling, in combination with amino acid mutations at putative contact sites. For example, see de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J.Mol.Biol.224:899-904; and Wlodaver et al., 1992, FEBS Lett.309:59-64. Furthermore, the identity of essential amino acids can be inferred from alignment with related polypeptides.

[0054] Substitutions, deletions, and / or insertions of single or multiple amino acids can be performed and tested using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; International Publication No. 95 / 17413; or International Publication No. 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Patent No. 5,223,409; International Publication No. 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0055] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutageneised polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutageneised DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for rapid determination of the importance of individual amino acid residues within the polypeptide. [Modes for carrying out the invention]

[0056] The present invention relates to an improved aerated dairy composition, wherein a protein deamidase and a second polypeptide derived from a propeptide of an optional protein deamidase proform are added to the dairy composition before aeration. The present invention also includes a method for producing an improved aerated dairy product. In some embodiments, the resulting aerated dairy product has an improved foam density, containing finer and more uniform bubbles. This improvement in foam density results in an aerated dairy product that is lighter, fluffier and / or has a smoother texture, and also exhibits improved foam stability, compared to a dairy composition not treated with protein deamidase before aeration. In some embodiments, the aerated dairy product is foamed milk having better stability, a smoother texture, and / or higher quality microfoam compared to foam produced from milk not treated with protein deamidase before aeration. This higher quality foam improves the creation of latte art in the final product containing the foamed milk, for example, creating latte art on the surface of a hot beverage or soup.

[0057] Deamidase In this invention, "protein deamidase," "deamidase," "mature protein deamidase," or "mature deamidase" refers to an enzyme that directly acts on the amide group of the side chain of amino acids constituting a protein, causing deamidation without cleaving the peptide bonds of the protein and cross-linked proteins, thereby releasing ammonia. A specific example of protein deamidase is protein glutaminase (EC 3.5.1.44), which directly acts on the amide group of the side chain of glutamine residues contained in a protein to release ammonia, thereby converting glutamine residues to glutamic acid residues. Another example of protein deamidase is protein asparaginase, which directly acts on the amide group of the side chain of asparagine residues contained in a protein to release ammonia, thereby converting asparagine residues to aspartic acid residues. In this invention, it is possible to use either protein glutaminase or protein asparaginase alone, or to use both in combination, as the protein deamidase. In some embodiments, the protein deamidase used in the present invention is protein glutaminase.

[0058] The protein deamidase used in the method of the present invention may be obtained from a microorganism of any genus. For the purposes of the present invention, the term "obtained from" as used herein in relation to a given source means that a polypeptide encoded by a polynucleotide is produced by the source or a strain into which a polynucleotide derived from the source has been inserted. In one embodiment, the polypeptide obtained from a given source is secreted extracellularly.

[0059] Protein deamidase can be obtained from the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides. Protein deamidase can be obtained from the culture broth of the above microorganisms and can be used in the present invention.

[0060] In some embodiments, the protein deamidase may be derived from the genus Chryseobacterium, such as Chryseobacterium viscerum, C. gambrini, C. culicis, C. defluvii, or C. proteolyticum. In some embodiments, the deamidase in the method of the present invention is Chryseobacterium viscerum (this strain was previously called Chryseobacterium sp-62563).

[0061] European Patent No. 1839491 discloses the cloning of protein glutaminase from Chryseobacterium proteolyticum expressed in Corynebacterium glutamicum. Protein glutaminase derived from Chryseobacterium proteolyticum is commercially available, for example, as AMANO PG500 (manufactured by AMANO Enzyme Inc.).

[0062] The protein deamidase in the method of the present invention acts on milk proteins such as casein protein and whey protein. Examples of casein proteins include alpha-s1-casein, alpha-s2-casein, beta-casein, and kappa-casein. Examples of whey proteins include alpha-lactalbumin and beta-lactoglobulin. In some embodiments, the deamidase acts mainly on casein protein.

[0063] Protein deamidases, such as those derived from species of the genus Chryseobacterium, are typically produced as inactive proforms (also referred to herein as "proforms" or "proform protein deamidases"), which are expressed as fusion proteins and contain an N-terminal propeptide domain, also called a "propeptide" or "propeptide derived from proform protein deamidase," located upstream of and strongly bound to the C-terminal deamidase domain. Because the propeptide is bound to the deamidase domain, the proform has reduced deamidase activity, thereby protecting the viability of the cell. By nature, the fusion protein is post-processed to remove the propeptide and release active mature deamidase outside the microbial cell. In recombinant expression systems, the fusion protein is secreted outside the host cell as an inactive proform containing the propeptide, which can then be cleaved and separated from the mature deamidase. The cleavage can be performed by site-specific endopeptidases such as glutamyl endopeptidase, trypsin or trypsin-like endopeptidase, or chymotrypsin or chymotorapsin-like endopeptidase.

[0064] In many deamidase proforms, the propeptide is difficult to separate from mature deamidase even after cleavage of the proform due to the high binding affinity of the propeptide to the deamidase domain. Due to its inhibitory effect, mature protein deamidase still bound to the propeptide exhibits low activity. Therefore, cleavage and removal of the propeptide from mature deamidase is highly desired in the art, and considerable resources have been expended to purify the propeptide from mature deamidase. However, it has been discovered that novel proforms of deamidase enzymes can be generated that can reduce the binding affinity of the propeptide to mature deamidase (International Publication No. 2023 / 170177, incorporated herein by reference). Unexpectedly, in the present invention, it has been found that the presence of these modified propeptides can provide beneficial effects on the activity of deamidase.

[0065] The protein deamidase of the methods and compositions of the present invention is a mature deamidase in which the propeptide is cleaved from the proform protein deamidase. In some embodiments, the propeptide derived from the same proform as the mature protein deamidase may still be present in the composition containing the mature deamidase. In some embodiments, the purified propeptide is separately added to the composition containing the mature protein deamidase.

[0066] The propeptide of the present invention includes a deamidase inhibitory domain that can interact with the deamidase domain of the proform. The deamidase inhibitory domain includes the amino acid sequence motif [I / M][L / I / V][S / T]AQ, which corresponds to amino acids 39-43 of SEQ ID NOs: 3, 6, and 9, amino acids 41-45 of SEQ ID NO: 12, and amino acids 40-44 of SEQ ID NO: 15. Amino acids 40-44 of SEQ ID NO: 12 are 15. The propeptide may also include the amino acid sequence motif [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ, which corresponds to amino acids 35-43 of SEQ ID NOs: 3, 6, and 9, amino acids 37-45 of SEQ ID NO: 12, and amino acids 36-44 of SEQ ID NO: 15. Amino acids 36-44 of SEQ ID NO: 12 are 15. After expressing the proform polypeptide in a recombinant expression system, the propeptide is cleaved using site-directed endopeptidase, leaving active mature deamidase. In some embodiments, the cleaved propeptide is not purified from the mature deamidase. Therefore, the propeptide is present in the composition together with the mature deamidase.

