Foamable dairy product

Abstract

The present invention provides a dairy composition having improved inflation properties wherein the dairy composition comprises a protein deamidase and optionally a second polypeptide derived from a propeptide in the proform of a protein deamidase. The invention further provides an improved method for producing an aerated dairy product from a dairy composition wherein a protein deamidase and optionally a second polypeptide derived from a propeptide in the form of a protein deamidase is added to the dairy composition.

Description

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

[0001] This invention relates to dairy products that have been treated to enhance their foaming properties. The invention also provides a method for treating dairy products to enhance their foaming properties. Background Technology

[0002] Cappuccino-style drinks are currently very popular, typically characterized by a mixture of coffee and milk topped with a layer of milk foam, often decorated with patterns or "latte art." The milk provides both the creamy texture of the drink and the smoothness of the foam. Whole milk is typically used for this beverage and is often the preferred choice. However, many health-conscious consumers prefer low-fat milk, especially milk containing less than 2% milk fat, or even skim milk containing very little milk fat. Unfortunately, it is difficult to produce the same quality foam using low-fat or skim milk as using whole milk. Furthermore, even whole milk can sometimes be difficult to foam to the desired consistency and texture. Summary of the Invention

[0003] This invention provides a dairy composition with improved aeration properties, wherein the dairy composition comprises a first polypeptide (which is a mature protein deamidase) and optionally a second polypeptide (which is a propeptide derived from the pro-deamidase form of a protein). The invention further provides an improved method for producing aerated dairy products from the dairy composition, wherein a protein deamidase and optionally a second polypeptide derived from the pro-deamidase form of the dairy composition are added to the dairy composition. Unexpectedly, compared to the method of adding only the mature protein deamidase, the presence of the propeptide in the dairy composition improves the foaming properties of the dairy composition, thereby producing an improved aerated dairy product.

[0004] The present invention also provides a method for improving the foaming of a dairy composition, wherein the dairy composition comprises a first polypeptide (which is a mature protein deamidase) and optionally a second polypeptide (which is a propeptide derived from the proprotein deamidase form). The present invention further provides a method for improving latte art made from a dairy composition, wherein the dairy composition comprises a first polypeptide (which is a mature protein deamidase) and optionally a second polypeptide (which is a propeptide derived from the proprotein deamidase form). sequence SEQ ID NO: 1: From visceral chrysogena ( Chryseobacterium viscerum The amino acid sequence of the proto-peptide of the deamidase, which contains the propeptide variant of F99G.

[0005] SEQ ID NO: 2: Amino acid sequence of mature deamidase derived from SEQ ID NO: 1.

[0006] SEQ ID NO: 3: The amino acid sequence of the precursor peptide from Visceral Chrysogenum, which contains F99 (wild type).

[0007] SEQ ID NO: 4: From *Aureobacterium praecox* ( Chryseobacterium gambrini The amino acid sequence of the original form of the deamidase polypeptide.

[0008] SEQ ID NO: 5: Amino acid sequence of mature deamidase derived from SEQ ID NO: 4.

[0009] SEQ ID NO: 6: Amino acid sequence of the precursor peptide derived from SEQ ID NO: 4.

[0010] SEQ ID NO: 7: From Culex pipiens ( Chryseobacterium culicis The amino acid sequence of the original form of the deamidase polypeptide.

[0011] SEQ ID NO: 8: Amino acid sequence of mature deamidase derived from SEQ ID NO: 7.

[0012] SEQ ID NO: 9: Amino acid sequence of the precursor peptide derived from SEQ ID NO: 7.

[0013] SEQ ID NO: 10: From sewage Chlorobacterium tumefaciens ( Chryseobacterium defluvii The amino acid sequence of the original form of the deamidase polypeptide.

[0014] SEQ ID NO: 11: Amino acid sequence of mature deamidase derived from SEQ ID NO: 10.

[0015] SEQ ID NO: 12: Amino acid sequence of the precursor peptide derived from SEQ ID NO: 10.

[0016] SEQ ID NO: 13: From *Cyclophorus pyogenes* ( Chryseobacterium proteolyticum The amino acid sequence of the original form of the deamidase polypeptide.

[0017] SEQ ID NO: 14: Amino acid sequence of mature deamidase derived from SEQ ID NO: 13.

[0018] SEQ ID NO: 15: Amino acid sequence of the precursor peptide derived from SEQ ID NO: 13.

[0019] SEQ ID NO: 16: F99G, a propeptide variant of SEQ ID NO: 3.

[0020] SEQ ID NO: 17: F99A, a precursor peptide variant of SEQ ID NO: 6.

[0021] SEQ ID NO: 18: F99G, a propeptide variant of SEQ ID NO: 9.

[0022] SEQ ID NO: 19: L101G, a precursor peptide variant of SEQ ID NO: 12.

[0023] SEQ ID NO: 20: L100G, a precursor peptide variant of SEQ ID NO: 15.

[0024] SEQ ID NO 21: A variant of SEQ ID NO: 4 containing a propeptide variant of SEQ ID NO: 17.

[0025] SEQ ID NO 22: A variant of SEQ ID NO: 7 containing a propeptide variant of SEQ ID NO: 18.

[0026] SEQ ID NO 23: A variant of SEQ ID NO: 10 containing a propeptide variant of SEQ ID NO: 19.

[0027] SEQ ID NO 24: A variant of SEQ ID NO: 13 containing a propeptide variant of SEQ ID NO: 20. definition Deamidase: The term "deamidase" refers to the activity of protein-glutamine glutaminase (also known as gamma-glutamyl peptide glutaminase), as described in EC 3.5.1.44, which catalyzes the hydrolysis of γ-amides of glutamines substituted at the carboxyl position or both the α-amino and carboxyl positions (e.g., L-glutamylglycine and L-phenylalanyl-L-glutamylglycine). Therefore, deamidases can deamidate glutamine residues in proteins to glutamate residues, and deamidases are also referred to as protein glutamine deamidases. Deamidases include the Cys-His-Asp catalytic triad (e.g., Cys-156, His-197, and Asp-217, as shown in Hashizume et al., “Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex”, Journal of Biological Chemistry, Vol. 286, No. 44, pp. 38691–38702) and belong to InterPro entry IPR041325.

[0028] Deamidase activity: Deamidase (protein glutaminase) activity was measured using the assay described in the examples. The activity assay consisted of two separate parts: (1) an enzymatic step in which ammonia was formed by the catalysis of the protein deamidase; and (2) a non-enzymatic assay step in which the ammonia formed in step (1) was derivatized into an indophenol blue compound with maximum absorption at 630 nm. The amount of enzyme that produces 1 μmol of ammonia per minute at 37 °C was defined as 1 unit (expressed in indophenol assay units: IPA (U)). Activity was determined relative to known intensity standards.

[0029] Deamidase inhibitory domain: The term "deamidase inhibitory domain" refers to an amino acid sequence that interacts with amino acid residues at the active site of a deamidase and inhibits or reduces deamidase activity. For example, in the presence of a deamidase inhibitory domain, deamidase activity can be reduced to less than 50%, preferably less than 40% (compared to deamidase activity in the absence of deamidase inhibitory activity).

[0030] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is the precursor of mRNA, which is processed through a series of steps (including splicing) to become mature, spliced ​​mRNA.

[0031] 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 typically defined by an open reading frame (OPG), which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0032] Control Sequences: The term "control sequence" refers to a nucleic acid sequence that participates in the regulation of polynucleotide expression in a particular organism, either in vivo or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from different genes) for the polynucleotide encoding a polypeptide, and is native or heterologous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding regions of polynucleotides encoding polypeptides.

[0033] Expression: The term “expression” refers to any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0034] Expression vector: An expression vector is a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, which is operatively linked to a suitable control sequence capable of influencing the expression of the DNA in a suitable host. Such control sequences may include promoters that influence transcription, optional operon sequences that control transcription, sequences encoding suitable ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0035] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of a polypeptide, wherein the "extended" polypeptide has deamidase activity.

[0036] Fragment: The term "fragment" refers to a polypeptide that has one or more amino acids missing from the amino and / or carboxyl termini of a mature polypeptide, wherein the fragment has deamidase activity.

[0037] Fusion polypeptide: The term "fusion polypeptide" is a polypeptide in which one polypeptide of the present invention is fused to the N-terminus and / or C-terminus of another polypeptide of the present invention. Fusion polypeptides are produced by fusing two or more polynucleotides encoding the polypeptide of the present invention together. Techniques for producing fusion polypeptides are known in the art and include linking the coding sequences of the polypeptides such that they conform to reading frames, and that the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intronomer techniques in which fusion polypeptides are produced post-translational (Cooper et al., 1993, EMBO J. [European Journal of Molecular Biology] 12: 2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779). Fusion polypeptides may further include a cleavage site between the two polypeptides. Thus, these fusion polypeptides may include a site-specific endopeptidase cleavage site, for example, within 20 amino acids, preferably within 10 amino acids, at the C-terminus of the first polypeptide. Examples of well-known site-specific endopeptidases include gamma-glutamyl endopeptidases (e.g., EC3.4.21.19 or EC 3.4.21.82), trypsin-like endopeptidases, and chymotrypsin-like endopeptidases (including intestinal peptidases).Cleavage sites and many other examples of corresponding endopeptidases include, but are not limited to, those disclosed in: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology] 3: 568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63: 3488-3493; Ward et al., 1995, Biotechnology [Biotechnology] 13: 498-503; and Contreras et al., 1991, Biotechnology [Biotechnology] 9: 378-381; Eaton et al., 1986, Biochemistry [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.

[0038] Heterogeneous: For host cells, the term "heterogeneous" means that the polypeptide or nucleic acid is not naturally present in the host cell. For polypeptides or nucleic acids, the term "heterogeneous" means that the control sequence (e.g., promoter) of the polypeptide or nucleic acid is not naturally associated with that polypeptide or nucleic acid; that is, the control sequence comes from a gene other than the gene encoding the mature polypeptide.

[0039] Host strain or host cell: A "host strain" or "host cell" is an organism in which an expression vector, bacteriophage, virus, or other DNA construct (including a polynucleotide encoding a target polypeptide, such as an amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a target polypeptide and / or fermenting sugars. The term "host cell" includes protoplasts produced by cells.

[0040] Introduction: In the context of inserting a nucleic acid sequence into a cell, the term “introduction” means “transfection,” “conversion,” or “transduction,” as is known in the art.

[0041] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other specific material or component that is separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.) found in nature and naturally associated with it. Isolated polypeptides include, but are not limited to, culture media containing secreted polypeptides.

[0042] Mature polypeptide: The term “mature polypeptide” or “mature protein” refers to a polypeptide in its mature form after processing at the N-terminus (e.g., removal of the signal peptide and / or propeptide).

[0043] Signal peptide: A signal peptide is an amino acid sequence attached to the N-terminal portion of a protein that promotes its secretion outside the cell. The mature form of extracellular proteins lacks the signal peptide, which is cleaved during the secretion process.

[0044] Natural: The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0045] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0046] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a way that does not originally exist in nature to contain a segment of nucleic acid or is synthesized and contains one or more control sequences that are operatively linked to the nucleic acid sequence.

[0047] Operationally linked: The term "operationally linked" means that specified components are in a relationship that allows them to function in the intended manner (including, but not limited to, juxtaposition). For example, a regulatory sequence is operationally linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

[0048] Purified: The term "purified" means nucleic acids or peptides that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation, where purified peptides or nucleic acids can form discrete bands). Purified nucleic acids or peptides 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%, about 99.8%, or more pure (e.g., molar weight percentage). In a relevant sense, a composition is enriched with the molecule when the concentration of the molecule increases significantly after the application of purification or enrichment techniques. The term “enrichment” refers to the presence of compounds, peptides, cells, nucleic acids, amino acids, or other specified materials or components in a composition at a relative or absolute concentration higher than that of the starting composition.

[0049] In one respect, as used herein, the term "purified" means that the polypeptide or cell is substantially free of components (especially insoluble components) from the producing organism. In another respect, the term "purified" means that the polypeptide is substantially free of insoluble components (especially insoluble components) from the natural organism from which the polypeptide was obtained. In one respect, the polypeptide is separated from some soluble components of the organism from which the polypeptide was recovered and the culture medium. The polypeptide can be purified (i.e., separated) by one or more of the following unit operation methods: filtration, precipitation, or chromatography.