[0067] In some embodiments, the propeptides are mutant variants having a lower binding affinity to the deamidase domain of their corresponding deamidases, and as a result, are more readily cleaved enzymatically after recombinant expression and secretion from host cells. Propeptide mutations affecting the binding affinity of the propeptide to mature deamidases are described in International Publication No. 2023 / 170177 (which is incorporated herein by reference in its entirety).

[0068] In some embodiments, the propeptide contains an amino acid change at a position corresponding to the position of SEQ ID NO: 3, selected from the group consisting of 23, 38, 39, 43, 45, 67, 69, 88, 91, 92, 94, 95, 96, 98, 99, 100, and 101. In further embodiments, the propeptide contains an amino acid change at a position corresponding to the position of SEQ ID NO: 3, selected from the group consisting of V23, F38, M39, Q43, Y45, E67, P69, T88, D91, I92, Y94, F95, K96, F98, F99, T100, and K101. In further embodiments, the propeptide is V23G,D,Y,S;F38A,C,D,G,N,T,V;M39D,E,F,G,H,K,N,P,Q,R,S,W,Y;Q43D,E,F,G,I,K,M,R,Y;Y45A,C,G,I,K,M,N,Q,R,S,T,V;E67D,K,N,P,W;P69D,F,G,H,K,L,M,Q,R,S,T,W,Y;T88F,I,K,L,P,R,V,W,Y;D91F,G,H,K,L,M,N, The propeptides of the present invention include amino acid changes at positions corresponding to the positions of SEQ ID NO: 3, selected from P,Q,R,S,Y;I92G,N,P,Q,S,T;Y94A,E,I,K,P,Q,R,T;F95A,D,E,G,H,I,K,L,M,N,R,S,T,V;K96C,F,I,P,V,Y;F98A,C,D,E,G,H,K,N,P,Q,R,S,T,W,Y;F99A,C,D,G,H,K,P,Q,R,V,W,Y;T100E,P,W; and / or K101E,P. The propeptides of the present invention may contain the amino acid sequences of SEQ ID NO: 3, 6, 9, 12, or 15-20, and may further contain any of the mutations described herein at positions corresponding to the positions in SEQ ID NO: 3.

[0069] In some embodiments, V23G,D,Y,S;F38A,C,D,G,N,T,V;M39D,E,F,G,H,K,N,P,Q,R,S,Y;Q43D,E,I,K,R;Y45A,C,G,I,K,M,N,Q,R,S,T,V;E67D,N,P;P69D,F,G,H,K,M,Q,R,S,T,W,Y;T88I,P,W;D91 In a further embodiment, the propeptide contains an amino acid change at a position corresponding to the position of SEQ ID NO: 3, selected from the group consisting of V23S, F38C, Y45G, Y45T, Y45I, Y45M, Y45R, P69D, P69G, P69L, P69M, P69Q, P69S, P69T, Y94P, F99A, F99A, F99G, and / or F99K.

[0070] In some embodiments, the propeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NOs. 16-20 are described. In another embodiment, the propeptide comprises the amino acid sequences of SEQ ID NOs. 16-20.

[0071] In some embodiments, the proform may contain the amino acid sequence of SEQ ID NOs: 1, 21, 22, 23, or 24, which can be cleaved to produce propeptides containing the amino acid sequences of SEQ ID NOs: 16-20, respectively, and mature protein deamidases containing the amino acid sequences of SEQ ID NOs: 2, 5, 8, 11, and 14, respectively.

[0072] In some embodiments, the methods and compositions of the present invention are described as follows: a) Proform protein deamidase containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 (where this proform is cleaved to have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 90% sequence identity to SEQ ID NO: 2) It may release a mature protein deamidase containing an amino acid sequence having 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and a propeptide containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16. b) Proform protein deamidase containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 21 (where this proform is cleaved to have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 90% sequence identity to SEQ ID NO: 5 It may release a mature protein deamidase containing an amino acid sequence having 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and a propeptide containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17. c) Proform protein deamidase containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22 (where this proform is cleaved to have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 90% sequence identity to SEQ ID NO: 8 It may release a mature protein deamidase containing an amino acid sequence having 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and a propeptide containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18. d) A proform protein deamidase containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23 (where this proform is cleaved to have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% sequence identity to SEQ ID NO: 11 It may release a mature protein deamidase containing an amino acid sequence having sequence identity of %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to SEQ ID NO: 19, and a propeptide containing an amino acid sequence having sequence identity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to SEQ ID NO: 19); or e) A proform protein deamidase containing an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24 (where this proform is cleaved to have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, (This process can release a mature protein deamidase containing an amino acid sequence with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and a propeptide containing an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20.) Includes.

[0073] In the method of the present invention, protein deamidase is added to whole milk, low-fat milk, or skim milk to improve the foaming properties of the milk. The resulting foamed milk has better stability, a higher density of microfoam, higher volume, better cohesiveness, and / or a smoother texture compared to foam produced from dairy compositions that have not been treated with deamidase. The ability of milk to foam and the quality of this foam depend on the proteins and fats present in the milk. Higher quality foam is typically produced using whole milk, while foam produced using low-fat milk or skim milk is less desirable. Surprisingly, the method and composition of the present invention can produce high-quality foam even with low-fat milk that contains no or substantially no milk fat, and even with skim milk. The quality of the foam and the resulting beverage is similar to or better than that of beverages prepared with whole milk without the addition of protein deamidase. Furthermore, in the method of the present invention, protein deamidase and the propeptide of protein deamidase are added to skim milk (also referred to as "skimmed milk") to improve the foaming properties of the milk. Unexpectedly, the method and composition of the present invention can produce higher quality foam using skim milk to which both protein deamidase and the propeptide of protein deamidase are added, compared to a method to which only protein deamidase is added.

[0074] It is well known in the art that latte art can be difficult to produce and relies on extremely high-quality milk foam with dense microfoam and high cohesiveness. Milk foam produced using low-fat or skim milk is generally considered to lack stability, volume, microfoam quantity, and / or sufficient cohesiveness to produce latte art. Furthermore, even whole milk can produce milk foam of inconsistent quality. Therefore, having a reliable dairy composition for latte art production is extremely desirable.

[0075] The compositions of the present invention, comprising milk and protein deamidase, and optionally a propeptide of protein deamidase, satisfy this requirement. The methods and compositions of the present invention produce high-density microfoam and highly cohesive milk foam suitable for latte art. Furthermore, the methods and compositions of the present invention, comprising low-fat milk or skim milk, also produce high-density microfoam and highly cohesive milk foam suitable for latte art.