[0050] Accordingly, peptides can be purified so that only small amounts of other proteins, particularly other peptides, are present. As used herein, the term "purified" can refer to the removal of other components, particularly other proteins and most particularly other enzymes, present in the cells from which the peptide originates. A peptide can be "substantially pure," meaning it is free from other components from the organism that produces the peptide (e.g., the host organism used to recombinantly produce the peptide). In one aspect, the peptide is at least 40% pure by weight of the total peptide material present in the formulation. In another aspect, the peptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total peptide material present in the formulation. As used herein, "substantially pure peptide" can mean a peptide formulation containing, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of other peptide material naturally or recombinantly associated with the peptide.

[0051] Therefore, it is preferred that the substantially pure polypeptide, based on the weight of the total polypeptide material present in the formulation, is at least 92% pure, 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 polypeptides of the present invention are preferably in a substantially pure form (i.e., the formulation is substantially free of other polypeptide materials naturally or recombinantly associated with the polypeptides of the present invention). For example, this can be achieved by preparing the polypeptides using well-known recombinant methods or classical purification methods.

[0052] Recombination: The term "recombination," used in its conventional sense, refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence group different from that found in nature. The term recombination refers to cells, nucleic acids, polypeptides, or vectors that have been modified from their natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) forms, or express natural genes at different levels or under different conditions compared to those found in nature. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0053] Recovery: The term "recovery" refers to the removal of peptides from at least one fermentation broth component selected from a list of cells, nucleic acids, or other specified materials, for example, by methods such as: harvesting peptides by peptide crystallization, by filtration (e.g., deep filtration (using filter aids or packed filter media, cloth filtration in a box filter, rotary drum filtration, drum filtration, rotary vacuum drum filter, candle filter, horizontal leaf filter, or the like, using sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, modular filtration, candle filtration, microfiltration, crossflow, dynamic crossflow, or ultrafiltration in dead-end operation)), or by centrifugation (using a sedimentation centrifuge, disc stack centrifuge, hyrdo cyclone, or the like), or by precipitation of peptides and using relevant solid-liquid separation methods to harvest peptides from broth media by particle size fractionation. Recovery encompasses the separation and / or purification of peptides.

[0054] Sequence identity: The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter “sequence identity”. For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of “longest identity”. This algorithm is implemented in the Needle procedure of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the non-simplified option must be specified on the command line. The output for the "longest identity" flag set by Niedel is calculated as follows: (identical residues × 100) / (alignment length - total number of vacancies in the alignment).

[0055] Variant: The term "variant" refers to a polypeptide with 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 1-5 amino acids (e.g., 1-3 amino acids, especially 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0056] Wild-type: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is natural or naturally occurring. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences).

[0057] Milk: The term "milk" (also known as "dairy milk") generally refers to milk from cows; however, it can also refer to milk from goats, sheep, buffalo, or camels. Milk is typically obtained in three forms, distinguished by their fat content. Whole milk has an average of 3.8% milk fat, with a minimum of 3.2%. Low-fat milk (also known as lite milk or reduced-fat milk) typically 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. Skim milk (also known as skimmed milk, nonfat milk, or fat-free milk) typically has 0.5% or less milk fat. In some embodiments, skim milk has 0.1% or less milk fat. In some embodiments, because skim milk has extremely low fat content, it may contain additional milk solids (such as lactose and / or protein) to optimize taste and texture.

[0058] Barista milk: The term "barista milk" refers to milk that may contain additional ingredients to create a better foam (with high cohesiveness for consistent pouring and latte art). Compared to standard whole milk, barista milk may contain more fat and / or more protein.

[0059] Dairy composition: The term “dairy composition” refers to a composition derived from milk, including cream, milk, butter, concentrated milk, evaporated milk, condensed milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or combinations thereof.

[0060] Dairy desserts: The term “dairy desserts” refers to ice cream, frozen custard, frozen yogurt, gelato, and frozen milk.

[0061] Aeration: The term "aeration" refers to the introduction of air into a material, such as a dairy composition. In this invention, aeration is typically achieved by whipping or beating the dairy composition, sometimes with the aid of steam. The product of aeration is a lighter, fluffier dairy composition. An aerated dairy composition may be referred to as foam. Examples of aerated dairy products include ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped cream, and whipped cream spreads.

[0062] Milk foam: The term "milk foam" (also known as "frothed milk" or "foam") refers to milk or whipped cream in which air is introduced, typically accompanied by heating. Foam can be produced by steam (such as by using the steam wand of an espresso machine). It can also be produced by stirring, shaking, or whipping. An electric milk frother can also be used to produce milk foam. Milk foam can be further characterized by "microfoam." Microfoam has tiny, uniform bubbles, can be glossy, and is slightly thickened. The viscosity of microfoam is similar to wet paint. Microfoam is preferred for latte art because it provides clarity and stability to the pattern and / or etching.

[0063] A key quality of milk foam is its "cohesiveness." Foam cohesiveness is determined by the interaction at the foam-liquid interface, where the foam adheres to the surface of the liquid milk. When pouring frothed milk, a lack of foam cohesiveness or poor foam cohesiveness causes the foam to lag behind the liquid milk, resulting in foam clumps remaining in the frother and then falling off. Such foam clumps are considered undesirable when preparing beverages containing frothed milk and can hinder, ruin, or destroy latte art. Foam with excellent cohesiveness pours smoothly from the frother, allowing the foam and liquid to pour out simultaneously or almost simultaneously, with little or no residual foam at the end of the pour.

[0064] Latte art: The term "latte art" (also known as coffee latte art, barista latte art, or coffee design) refers to a specific pattern or design intentionally created on top of milk or whipped cream foam (typically on top of the milk foam in hot beverages such as lattes, cappuccinos, or hot chocolate). Latte art can also be created for other hot liquids containing milk or whipped cream (such as cream-based soups like pumpkin soup or tomato soup). Latte art is created by pouring microfoam into, for example, a cup of espresso in a specific manner to create a pattern or design. Common latte art includes tulips, leaves, and heart shapes. Latte art can also use engraving, where an image is drawn on top of the milk foam of a hot beverage using a thin stick (such as a toothpick). With engraving, latte art can be any drawn image, including faces and animals. Templates can be used to assist with engraving. Consistently creating latte art is particularly difficult, partly due to the conditions required for frothing the milk. The variability of milk makes it difficult to predict whether the resulting milk foam will have the high density of microfoam and high cohesion required to create latte art. Variant naming conventions: For the purposes of this invention, the polypeptide disclosed in SEQ ID NO: 5 is used to determine the corresponding amino acid position in another propeptide and / or deamidase. The amino acid sequence of the other propeptide and / or deamidase is compared with the polypeptide disclosed in SEQ ID NO: 5, and based on this comparison, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) is used. J. Mol. Biol [Journal of Molecular Biology] 48: 443-453) determines the amino acid position numbers corresponding to any amino acid residues in the polypeptide disclosed in SEQ ID NO: 5 using an algorithm such as the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ). Trends Genet. The procedure implemented in Niedel's program (preferably version 5.0.0 or later) is described in [Genetic Trends] 16: 276-277. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0065] In describing variants, the following nomenclature is provided for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0066] replace For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg + Ser411Phe" or "G205R + S411F" represent the substitution of glycine (G) at position 205 and serine (S) at position 411 with arginine I and phenylalanine (F), respectively.

[0067] Missing For amino acid deletions, use the following nomenclature: original amino acid, position, Accordingly, the deletion of glycine at position 195 will be represented as "Gly195". "or "G195 Multiple missing items are separated by a plus sign ("+"), for example, "Gly195". + Ser411 "or "G195 + S411 ".

[0068] insert. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, inserting lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, inserting lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0069] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be: Multiple changes Variants containing multiple changes are separated by a plus sign ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" means that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0070] Different changes Where different changes can be introduced at a single position, the different changes are separated by commas. For example, "Arg170Tyr,Glu" or "R170Y,E" represents the substitution of arginine at position 170 with either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala + Arg170Gly,Ala" or "Y167G,A + R170G,A" represents the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

[0071] In the context of this invention, the term "variant" means a polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids (e.g., 1-5 amino acids) adjacent to and immediately following an amino acid occupying a position.

[0072] Amino acid alterations can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function (such as polyhistidine fragments, antigenic epitopes, or binding domains).

[0073] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not typically alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common 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.

[0074] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, these amino acid alterations can affect the peptide's thermal stability, change its substrate specificity, and alter its optimal pH.

[0075] 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 at each residue in the molecule, and the endopeptidase activity of the resulting mutant molecule is tested to identify amino acid residues essential for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acid at the putative contact site. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[0076] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, one or more amino acid substitutions, deletions, and / or insertions can be made and tested, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7:127).

[0077] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide. Detailed Implementation

[0078] This invention relates to an improved aerated dairy composition, wherein a protein deamidase and optionally a second polypeptide derived from a propeptide in the form of a protein deamidase are added to the dairy composition prior to aeration. The invention also includes a method for producing the improved aerated dairy product. In some embodiments, the resulting aerated dairy product has an improved foam density, comprising smaller, more uniform bubbles. This improvement in foam density produces an aerated dairy product with a lighter, fluffier, and / or smoother texture and improved foam stability compared to a dairy composition not treated with a protein deamidase prior to aeration. In some embodiments, the aerated dairy product is foamed milk, wherein the foam has better stability, a smoother texture, and / or higher quality microfoam compared to foam produced from milk not treated with a protein deamidase. This higher quality foam improves the creation of latte art in the final product containing foamed milk, such as latte art on the surface of hot beverages or soups.

[0079] Deamidase In this invention, "protein deamidase," "deamidase," "mature protein deamidase," or "mature deamidase" refers to an enzyme that acts directly on the amide group of the side chain of the amino acid constituting a protein to cause deamidation and release ammonia without cleaving the peptide bonds of the protein and / or cross-linking the protein. Specific examples of protein deamidases include protein glutaminase (EC 3.5.1.44), which acts directly on the amide group of the side chain of glutamine residues contained in a protein, thereby releasing ammonia and thus converting the glutamine residues to glutamate residues. Another example of a protein deamidase is protein asparaginase, which acts directly on the amide group of the side chain of asparagine residues contained in a protein, thereby releasing ammonia and thus converting the asparagine residues to aspartic acid residues. In this invention, either protein glutaminase or protein asparaginase, or a combination of both, can be used as the protein deamidase. In some embodiments, the protein deamidase used in this invention is protein glutaminase.

[0080] The protein deamidases used in the methods of this invention can be obtained from microorganisms of any genus. For the purposes of this invention, the term "obtained from," as used herein in conjunction with a given source, should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0081] Protein deamidases can be obtained from *Cryptotympany* spp. ( Chryseobacterium genus), Flavobacterium ( Flavobacterium genus), Stenotrophomonas ( Empedobacter genus), Sphingosine Bacillus ( Sphingobacterium genus), Chlorella ( Aureobacterium genus) or genus of mushrooms ( Myroides (genus). Protein deamidases can be obtained from the culture medium of the above-mentioned microorganisms and are used in this invention.

[0082] In some embodiments, the protein deamidase may be derived from the genus *Chlorella*, such as *Chlorella viviparum*, *Chlorella brevis*, *Chlorella cunea*, *Chlorella vulgaris*, or *Chlorella pyrenoidosa*. In some embodiments, the deamidase in the method of the present invention is derived from or obtained from *Chlorella viviparum* (formerly known as *Chlorella* species 62563).

[0083] EP 1839491 discloses the presence of Corynebacterium glutamicum ( Corynebacterium glutamicum This is a clone of a protein glutaminase from *Chlorella vulgaris* expressed in [the sample / sample]. Protein glutaminases derived from *Chlorella vulgaris* can be commercially available, for example, as Amano PG500 (manufactured by Amano Enzyme Inc.).

[0084] The protein deamidase in the method of this invention acts on milk proteins, such as casein and whey protein. Casein includes α-s1-casein, α-s2-casein, β-casein, and κ-casein. Whey protein includes α-lactalbumin and β-lactoglobulin. In some embodiments, the deamidase primarily acts on casein.

[0085] Protein deamidases (such as those derived from *Chlorella* species) are typically produced in an inactive proto-form (also referred to herein as the "proto-form" or "proto-form protein deamidase"), expressed as a fusion protein containing an N-terminal propeptide domain (also referred to herein as the "propeptide" or "propeptide derived from the proto-form protein deamidase"), which is upstream of and tightly bound to the C-terminal deamidase domain. Due to the binding of the propeptide to the deamidase domain, the proto-form exhibits reduced deamidase activity, thus protecting cell viability. Essentially, the fusion protein is post-processed to remove the propeptide and release the active, mature deamidase outside the microbial cell. In recombinant expression systems, the fusion protein is secreted outside the host cell as an inactive proto-form containing the propeptide, which can then be cleaved to separate it from the mature deamidase. It can be cleaved by site-specific endopeptidases (such as gamma-glutamyl endopeptidase, trypsin or trypsin-like endopeptidase, or chymotrypsin or chymotrypsin-like endopeptidase).