[0076] In some embodiments, the texture of the beverage itself is also improved, resulting in a creamier and smoother texture compared to similar beverages prepared with low-fat or skim milk without added protein deamidase.

[0077] As described above, in some embodiments, a first polypeptide, i.e., mature protein deamidase, and a second polypeptide are added to milk (where the second polypeptide is a propeptide of deamidase). In some embodiments, the second polypeptide is a propeptide resulting from the cleavage of the proform to produce the first polypeptide. In some embodiments, the second polypeptide is a mutant propeptide variant. In further embodiments, the second polypeptide is a mutant propeptide variant that binds less strongly to mature deamidase compared to the natural propeptide. In some embodiments, the second polypeptide is a mutant propeptide variant derived from the same species as the deamidase added to the dairy composition. In other embodiments, the second polypeptide is a mutant propeptide variant derived from a different species than the deamidase added to the dairy composition, where the dairy composition may include milk. In some embodiments, the second polypeptide is synthetic and unrelated to the deamidase added to the dairy composition. In some embodiments, the second polypeptide comprises the amino acid sequence motif [I / M][L / I / V][S / T]AQ and / or [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NOs.

[0078] In some embodiments, the second polypeptide contains an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3. In a further embodiment, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and having glycine at a position corresponding to position 99 of SEQ ID NO: 3.

[0079] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and having glycine at the position corresponding to position 99 of SEQ ID NO: 3. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0080] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and having alanine at the position corresponding to position 99 of SEQ ID NO: 6. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 17.

[0081] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 9, and having glycine at the position corresponding to position 99 of SEQ ID NO: 9. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

[0082] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and having glycine at the position corresponding to position 101 of SEQ ID NO: 12. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 19.

[0083] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 15, and having glycine at the position corresponding to position 100 of SEQ ID NO: 15. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

[0084] Those skilled in the art will recognize that the amount of protein deamidase provided to the dairy composition is sufficient to obtain the desired improved aerated dairy product, and that sufficient amount is partially determined by the incubation temperature and incubation time with the enzyme. In some embodiments, the protein deamidase is provided to the dairy composition of the present invention in an amount of 0.01 to 20 IPA(U) per gram of protein substrate or per gram of milk protein (also referred to as per gram of protein). In further embodiments, the protein deamidase is provided in amounts of 0.1-15 IPA(U) / g protein; 0.5-11 IPA(U) / g protein; 1.0-10 IPA(U) / g protein; 1.0-8.0 IPA(U) / g protein; 2.0-10.0 IPA(U) / g protein; 2.0-8.0 IPA(U) / g protein; 2.0-7.0 IPA(U) / g protein; or 2.5-7.0 IPA(U) / g protein. In some embodiments, at least 1.0, at least 2.0, at least 2.5, at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.5, or at least 7.0 IPA(U) / g substrate protein is added to the dairy composition in the method of the present invention. In some embodiments, protein deamidase is added to the dairy composition of the present invention at concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 IPA(U) / g protein.

[0085] The amount of protein deamidase used in the method of the present invention, whether as a proform, propeptide, or / or mature protein deamidase, does not significantly contribute to the total amount of soluble protein in the dairy composition of the present invention and is unlikely to be a factor in the observed increase in foaming ability of the dairy composition. The weight of milk protein in each reaction is at least 1000 times higher than the total weight of the added enzyme protein and propeptide.

[0086] Those skilled in the art will recognize that the foaming improvements described herein are similarly applicable to aeration methods of dairy compositions for producing other aerated or whipped dairy products. Similar to foamed milk, the presence of fat in dairy compositions has a positive effect on the aeration and quality of aerated dairy products. Introducing air throughout the dairy product results in a smooth, melt-in-the-mouth texture. As described above for milk foam, fat is a crucial component for successful aeration of dairy compositions. Therefore, it is difficult to produce the desired smooth texture in low-fat or fat-free aerated dairy products such as dairy desserts, whipped cream cheese, whipped yogurt, whipped butter, or whipped butter spreads (where the butter spread includes both butter and additional vegetable oils such as canola, olive, or vegetable oil).

[0087] The present invention includes a method for producing an improved aerated dairy product, comprising adding protein deamidase to a dairy product composition and aerating the dairy product composition to produce an aerated dairy product, wherein the aerated dairy product is improved compared to similar methods in which protein deamidase is not added to the dairy product composition. The present invention also includes a method for producing an improved aerated dairy product, comprising adding protein deamidase and a propeptide of deamidase to a dairy product composition and aerating the dairy product composition to produce an aerated dairy product, wherein the aerated dairy product is improved compared to similar methods in which protein deamidase is added but the dairy product composition does not contain a propeptide of deamidase.

[0088] In some embodiments, the dairy composition of the present invention comprises cream, milk, butter, concentrated milk, condensed milk, evaporated milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof. In some embodiments, the milk, butter, concentrated milk, condensed milk, evaporated milk, reconstituted milk, fermented milk, milk protein concentrate, and / or whey protein concentrate are low-fat milk or skim milk or are prepared therefrom.

[0089] In some embodiments, the methods and compositions of the present invention include dairy compositions with a low milk fat content, such as dairy compositions derived from low-fat or skim milk. In some embodiments, milk, butter, concentrated milk, condensed milk, evaporated milk, reconstituted milk, fermented milk, milk protein concentrate, and / or whey protein concentrate contain 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 of fat.

[0090] In some embodiments, protein deamidase and optionally deamidase propeptides are added to the dairy composition immediately before aeration. In some embodiments, the dairy composition may be further treated before aeration. In some embodiments, additional components are added to the dairy composition before or after the addition of protein deamidase and optionally deamidase propeptides.

[0091] Dairy compositions can be heat-treated. Heat treatment may be low-temperature long-time (LTLT), high-temperature short-time (HTST), high-temperature short-time (HHST), ultra-high temperature (UHT), ultra-high temperature pasteurization (UP), or any other form of heat treatment known in the art. In some embodiments, protein deamidase is added to the dairy composition before heat treatment. In other embodiments, protein deamidase is added to the dairy composition after heat treatment. In some embodiments, the dairy composition may be heat-treated both before and after the addition of deamidase as part of a process for producing the resulting dairy product.

[0092] In some embodiments, the dairy composition comprises butter and optionally vegetable oil, and the aerated dairy product is whipped butter or whipped spreadable butter. In some embodiments, the dairy composition comprises fermented milk, and the aerated dairy product is whipped yogurt or frozen yogurt dairy dessert. In some embodiments, the aerated dairy product is a dairy dessert such as ice cream, frozen custard, frozen yogurt, gelato, or ice milk. In some embodiments, the aerated dairy product contains 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 of fat.

[0093] Similar to the milk frothing method described above, in some embodiments, deamidase and a second polypeptide are added to the dairy composition. This second polypeptide may be a propeptide of deamidase. In some embodiments, the second polypeptide is a mutant propeptide variant. In further embodiments, the second polypeptide is a mutant propeptide variant that binds less strongly to mature deamidase compared to the natural propeptide. In some embodiments, the second polypeptide is a mutant propeptide variant derived from the same species as the deamidase added to the dairy composition. In other embodiments, the second polypeptide is a mutant propeptide variant derived from a different species than the deamidase added to the dairy composition. In some embodiments, the second polypeptide is synthetic and unrelated to the deamidase added to the dairy composition. In some embodiments, the second polypeptide comprises the amino acid sequence motif [I / M][L / I / V][S / T]AQ and / or [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NOs.

[0094] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and having glycine at the position corresponding to position 99 of SEQ ID NO: 3. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0095] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and having alanine at the position corresponding to position 99 of SEQ ID NO: 6. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 17.

[0096] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 9, and having glycine at the position corresponding to position 99 of SEQ ID NO: 9. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

[0097] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and having glycine at the position corresponding to position 101 of SEQ ID NO: 12. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 19.

[0098] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 15, and having glycine at the position corresponding to position 99 of SEQ ID NO: 15. In further embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

[0099] The aeration rate of a dairy composition can be adjusted by controlling the amount of deamidase, reaction time, reaction temperature, and other factors known to those skilled in the art. Similarly, the degree of foaming ability of the milk or the texture of the foam can be adjusted by controlling the amount of deamidase, reaction time, reaction temperature, and other factors known to those skilled in the art. Furthermore, the aeration rate of a dairy composition or the foaming ability of the milk can be adjusted by the speed, time, and / or intensity of mechanical aeration or frothing.