[0086] For many proto-deamidases, the propeptide is difficult to separate from the mature deamidase even after cleavage, due to its high binding affinity to the deamidase domain. Mature protein deamidases still bound to their propeptides exhibit reduced activity due to their inhibitory effect. Therefore, there is a strong desire in the art to remove the cleaved propeptide from mature deamidases, and considerable resources have been devoted to purifying the propeptide from mature deamidases. However, novel proto-deamidases have been discovered in which the binding affinity of the propeptide to the mature deamidase can be reduced (WO 2023 / 170177, which is incorporated herein by reference). Unexpectedly, it has been found in this invention that the presence of these modified propeptides can provide beneficial effects on the activity of the deamidase.

[0087] The protein deamidase in the methods and compositions of the present invention is a mature deamidase, wherein the propeptide has been cleaved from the proto-protein deamidase. In some embodiments, the propeptide derived from the same proto-protein deamidase as the mature deamidase may still be present in the composition containing the mature deamidase. In some embodiments, the purified propeptide is added separately to the composition containing the mature protein deamidase.

[0088] The propeptide of this invention comprises a deamidase inhibitory domain that interacts with the original deamidase domain. The deamidase inhibitory domain of the propeptide comprises the amino acid sequence motif [I / M][L / I / V][S / T]AQ, corresponding to amino acids 39-43 of SEQ ID NO: 3, 6, and 9, amino acids 41-45 of SEQ ID NO: 12, and amino acids 40-44 of SEQ ID NO: 15. The propeptide also comprises the amino acid sequence motif [K / R][V / I / L][S / A / N]X[I / M][L / I / V][S / T]AQ, corresponding to amino acids 35-43 of SEQ ID NO: 3, 6, and 9, amino acids 37-45 of SEQ ID NO: 12, and amino acids 36-44 of SEQ ID NO: 15. After expression of the original polypeptide in a recombinant expression system, the propeptide is cleaved using a site-specific endopeptidase, leaving an active, mature deamidase. In some embodiments, the cleaved propeptide is not purified from the mature deamidase. Therefore, the propeptide is present in the composition together with the mature deamidase.

[0089] In some embodiments, the propeptide is a mutant variant that has a lower binding affinity to the deamidase domain of its corresponding deamidase, making it more readily cleaved enzymatically after recombinant expression and secretion from the host cell. Propeptide mutations affecting the binding affinity of the propeptide to the mature deamidase are described in WO 2023 / 170177 (which is incorporated herein by reference in its entirety).

[0090] In some embodiments, the propeptide comprises amino acid variations at the position corresponding to 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 other embodiments, the propeptide comprises amino acid variations at the position corresponding to 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 other embodiments, the propeptide comprises amino acid variations at the position corresponding to SEQ ID NO: 3 selected from the group consisting of: The position 3 contains amino acid changes: 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; D9 1F,G,H,K,L,M,N,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. It should be understood that the propeptides of the present invention may comprise the amino acid sequence of SEQ ID NO: 3, 6, 9, 12, or 15-20, and further include any mutations described herein at positions corresponding to those in SEQ ID NO: 3.

[0091] In some embodiments, the propeptide comprises amino acid variations at positions corresponding to those selected from SEQ ID NO: 3: 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;D91G,H,K,N,P,Q,R,S;I92G,P,S;Y94A,E,I,P,Q,R,T;F95A,D,E, G,H,K,N,R,S,T,V; K96P; F98D,E,G,N,P,Q,S; F99A,C,D,G,H,K,P,Q,R; and / or T100P,W. In another embodiment, the propeptide comprises an amino acid variation at the position corresponding to 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, F99G, and / or F99K.

[0092] 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 NO: 16-20. In other embodiments, the propeptide comprises the amino acid sequence of SEQ ID NO: 16-20.

[0093] In some embodiments, the original form may contain the amino acid sequence of SEQ ID NO: 1, 21, 22, 23 or 24, which may be cleaved to produce a propeptide containing the amino acid sequences of SEQ ID NO: 16-20 and a mature protein deamidase containing the amino acid sequences of SEQ ID NO: 2, 5, 8, 11 and 14.

[0094] In some embodiments, the methods and compositions of the present invention comprise: a) A proto-protein deamidase comprising 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: 1, wherein the proto-protein deamidase can be cleaved to release a mature protein deamidase comprising 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 a propeptide comprising 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) A proto-protein deamidase comprising 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: 21, wherein the proto-protein deamidase can be cleaved to release a mature protein deamidase comprising 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 a propeptide comprising 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) A proto-protein deamidase comprising 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: 22, wherein the proto-protein deamidase can be cleaved to release a mature protein deamidase comprising 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 a propeptide comprising 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 proto-protein deamidase comprising 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: 23, wherein the proto-protein deamidase can be cleaved to release a mature protein deamidase comprising 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 a propeptide comprising 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: 19; or e) A proto-protein deamidase comprising 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: 24, wherein the proto-protein deamidase can be cleaved to release a mature protein deamidase comprising 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 a propeptide comprising 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: 20.

[0095] In the method of this invention, a protein deamidase is added to whole milk, low-fat milk, or skim milk to improve the foaming properties of the milk. Compared to foam produced from dairy compositions not treated with deamidase, the resulting foamed milk exhibits better stability, higher microfoam density, higher volume, higher cohesion, and / or a smoother texture. The ability of milk to foam and the quality of this foam depend on the protein and fat present in the milk. Higher quality foam is typically produced from whole milk, while foam produced from low-fat or skim milk is less desirable. Surprisingly, the method and compositions of this invention are capable of producing high-quality foam using low-fat milk and even skim milk (with little or no milk fat). The high quality of the foam and the resulting beverage are similar to or superior to beverages prepared with whole milk without added protein deamidase. Furthermore, in the method of this invention, a protein deamidase and its precursor peptide are added to skim milk (also known as "skimmed milk") to improve the foaming properties of the milk. Surprisingly, compared to methods that only add protein deamidase, the methods and compositions of the present invention (in which both protein deamidase and the protein deamidase precursor are added) are able to produce higher quality foam with skim milk.

[0096] It is well known in the art that latte art can be difficult to produce and relies on extremely high-quality milk foam with high microfoam density and cohesion. It is generally accepted that milk foam produced from low-fat or skim milk does not possess the stability, volume, number of microfoams, and / or sufficiently high cohesion required for latte art production. Furthermore, even whole milk can produce inconsistent quality milk foam. Therefore, a dairy composition that is highly reliable for latte art production is highly desirable.

[0097] The compositions of the present invention, comprising milk and protein deamidase and optionally a propeptide of the protein deamidase, meet this requirement. The methods and compositions of the present invention produce milk foam with high microfoam density and high cohesion suitable for making latte art. Additionally, the methods and compositions of the present invention, comprising low-fat or skim milk, also produce milk foam with high microfoam density and high cohesion suitable for making latte art.

[0098] In some embodiments, the texture of the beverage itself is also improved, making it richer and smoother compared to similar beverages prepared with low-fat or skim milk without added protein deamidase.

[0099] As stated above, in some embodiments, a first polypeptide (i.e., a mature protein deamidase) and a second polypeptide are added to milk, wherein the second polypeptide is a propeptide of the deamidase. In some embodiments, the second polypeptide is a propeptide obtained by cleaving the original form to produce the first polypeptide. In some embodiments, the second polypeptide is a mutated propeptide variant. In other embodiments, the second polypeptide is a mutated propeptide variant that binds less tightly to the mature deamidase compared to the natural propeptide. In some embodiments, the second polypeptide is a mutated variant of a propeptide derived from the same species as the deamidase added to the dairy composition. In other embodiments, the second polypeptide is a mutated variant of a propeptide derived from a different species than the deamidase added to the dairy composition, wherein the dairy composition may contain milk. In some embodiments, the second polypeptide is synthetic and is independent of 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 SEQ ID NO: 3, 6, 9, 12, or 15.

[0100] 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 SEQ ID NO: 3. In another 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 SEQ ID NO: 3, and contains glycine at position 99 corresponding to SEQ ID NO: 3.

[0101] 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 SEQ ID NO: 3, and having glycine at position 99 corresponding to SEQ ID NO: 3. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0102] 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 SEQ ID NO: 6, and having an alanine residue at position 99 corresponding to SEQ ID NO: 6. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 17.

[0103] 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 SEQ ID NO: 9, and having glycine at position 99 corresponding to SEQ ID NO: 9. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

[0104] 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 SEQ ID NO: 12, and having glycine at position 101 corresponding to SEQ ID NO: 12. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 19.

[0105] 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 SEQ ID NO: 15, and having glycine at position 100 corresponding to SEQ ID NO: 15. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

[0106] Those skilled in the art will recognize that the amount of protein deamidase provided to the dairy composition will be sufficient to obtain the desired improved aerated dairy product, and that sufficient amount depends in part on the temperature and duration of incubation with the enzyme. In some embodiments, the protein deamidase is provided to the dairy composition of the present invention in an amount between 0.01 and 20 IPA (U) / g protein substrate or per gram of milk protein (also referred to as per gram of protein). In other embodiments, the protein deamidase is provided in amounts between the following ranges: 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, in the method of the present invention, 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 some embodiments, protein deamidase is provided to the dairy composition of the present invention at 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.

[0107] The amount of protein deamidases (as protopeptides, propeptides, and / or mature protein deamidases) used in the methods of this invention does not significantly contribute to the total amount of soluble protein in the dairy compositions of this invention, and is unlikely to be the cause of the observed increase in foaming properties of the dairy compositions. The weight of milk protein in each reaction is at least 1000 times the combined weight of the added enzyme protein and propeptide.

[0108] Those skilled in the art will recognize that the improved foaming techniques shown herein are similarly applicable to aeration methods of dairy compositions for the production of other aerated or whipped dairy products. Similar to foamed milk, the presence of fat in the dairy composition has a positive impact on the aeration of the dairy composition and the quality of the aerated dairy product. Introducing air throughout the dairy product produces a smooth, melt-in-your-mouth texture. As described above regarding milk foam, fat is a key component for successfully introducing air into the dairy composition. Therefore, it is difficult to produce the desired smooth texture in low-fat or non-fat aerated dairy products such as dairy desserts, whipped cream cheese, whipped yogurt, whipped cream, or whipped cream spreads (where the spread contains both cream and another vegetable oil, such as canola oil, olive oil, or other vegetable oils).

[0109] The method of the present invention includes a method for producing an improved aerated dairy product, the method comprising adding a 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 the protein deamidase is not added to the dairy product composition. The method of the present invention also includes a method for producing an improved aerated dairy product, the method comprising adding a protein deamidase and a deamidase precursor 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 a protein deamidase is added but the deamidase precursor is not present in the dairy product composition.

[0110] 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 made from low-fat milk or skim milk.

[0111] In some embodiments, the methods and compositions of the present invention comprise dairy compositions having a low amount of milk fat, such as dairy compositions derived from low-fat or skim milk. In some embodiments, milk, cream, 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 fat.

[0112] In some embodiments, a protein deamidase and optionally a propeptide of the deamidase are added to the dairy composition prior to aeration. In some embodiments, the dairy composition may be further processed prior to aeration. In some embodiments, additional ingredients are added to the dairy composition before or after the addition of the protein deamidase and optionally a propeptide of the deamidase.

[0113] The dairy product composition may be heat-treated. The heat treatment may be Low Temperature Long Time (LTLT), High Temperature Short Time (HTST), Higher Heat Shorter Time (HHST), Ultra-High Temperature (UHT), Ultra Pasteurized (UP), or any other form of heat treatment known in the art. In some embodiments, a protein deamidase is added to the dairy product composition prior to heat treatment. In other embodiments, the protein deamidase is added to the dairy product composition after heat treatment. In some embodiments, the dairy product composition may be heat-treated both before and after the addition of the deamidase (as part of the process of producing the resulting dairy product).

[0114] In some embodiments, the dairy composition comprises cream and optionally vegetable oil, and the aerated dairy product is whipped cream or whipped cream spread. In some embodiments, the dairy composition comprises fermented milk, and the aerated dairy product is whipped yogurt or frozen yogurt, a dairy dessert. In some embodiments, the aerated dairy product is a dairy dessert, such as ice cream, frozen custard, frozen yogurt, gelato, or frozen 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 fat.