[0100] Preferred Embodiment 1. A method for producing improved aerated dairy products, a) A step to obtain a dairy product composition; b) A step of adding a first polypeptide to a dairy product composition, wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase; c) a step of optionally adding a second polypeptide to a dairy product composition, wherein the second polypeptide is a propeptide derived from proform protein deamidase; and d) A process of producing an aerated dairy product by passing aeration through a dairy product composition. Includes, A method for producing aerated dairy products is improved compared to similar methods in which a first polypeptide and an optional second polypeptide are not added to the dairy product composition. 2. The method of Embodiment 1, wherein the dairy product composition comprises cream, milk, butter, concentrated milk, condensed milk, evaporated milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof. 3. The method of Embodiment 1 or 2, wherein the dairy product composition contains 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 of fat. 4. The aerated dairy product, compared to an aerated dairy product manufactured without the addition of protein deamidase, has improved foam density, a lighter, fluffier and / or smoother texture, and / or improved foam stability, according to any one of Embodiments 1 to 3. 5. The aerated dairy product is ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped butter, or whipped butter spread, according to any one of Embodiments 1 to 4. 6. The aerated dairy product contains 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 of fat, according to any one of Embodiments 1 to 5. 7. A method for producing improved foam from a dairy product composition, a) A step to obtain a dairy product composition; b) A step of adding a first polypeptide to the dairy product composition, wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase; c) a step of optionally adding a second polypeptide to a dairy product composition, wherein the second polypeptide is a propeptide derived from proform protein deamidase; and d) A step of passing air through the dairy composition to generate foam. Includes, A method wherein the foam of a dairy product composition is improved compared to a similar method in which a first polypeptide and an optional second polypeptide are not added to the dairy product composition. 8. A method for improving latte art produced from a dairy product composition, a) A step to obtain a dairy product composition; b) A step of adding a first polypeptide to a dairy product composition, wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase; c) a step of optionally adding a second polypeptide to a dairy product composition, wherein the second polypeptide is a propeptide derived from proform protein deamidase; and d) A step of passing air through the dairy product composition to generate foam; and e) The process of creating latte art using a frothed dairy product composition. Includes, Latte art is improved compared to similar methods in which a first polypeptide and an optional second polypeptide are not added to the dairy composition. 9. Compared to foam produced from dairy compositions not treated with deamidase, it has better stability, a higher density of microfoam, higher volume, better cohesiveness, and / or a smoother texture. Method of Embodiment 7 or 8. 10. A dairy product composition comprising cream, milk, concentrated milk, condensed milk, reconstituted milk, milk protein concentrate, whey protein concentrate, or a combination thereof, according to any one of Embodiments 7 to 9. 11. The dairy product composition is whole milk, low-fat milk, or skim milk, according to any one of Embodiments 7 to 9. 12. The method of Embodiment 11, wherein the milk contains 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 of milk fat. 13. Any one of Embodiments 1 to 12, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 5, 8, 11, or 14. 14. The first polypeptide comprises the amino acid sequence of SEQ ID NOs: 2, 5, 8, 11, or 14, according to any one of Embodiments 1 to 13. 15. Deamidase is added in an amount ranging from 0.01 to 15.0 IPA(U) / g protein; 0.1 to 11.0 IPA(U) / g protein; 0.5 to 7.0 IPA(U) / g protein; or 2.5 to 7.0 IPA(U) / g protein, according to any one of Embodiments 1 to 14. 16. Any one of Embodiments 1 to 15, wherein the dairy composition is heat-treated before treatment with deamidase. 17. The heat treatment is carried out at 60-140°C, according to the method of Embodiment 16. 18. The heat treatment is low-temperature long-time (LTLT), high-temperature short-time (HTST), high-temperature short-time (HHST), ultra-high temperature (UHT), or ultra-high temperature sterilization (UP) treatment, according to the method of Embodiment 16 or 17. 19. Any one of Embodiments 1 to 15, wherein the dairy composition is not heat-treated before treatment with deamidase. 20. A method according to any one of Embodiments 1 to 19, wherein the second polypeptide is a propeptide derived from proform protein deamidase and comprises the amino acid sequence motif [I / M][L / I / V][S / T]AQ and / or the amino acid sequence motif [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ. 21. The second polypeptide is, a) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and having glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and having alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 9, and having glycine at the position corresponding to position 99 of SEQ ID NO: 9; d) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and having glycine at the position corresponding to position 101 of SEQ ID NO: 12; or e) Any one of Embodiments 1 to 20, which is a propeptide comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 15, and having glycine at a position corresponding to position 100 of SEQ ID NO: 15. 22. The second polypeptide comprises the amino acid sequence of SEQ ID NOs: 16, 17, 18, 19, and 20, according to any one of Embodiments 1 to 21. 23. Any one of Embodiments 1 to 22, wherein the first polypeptide and the second polypeptide are derived from the same proform protein deamidase. 24. Any one of Embodiments 1 to 23, wherein the proform protein deamidase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 21, 22, 23, or 24. 25. The first and second polypeptides are derived from proforms containing amino acid sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 21, 22, 23, or 24, and as a result, the first polypeptides each have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, or 92% sequence identity to SEQ ID NOs: 2, 5, 8, 11, or 14. A method of any one of Embodiments 1 to 24, wherein the first is a mature protein deamidase containing an amino acid sequence having 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity, and the second polypeptide is a propeptide containing amino acids having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 16, 17, 18, 19, or 20. 26. a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; d) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and the optionally selected second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12, and having glycine at the position corresponding to position 101 of SEQ ID NO: 12; or e) Any one of Embodiments 1 to 25, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 14, and the optionally selected second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 15, and has glycine at a position corresponding to position 100 of SEQ ID NO: 15. 27. Any one of Embodiments 1 to 22 and 26, wherein the first polypeptide and the second polypeptide are not derived from the same proform protein deamidase. 28. The method of Embodiment 27, wherein the second polypeptide is a propeptide expressed and purified without a proform intermediate. 29. Any one of Embodiments 1 to 28, wherein the performance of the deamidase is increased by the addition of a second polypeptide. 30. A dairy product composition comprising a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase) and optionally a second polypeptide (wherein the second polypeptide is a propeptide derived from a proform protein deamidase). 31. a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; d) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and the optionally selected second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12, and having glycine at the position corresponding to position 101 of SEQ ID NO: 12; or e) The dairy product composition of Embodiment 30, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14, and the optionally selected second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 15, and has glycine at the position corresponding to position 100 of SEQ ID NO: 15. 32. The dairy composition of Embodiment 30 or 31, having improved air permeability or foaming properties compared to a dairy composition that does not contain a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from proform protein deamidase) and an optional second polypeptide (wherein the second polypeptide is a propeptide derived from proform protein deamidase). 33. A dairy product composition comprising cream, milk, butter, concentrated milk, evaporated milk, condensed milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof, any one of the dairy product compositions of Embodiments 30 to 32. 34. A dairy product composition according to any of embodiments 30 to 33, which is processed to produce an aerated dairy product such as ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, or whipped butter. 35. A dairy product composition according to any of Embodiments 30 to 33, wherein the dairy product composition is whole milk, low-fat milk, or skim milk. 36. The dairy composition of Embodiment 35, which is processed to produce barista milk. 37. A dairy product composition of any one of embodiments 30 to 36, wherein the first polypeptide and the second polypeptide are not derived from the same proform protein deamidase. 38. The dairy composition of Embodiment 37, wherein the second polypeptide is a propeptide expressed and purified without a proform intermediate. 39. Aerated dairy product manufactured by any one of the methods of Embodiments 1-7 and 13-29. 40. The aerated dairy product of Embodiment 39 is ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped butter, or whipped butter spread. 41. The aerated milk product of Embodiment 39 or 40, having improved foam density, a lighter, fluffier and / or smoother texture, and / or improved foam stability, compared to an aerated milk product produced without the addition of protein deamidase and an optional second polypeptide (wherein the second polypeptide is derived from deamidase propeptide). 42. Foam generated by any of the methods in Embodiments 8 to 29. 43. The foam of Embodiment 42, having better stability, a higher density microfoam, and / or a smoother texture compared to foam produced from milk that has not been treated with a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from proform protein deamidase) and an optional second polypeptide (wherein the second polypeptide is a propeptide derived from proform protein deamidase). 44. Use of a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase) and an optional second polypeptide (wherein the second polypeptide is a propeptide derived from a proform protein deamidase) for producing a dairy product composition having improved air permeability or foaming properties. 45. Use of Embodiment 44, a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; d) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and the optionally selected second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12, and having glycine at the position corresponding to position 101 of SEQ ID NO: 12; or e) Use of Embodiment 44, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14, and the optionally selected second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 15, and has glycine at a position corresponding to position 100 of SEQ ID NO: 15. 46. ​​Use of the protein deamidase comprising the amino acids of SEQ ID NOs: 2, 5, 8, 11, or 14 in Embodiment 44 or 45. 47. Use of any of Embodiments 44 to 46, wherein the dairy composition includes cream, milk, butter, concentrated milk, condensed milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof. 48. A method for producing improved aerated dairy products, a) A step to obtain a dairy product composition; b) A step of adding a first polypeptide to a dairy product composition, wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase; c) optionally, a step of adding a second polypeptide to a dairy product composition, wherein the second polypeptide is a propeptide derived from the same proform protein deamidase; and d) A process of producing an aerated dairy product by passing aeration through a dairy product composition. Includes, A method for producing aerated dairy products is improved compared to similar methods in which a first polypeptide and an optional second polypeptide are not added to the dairy product composition. 49. The method of Embodiment 48, wherein the aerated dairy product composition produced by adding the first polypeptide and the second polypeptide is improved compared to a similar method in which the first polypeptide is added to the dairy product composition but the second polypeptide is not. 50. The method of Embodiment 48 or 49, wherein the dairy composition comprises cream, milk, butter, concentrated milk, condensed milk, evaporated milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof. 51. Any one of Embodiments 48 to 50, wherein the mature protein deamidase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 5, 8, or 11. 52. Any one of Embodiments 48 to 51, wherein the second polypeptide is a propeptide containing a mutation that reduces its binding affinity to the deamidase domain compared to the parent propeptide. 53. The second polypeptide is, a) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and having glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and having alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) An amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 9, and having glycine at the position corresponding to position 99 of SEQ ID NO: 9; or d) Any one of Embodiments 48 to 52, wherein the propeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and has glycine at a position corresponding to position 101 of SEQ ID NO: 12. 54. The second polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 17, 18, or 19, according to any one of the embodiments 48 to 53. 55. A method which is either Embodiment 48 or 54, a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; or d) Any one of Embodiments 48 to 54, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and the optionally selected second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12, and has glycine at a position corresponding to position 101 of SEQ ID NO: 12. 56. A dairy product composition comprising a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase) and optionally a second polypeptide (wherein the second polypeptide is a propeptide derived from the same proform protein deamidase). 57. A dairy product composition comprising a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase) and a second polypeptide (wherein the second polypeptide is a propeptide derived from the same proform protein deamidase). 58. A dairy product composition of Embodiment 56 or 57, a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; or d) The dairy product composition of Embodiment 56 or 57, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12, and has glycine at a position corresponding to position 101 of SEQ ID NO: 12. 59. A dairy product composition comprising cream, milk, butter, concentrated milk, evaporated milk, condensed milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof, any one of the dairy product compositions of Embodiments 56 to 58. 60. A dairy product composition, any one of embodiments 56 to 59, which is processed to produce an aerated dairy product such as ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, or whipped butter. 61. Aerated dairy product manufactured by any of the methods of Embodiments 48 to 55. 62. The aerated dairy product of Embodiment 61 is ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped butter, or whipped butter spread. 63. Use of a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase) and an optional second polypeptide (wherein the second polypeptide is a propeptide derived from the same proform protein deamidase) for producing a dairy product composition having improved air permeability or foaming properties. 64. Use of a first polypeptide (wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase) and a second polypeptide (wherein the second polypeptide is a propeptide derived from the same proform protein deamidase) for producing a dairy product composition having improved air permeability or foaming properties. 65. Use of Embodiment 62 or 63, a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; or d) Use of Embodiment 62 or 63, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11, and the second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12, and has glycine at a position corresponding to position 101 of SEQ ID NO: 12.

[0101] The invention described herein and claimed is not to be limited to the scope of the specific embodiments disclosed herein, for these embodiments are intended to illustrate several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention, and one or more of the embodiments.

[0102] Various references are cited herein, and their disclosures are incorporated in their entirety by reference. The present invention will be further illustrated by the following embodiments, which should not be construed as limiting the scope of the invention. [Examples]

[0103] Materials and methods Example 1: Activation and purification of protein deamidase Proform purification: The proform of the protein deamidase was expressed in a heterologous expression system, and the cell aggregate was removed by centrifugation followed by bacterial filtration. The proform was purified using standard protein purification techniques known to those skilled in the art. The purity of the final sample was confirmed by SDS-PAGE. These samples were used for activation.

[0104] Activation and Purification of Mature Form: Specific glutamyl endopeptidase was added to a purified proform and incubated at room temperature with gentle shaking. The progress of the activation process was checked using standard SDS-PAGE (shift from approximately 30 kDa to approximately 20 kDa). Once the activation process was complete, the mature enzyme was separated from the propeptide, and the sample was buffer-exchanged using a Sephadex 25 resin-packed column according to a standard protocol to inactivate / remove the glutamyl endopeptidase. The eluted fraction was confirmed by SDS-PAGE, and the fraction containing the mature active molecule was pooled. The resulting sample was loaded onto a cation exchanger, Source 15S, calibrated with the same buffer, and eluted using a linear gradient (0-100% in 20 CV). The peak fraction was analyzed by SDS-PAGE, and the relevant fraction (mature deamidase) was pooled. The concentration was determined using A280 measurement on a Nanodrop instrument in combination with the theoretical extinction coefficient. The sample was frozen at -18°C until further use.

[0105] Propeptide purification: The propeptide of the protein deamidase was cloned and expressed in a heterologous expression system. At the end of fermentation, the cell aggregate was removed by centrifugation followed by bacterial filtration. The obtained sample was buffer-exchanged using a Sephadex G-25 resin-packed column according to a standard protocol. The peak fraction was pooled and loaded onto an anion exchange column (Source 15Q) equilibrated with the same buffer. The pass-through and wash fractions were pooled and loaded onto a Source 15Q column. The pass-through and wash fractions were pooled and concentrated using a pressurized UF cell with a 5 MWCO membrane. The concentration of the resulting retention solution was determined using A280 measurement on a Nanodrop instrument combined with the theoretical extinction coefficient.

[0106] Example 2: Skim milk treated with deamidase and frothed Enzyme treatment: 4 x 250 g of pasteurized skim milk (Egelykke, Arla Foods, Viby, Denmark; 3.5 g protein / 100 g; 0.1 g fat / 100 g; 4.7 g carbohydrates / 100 g) was weighed into blue-capped bottles and heated to 55°C in a water bath. Protease containing the amino acid sequence of SEQ ID NO: 2 (derived from C. viscerum; 350 IPA(U) / g) was added at concentrations of 0, 1.4, 3.4, or 6.8 IPA(U) / g of milk protein and incubated for 30 minutes. After enzymatic treatment, the bottles were transferred to a 75°C water bath and held at 72°C for 15 seconds. The bottles were then cooled on ice and stored at 5°C for at least 90 minutes.

[0107] Frothing in a Milk Frother: 120 g (+ / - 1 g) of cold (5°C) enzyme-treated milk was weighed. Four 120 g samples, each covering the dose-response levels of 0, 1.4, 3.4, and 6.8 IPA(U) / g milk protein, were heated to 50°C or 60°C. The heated samples were added to a Milk Frother (Severin SM3587 Spuma 700 plus; Severin Elektrogerate GmbH, Sundern, Germany) and stirred for 60 seconds without further heating. The frothed milk was transferred to a 500 ml measuring cup, and the total volume, foam volume, and liquid volume were measured. After 10 minutes, the total volume, foam volume, and liquid volume were measured again at room temperature to assess foam stability. The sample used with 0 IPA(U) / g protein served as a control sample.

[0108] [Table 2]

[0109] The addition of the protein deamidase of Sequence ID No. 2 significantly increased the volume of bubbles at both 50°C and 60°C.

[0110] In addition to volume, foam quality was evaluated. The fluffiness of the foam (which can range from larger bubbles that are clearly visible as individual bubbles to homogeneous microfoam with a silky appearance) was analyzed, as was its cohesiveness. Foam cohesiveness was evaluated while transferring the frothed milk from the Milk Frother to a measuring cup. In the quality evaluation, foam cohesiveness was evaluated as follows: None (foam and liquid milk separated, the liquid phase lacked fluffiness, and it was not possible to create latte art); Poor (foam and liquid milk separated, and the liquid phase had some fluffiness that could produce latte art, although small spherical clumps of foam may have formed); Good (foam and liquid milk were partially integrated, and the liquid phase had good fluffiness that could produce good latte art); and Very Good (foam and liquid milk were completely integrated, the liquid phase had high fluffiness, and it was possible to produce very good latte art).

[0111] [Table 3]

[0112] The addition of the protein deamidase of Sequence ID No. 2 improved foam volume and quality at both 50°C and 60°C. A positive correlation was observed between the enzyme dose and foam volume, resulting in the formation of microfoam with a silky appearance and good foam cohesion.

[0113] Example 3: Semi-skimmed milk and whole milk treated with deamidase to create foam. Enzyme treatment: 4 x 500 g of pasteurized semi-skimmed milk (Arla Foods, Viby, Denmark; 3.5 g protein / 100 g; 1.5 g fat / 100 g; 4.6 g carbohydrates / 100 g) and 4 x 500 g of pasteurized whole milk (Arla Foods; 3.4 g protein / 100 g; 3.5 g fat / 100 g; 4.6 g carbohydrates / 100 g) were weighed into blue-capped bottles and heated to 55°C in a water bath. Mature protein deamidase containing the amino acid sequence of SEQ ID NO: 2 was added at concentrations of 0, 1.4, 3.4, and 6.8 IPA(U) / g and incubated for 30 minutes. After enzyme treatment, the bottles were transferred to a 75°C water bath and held at 72°C for 15 seconds. The bottles were then cooled on ice and stored at 5°C for at least 90 minutes.