[0115] Similar to the methods described above for foaming milk, in some embodiments, a deamidase and a second polypeptide are added to the dairy product composition. This second polypeptide may be a propeptide of the deamidase. In some embodiments, the second polypeptide is a mutated propeptide variant. In other embodiments, the second polypeptide is a mutated propeptide variant that binds less tightly to the mature deamidase compared to the natural propeptide. In some embodiments, the second polypeptide is a mutated variant of a propeptide derived from the same species as the deamidase added to the dairy product composition. In other embodiments, the second polypeptide is a mutated variant of a propeptide derived from a different species than the deamidase added to the dairy product composition. In some embodiments, the second polypeptide is synthetic and independent of the deamidase added to the dairy product 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 SEQ ID NO: 3, 6, 9, 12, or 15.

[0116] 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 SEQ ID NO: 3, and having glycine at position 99 corresponding to SEQ ID NO: 3. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0117] 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 SEQ ID NO: 6, and having an alanine residue at position 99 corresponding to SEQ ID NO: 6. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 17.

[0118] 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 SEQ ID NO: 9, and having glycine at position 99 corresponding to SEQ ID NO: 9. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

[0119] 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 SEQ ID NO: 12, and having glycine at position 101 corresponding to SEQ ID NO: 12. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 19.

[0120] 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 SEQ ID NO: 15, and having glycine at position 99 corresponding to SEQ ID NO: 15. In another embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

[0121] The aeration level 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 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. Additionally, the aeration or foaming properties of the dairy composition can be adjusted by the speed, time, and / or intensity of mechanical aeration or foaming. Preferred embodiments 1. A method for producing improved aerated dairy products, the method comprising: a) Obtain a dairy composition; b) Add a first polypeptide to the dairy composition, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase. c) Optionally, a second polypeptide is added to the dairy composition, wherein the second polypeptide is a propeptide derived from the original form of a protein deamidase; and d) Aerating the dairy composition to produce aerated dairy products; The aerated dairy product is an improvement over similar methods that do not include the first polypeptide and optionally the second polypeptide in the dairy product composition.

[0122] 2. The method as described in Example 1, 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.

[0123] 3. The method as described in any of the foregoing embodiments, wherein the dairy composition comprises 3.8% or less, 3.25% or less, 3% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.25% or less, or 0.15% or less fat.

[0124] 4. The method as described in any of the foregoing embodiments, wherein the aerated dairy product has improved foam density, a lighter, fluffier and / or smoother texture, and / or improved foam stability compared to aerated dairy products produced without the addition of protein deamidase.

[0125] 5. The method as described in any of the foregoing embodiments, wherein the aerated dairy product is ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped cream, or whipped cream spread.

[0126] 6. The method as described in any of the foregoing embodiments, wherein 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 fat.

[0127] 7. A method for generating improved foam in a dairy composition, the method comprising: a) Obtain a dairy composition; b) Add a first polypeptide to the dairy composition, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase. c) Optionally, a second polypeptide is added to the dairy composition, wherein the second polypeptide is a propeptide derived from the original form of a protein deamidase; and d) Aerate the dairy composition to produce foam; The foaming of the dairy composition is improved compared to similar methods that do not include the first polypeptide and optionally the second polypeptide in the dairy composition.

[0128] 8. A method for improving latte art made from a dairy product composition, the method comprising: a) Obtain a dairy composition; b) Add a first polypeptide to the dairy composition, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase. c) Optionally, a second polypeptide is added to the dairy composition, wherein the second polypeptide is a propeptide derived from the original form of a protein deamidase; and d) Aerating the dairy composition to produce foam; and e) Using the foamed dairy composition to create latte art; The latte art is improved compared to similar methods that do not include the first polypeptide and optionally the second polypeptide in the dairy composition.

[0129] 9. The method as described in Example 7 or 8, wherein the foam has better stability, higher microfoam density, higher volume, higher cohesion, and / or smoother texture compared to foam produced from a dairy composition not treated with deamidase.

[0130] 10. The method of any one of Examples 7 to 9, wherein the dairy composition is cream, milk, concentrated milk, condensed milk, reconstituted milk, milk protein concentrate, whey protein concentrate, or a combination thereof.

[0131] 11. The method of any one of Examples 7 to 9, wherein the dairy composition is whole milk, low-fat milk or skim milk.

[0132] 12. The method of Example 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 milk fat.

[0133] 13. The method as described in any of the foregoing embodiments, wherein the first polypeptide is a mature protein deamidase comprising 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, 5, 8, 11, or 14.

[0134] 14. The method as described in any of the preceding embodiments, wherein the first polypeptide is a mature protein deamidase comprising the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 or 14.

[0135] 15. The method as described in any of the foregoing embodiments, wherein the deamidase is added in amounts ranging from: 0.01-15.0 IPA (U) / g protein; 0.1-11.0 IPA (U) / g protein; 0.5-7.0 IPA (U) / g protein; or 2.5-7.0 IPA (U) / g protein.

[0136] 16. The method as described in any of the foregoing embodiments, wherein the dairy composition is heat-treated prior to treatment with the deamidase.

[0137] 17. The method as described in Example 16, wherein the heat treatment is performed at 60°C-140°C.

[0138] 18. The method as described in Example 16 or 17, wherein the heat treatment is a low-temperature long-time sterilization (LTLT), high-temperature short-time sterilization (HTST), high-temperature instantaneous sterilization (HHST), ultra-high-temperature instantaneous sterilization (UHT), or ultra-pasteurization (UP).

[0139] 19. The method as described in any one of Examples 1 to 15, wherein the dairy composition is not heat-treated prior to treatment with the deamidase.

[0140] 20. The method of any of the foregoing embodiments, wherein the second polypeptide is a propeptide derived from the original form of a 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.

[0141] 21. The method of any of the preceding claims, wherein the second polypeptide is a propeptide, the propeptide comprising: 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 SEQ ID NO: 3, and having glycine at position 99 corresponding to 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 SEQ ID NO: 6, and having alanine at position 99 corresponding to 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 SEQ ID NO: 9, and having glycine at position 99 corresponding to 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 SEQ ID NO: 12, and containing glycine at position 101 corresponding to SEQ ID NO: 12; or e) 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 having glycine at position 100 corresponding to SEQ ID NO: 15.

[0142] 22. The method as described in any of the preceding embodiments, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 17, 18, 19, 20.

[0143] 23. The method as described in any of the foregoing embodiments, wherein the first polypeptide and the second polypeptide are derived from the same proto-protein deamidase.

[0144] 24. The method as described in any of the foregoing embodiments, wherein the original form 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 NO: 1, 21, 22, 23, or 24.