[0114] Foaming in a Milk Frother: 120g (+ / -1g) of semi-skimmed or whole milk treated with cold (5°C) enzymes was weighed. Three 120g samples, each covering the dose-response levels of 0, 1.4, 3.4, and 6.8 IPA(U) / g milk protein, were heated to 40°C, 50°C, or 60°C, respectively. The heated samples were added to a Milk Frother (Severin SM3587 Spuma 700 plus) and stirred for 60 seconds without further heating. The frothed milk was transferred to a 500ml measuring cup, and the total volume, foam volume, and liquid volume were measured. After 10 minutes, the total volume, foam volume, and liquid volume were measured again at room temperature to assess foam stability.

[0115] The volume results obtained by frothing semi-skimmed milk and whole milk are shown in Tables 3 and 5 below, respectively. The quality of the foam produced from semi-skimmed milk and whole milk in terms of fluffiness and cohesiveness is described in Tables 4 and 6, respectively. The sample used with 0 IPA(U) / g protein is the control sample.

[0116] [Table 4]

[0117] [Table 5]

[0118] [Table 6]

[0119] [Table 7]

[0120] The addition of the protein deamidase of Sequence ID No. 2 dramatically improved the frothing ability in both semi-skimmed and whole milk. The milk quality improved, with increased fluffiness, resulting in more microfoam production, a silky appearance, and excellent consistency.

[0121] Example 4: Skim milk treated with deamidase, purified deamidase, and commercially available deamidase, and then frothed. The protein deamidase of the present invention is generated from a proform, which is an inactive polypeptide. The proform is cleaved to release a propeptide from the mature protein deamidase. In this example, samples containing mature protein deamidase with or without the propeptide are assayed. The molar concentration of the added propeptide is no more than twice the molar concentration of the mature protein deamidase.

[0122] Enzyme treatment: 8 × 120 g (+ / - 1 g) of skim milk (Egelykke, Arla Foods; 3.5 g protein / 100 g; 0.1 g fat / 100 g; 4.7 g carbohydrates / 100 g) was weighed into a blue-capped bottle and heated to 55°C in a water bath. The control sample did not contain any protein deamidase or propeptide added to the milk. Mature protein deamidase containing the amino acid sequence of SEQ ID NO: 2, with or without the propeptide containing SEQ ID NO: 2, or PG500 (Amano Enzyme Europe Ltd., Oxfordshire, UK; 300.5 IPA(U) / g) was added at concentrations of 0, 1.3, or 3.4 IPA(U) / g of milk protein, and incubated for 30 minutes. After enzyme treatment, the bottles were transferred to a 75°C water bath and held at 72°C for 15 seconds. A 200 μl subsample of the inactivated sample was retained for ammonium analysis. The vial was then cooled on ice and stored at 5°C for at least 90 minutes.

[0123] Foaming in a Milk Frother: Eight samples of cold (5°C) enzyme-treated skim milk were heated in a Milk Frother (Severin SM3587 Spuma 700 plus) with stirring for 80 seconds. The frothed milk was transferred to a 250 ml measuring cup, and the total volume, foam volume, and liquid volume were measured. After 10 minutes, the total volume, foam volume, and liquid volume were measured again at room temperature to evaluate foam stability.

[0124] Ammonium assay: The activity assay consists of two distinct and isolated parts: 1) an enzymatic step in which ammonia is formed by the catalytic action of protein deamidase; and 2) a non-enzymatic detection step in which the ammonia formed in step (1) is derivatized to an indophenol blue compound having an absorption maximum at 630 nm.

[0125] In step (1), ammonia is generated by the deamidation of protein deamidase. In step (2), the generated ammonia reacts with phenol under alkaline conditions to form dioxyphenylamine. The reaction is catalyzed by sodium pentacyanonitrosylferrate(III) (sodium nitroprusside). "Coloring reagent solution A" contains phenol and sodium nitroprusside. "Color development reagent solution B" provides alkaline reaction conditions. The intermediate is then oxidized by adding sodium hypochlorite ("color development reagent solution C") to form indophenol blue. This compound absorbs visible light at 630 nm. The enzyme activity is then calculated using a standard curve.

[0126] Assay procedure: Step (1) Enzyme step involving ammonia formation reagent: Assay dilution solution: 0.2 M sodium phosphate buffer, 0.01% Triton® X-100, pH 6.5 Assay buffer: Same as above. Used to prepare stock and diluted samples of protein deamidase (hereinafter referred to as "enzyme"). Substrate solution: 30 mM Z-Gln-Gly (Merck C6154-1G) in the assay dilution solution (check pH after dissolution). Stop liquid: 0.4M TCA Standard: NH4Cl (Ammonium standard for IC, Merck 59755-100ML, 1000mg / L NH4 in water) + Dilute the assay solution with the assay dilution solution (see also the section on "Standard Curve"). The enzyme product is dissolved / diluted in the assay buffer to prepare a suitable dilution that yields a linear assay response.

[0127] incubation: 1. Add 10 μL of diluted enzyme sample to the wells of a 96-well microtiter plate (MTP) in a triple-row configuration. 2. Add 100 μL of substrate solution to each well. 3. For blank samples, add 100 μL of 0.4 M TCA solution. 4. Use a transparent plate sealer to seal the plate. 5. Incubate the plate in a thermomixer with a lid heating function at 37°C for 10 minutes at 500 rpm. 6. To stop the reaction, carefully add 100 μL of 0.4 M TCA solution (except for blank samples that already contain TCA). Total reaction volume: 210 μL

[0128] Step (2) Ammonia detection step reagent: Colorimetric reagent A: 4% (w / v) phenol, 0.015% (w / v) sodium pentacyanonitrosyl iron(III) dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO]·2H2O). Colorimetric reagent B: 5% (w / v) potassium hydroxide. Colorimetric reagent C: 28% (w / v) potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25ml, less than 5% usable Cl2).

[0129] incubation: 1. Transfer 15 μL from each well in step (1) to a new 96-well MTP. Transfer 2.45 μL of Milli-Q water to each well. 3. Add 30 μL of chromogenic reagent B to each well (mix by gently shaking on the workbench). 4. Add 60 μL of chromogenic reagent A to each well (mix by gently shaking on the workbench). 5. Add 60 μL of chromogenic reagent C to each well (mix by gently shaking on the workbench). 6. Color development: Carefully seal the plate and leave it on the workbench for 30 minutes. 7. Carefully transfer the MTP to a plate reader and measure its absorbance at 630 nm. Total reaction volume: 210 μL

[0130] Standard curve: Standard stock solution: 1000mg NH4 + / L. In the ammonia detection step, a standard curve is created by adding a diluted ammonium standard substance to the assay dilution buffer. Specifically, 15 μL of diluted ammonia standard substance is mixed with 45 μL of water, and then the chromogenic reagents are added in the order B, A, and C described above.

[0131] The amount of enzyme that produces 1 μmol of ammonia per minute at 37°C is defined as 1 unit (indophenol assay unit; IPA(U)):

number

number

number

[0132] Table 7 shows the volume results and ammonium assay results for all tested protein deaminases. The propeptide added to the sample contains the amino acid sequence of SEQ ID NO: 16. Table 8 describes the quality of the generated foam in terms of fluffiness and cohesiveness. The control sample did not contain protein deamidase added to milk.

[0133] [Table 8]

[0134] The quality of the foam was evaluated in terms of fluffiness (the size of the bubbles ranging from clearly visible to homogeneous microfoam with a silky appearance) and cohesion (the ability to create latte art and avoid small, spherical clumps of foam) while transferring it from the Frother to a measuring cup.

[0135] [Table 9]

[0136] Preparations containing both mature protein deamidase and propeptide surprisingly performed better than purified mature form and commercially available Amano PG500 in terms of higher foaming, higher ammonium release, and improved foam quality.

[0137] Example 5: Deamidase, purified deamidase, and skim milk frothed using FoamScan. Purification of protein deamidase: Protein deamidase proforms containing the amino acid sequences of SEQ ID NOs: 1, 21, 23, or 24 were purified according to Example 1. As described in Example 1, the proform polypeptide was enzymatically cleaved to produce mature protein deamidase. In samples containing propeptides, no further purification steps were performed to remove the propeptides. In samples without propeptides, the propeptides were removed as described in Example 1.

[0138] Enzyme treatment: 80g of skim milk (Egelykke, Arla Foods Group, Denmark; 3.5g protein / 100g; 0.1g fat / 100g; 4.7g carbohydrates / 100g) was weighed into a blue-capped bottle and heated to 55°C in a water bath. Protein deamidase containing the proform amino acid sequence of SEQ ID NOs. 1, 21, 23, or 24 was added at a concentration of 0 (blank control) or 3.4 IPA(U) / g milk protein and incubated for 30 minutes. After enzyme treatment, the bottle was transferred to a 75°C water bath and held at 72°C for 15 seconds. The bottle was then cooled on ice and stored at 5°C.

[0139] Foaming in FOAMSCAN®: 60 mL of enzyme-treated milk and an enzyme-free control milk were applied to a tube in a FOAMSCAN® (Teclis Scientific, Lyon, France) according to the manufacturer's instructions protocol, with the following settings: initial liquid volume = 60 mL, nitrogen gas flow rate = 100 mL / min, final foaming time = 90 seconds, and total gas volume = 150 mL (i.e., final foaming time). Foam was recorded for at least 300 seconds to measure the half-life (t1 / 2) of the foam volume after the nitrogen gas flow was stopped. The final foam volume (mL) and t1 / 2 of the foam volume (seconds) were recorded, and the results are shown in Table 9.

[0140] Ammonium assay: This was carried out as described in Example 4.

[0141] Table 9 shows the volume results and ammonium assay results for all tested protein deaminases. The control sample did not contain protein deaminase added to milk. The generated ammonium is shown after subtracting the blank.

[0142] [Table 10]

[0143] As shown in Table 9, protein deamidases derived from various Chryseobacterium species improve the foaming of skim milk compared to samples without enzyme addition. Surprisingly, the final foam volume is higher in samples with propeptides present compared to enzyme treatments where propeptides are removed. The half-life of the foam volume is also extended in enzyme treatments where propeptides are present. Furthermore, the measured ammonia produced correlates with the final foam volume. In conclusion, the presence of propeptides has a significant effect on the milk foam produced, and the presence of propeptides increases the efficiency of the enzyme.

Claims

1. A method for producing improved aerated dairy products, a) Steps to obtain a dairy product composition; b) A step of adding a first polypeptide to the dairy product composition, wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase; c) a step of optionally adding a second polypeptide to the dairy product composition, wherein the second polypeptide is a propeptide derived from the same proform protein deamidase; and d) A step of producing an aerated dairy product by passing the dairy product composition through it; Includes, The aerated dairy product is an improved method compared to similar methods in which the first polypeptide and the optionally selected second polypeptide are not added to the dairy product composition.

2. The method according to claim 1, wherein the dairy product composition comprises cream, milk, butter, concentrated milk, condensed milk, evaporated milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof.

3. The method according to claim 1 or 2, wherein the mature protein deamidase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 2, 5, 8, or 11.

4. The method according to any one of claims 1 to 3, wherein the second polypeptide is a propeptide containing a mutation that reduces its binding affinity to the deamidase domain compared to the parent propeptide.

5. The second polypeptide described above is a) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and having glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and having alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 9, and having glycine at the position corresponding to position 99 of SEQ ID NO: 9; or d) The method according to any one of claims 1 to 4, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and having glycine at a position corresponding to position 101 of SEQ ID NO:

12.

6. The method according to any one of claims 1 to 5, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 17, 18, or 19.

7. a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 2, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 5, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; or d) The method according to any one of claims 1 to 6, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 11, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and has glycine at a position corresponding to position 101 of SEQ ID NO:

12.

8. A dairy product composition comprising a first polypeptide, wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase, and optionally a second polypeptide, wherein the second polypeptide is a propeptide derived from the same proform protein deamidase.

9. a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 2, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 5, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; or, d) The dairy composition according to claim 8, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 11, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and has glycine at a position corresponding to position 101 of SEQ ID NO:

12.

10. The dairy product composition according to claim 8 or 9, wherein the dairy product composition comprises cream, milk, butter, concentrated milk, evaporated milk, condensed milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof.

11. The dairy product composition according to any one of claims 8 to 10, wherein the dairy product composition is processed to produce an aerated dairy product such as ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, or whipped butter.

12. Aerosolized dairy product manufactured by the method described in any one of claims 1 to 7.

13. The aerated dairy product according to claim 12, wherein the aerated dairy product is ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped butter, or whipped butter spread.

14. The use of a first polypeptide for producing a dairy product composition having improved air permeability or foaming properties, wherein the first polypeptide is a mature protein deamidase derived from a proform protein deamidase, and an optional second polypeptide, wherein the second polypeptide is a propeptide derived from the same proform protein deamidase.

15. a) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 2, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 3, and has glycine at the position corresponding to position 99 of SEQ ID NO: 3; b) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 5, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 6, and has alanine at the position corresponding to position 99 of SEQ ID NO: 6; c) The first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 9, and has glycine at the position corresponding to position 99 of SEQ ID NO: 9; or d) The use according to claim 14, wherein the first polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 11, and the optional second polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to SEQ ID NO: 12, and has glycine at a position corresponding to position 101 of SEQ ID NO: 12.

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