[0145] 25. The method as described in any of the foregoing embodiments, wherein the first polypeptide and the second polypeptide are derived from the original form comprising 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 NO: 1, 21, 22, 23, or 24, such that the first polypeptide is a mature protein deamidase comprising 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 NO: 2, 5, 8, 11, or 14, respectively, and the second polypeptide comprises amino acids having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 2, 5, 8, 11, or 14, respectively, and the second polypeptide is a mature protein deamidase comprising amino acid sequences ... The 16th, 17th, 18th, 19th, or 20th amino acids have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the propeptide.

[0146] 26. The method as described in one of the foregoing embodiments, wherein: 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 having glycine at position 99 corresponding to 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 having an alanine at position 99 corresponding to 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 having glycine at position 99 corresponding to 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 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: 12 and having glycine at position 101 corresponding to SEQ ID NO: 12; or e) 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 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: 15 and having glycine at position 100 corresponding to SEQ ID NO: 15.

[0147] 27. The method of any one of Examples 1-22 and 26, wherein the first polypeptide and the second polypeptide are not derived from the same proto-protein deamidase.

[0148] 28. The method as described in Example 27, wherein the second polypeptide is a propeptide expressed and purified without using the original form intermediate.

[0149] 29. The method as described in any of the foregoing embodiments, wherein the addition of the second polypeptide improves the performance of the deamidase.

[0150] 30. A dairy composition comprising a first polypeptide and optionally a second polypeptide, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase, and wherein the second polypeptide is a propeptide derived from the original form of a protein deamidase.

[0151] 31. The dairy composition as described in Example 30, wherein: 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 having glycine at position 99 corresponding to 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 having an alanine at position 99 corresponding to 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 having glycine at position 99 corresponding to 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 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: 12 and having glycine at position 101 corresponding to SEQ ID NO: 12; or e) 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 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: 15 and having glycine at position 100 corresponding to SEQ ID NO: 15.

[0152] 32. The dairy composition as described in Example 30 or 31, wherein the dairy composition has improved aeration or foaming properties compared to a dairy composition not containing the first polypeptide and optionally the second polypeptide, wherein the first polypeptide is a mature protein deamidase derived from the original form of protein deamidase, and wherein the second polypeptide is a propeptide derived from the original form of protein deamidase.

[0153] 33. The dairy composition of any one of Examples 30 to 32, wherein the dairy 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.

[0154] 34. The dairy composition as described in any one of Examples 30 to 33, wherein the dairy composition is processed to produce aerated dairy products, such as ice cream, whipped cream, whipped yogurt, whipped cream cheese, frothy milk, or whipped cream.

[0155] 35. The dairy composition as described in any one of Examples 30 to 33, wherein the dairy composition is whole milk, low-fat milk or skim milk.

[0156] 36. The dairy composition as described in Example 35, wherein the dairy composition is processed to produce barista milk.

[0157] 37. The dairy composition of any one of Examples 30-36, wherein the first polypeptide and the second polypeptide are not derived from the same proto-protein deamidase.

[0158] 38. The dairy composition as described in Example 37, wherein the second polypeptide is a propeptide expressed and purified without using the original form intermediate.

[0159] 39. An aerated dairy product produced by the method described in any one of Examples 1-7 and 13-29.

[0160] 40. The aerated dairy product as described in Example 39, wherein the aerated dairy product is ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped cream, or whipped cream spread.

[0161] 41. An aerated dairy product as described in Example 39 or 40, wherein, compared with an aerated dairy product produced without the addition of a protein deamidase and optionally a second polypeptide, the aerated dairy product has improved foam density, a lighter, fluffier and / or smoother texture, and / or improved foam stability, wherein the second polypeptide is derived from a deamidase precursor peptide.

[0162] 42. A foam produced by any one of Examples 8-29.

[0163] 43. The foam as described in Example 42, wherein the foam has better stability, higher microfoam density and / or smoother texture compared to foam produced from milk not treated with the first polypeptide and optionally the second polypeptide, wherein the first polypeptide is a mature protein deamidase derived from the original form of protein deamidase, and wherein the second polypeptide is a propeptide derived from the original form of protein deamidase.

[0164] 44. Use of a first polypeptide and optionally a second polypeptide for the production of a dairy composition having improved aeration or foaming properties, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase, and wherein the second polypeptide is a propeptide derived from the original form of a protein deamidase.

[0165] 45. The use as described in Example 44, wherein: 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 having glycine at position 99 corresponding to 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 having an alanine at position 99 corresponding to 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 having glycine at position 99 corresponding to 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 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: 12 and having glycine at position 101 corresponding to SEQ ID NO: 12; or e) 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 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: 15 and having glycine at position 100 corresponding to SEQ ID NO: 15.

[0166] 46. ​​Use as described in Examples 44 or 45, wherein the protein deamidase comprises the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 or 14.

[0167] 47. Use as described in any one of Examples 44-46, wherein the dairy composition comprises cream, milk, butter, concentrated milk, condensed milk, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof.

[0168] 48. A method for producing improved aerated dairy products, the method comprising: a) Obtain a dairy composition; b) Add a first polypeptide to the dairy composition, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase. c) Optionally, a second polypeptide is added to the dairy composition, wherein the second polypeptide is a propeptide derived from the same protoform protein deamidase; and d) Aerating the dairy composition to produce aerated dairy products; The aerated dairy product is an improvement over similar methods that do not include the first polypeptide and optionally the second polypeptide in the dairy product composition.

[0169] 49. The method as described in Example 48, wherein the aerated dairy composition produced by adding the first polypeptide and the second polypeptide is improved compared to a similar method of adding the first polypeptide to the dairy composition without adding the second polypeptide.

[0170] 50. The method as described in Examples 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.

[0171] 51. The method of any one of Examples 48-50, 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 NO: 2, 5, 8, or 11.

[0172] 52. The method of any one of Examples 48-51, wherein the second polypeptide is a mutated propeptide that, compared with the parental propeptide, reduces the binding affinity of the propeptide to the deamidase domain.

[0173] 53. The method of any one of Examples 48-52, wherein the second polypeptide is a propeptide, the propeptide comprising: 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 SEQ ID NO: 3, and having glycine at position 99 corresponding to 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 SEQ ID NO: 6, and having alanine at position 99 corresponding to 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 SEQ ID NO: 9, and containing glycine at position 99 corresponding to SEQ ID NO: 9; or 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 SEQ ID NO: 12, and having glycine at position 101 corresponding to SEQ ID NO: 12.

[0174] 54. The method of any one of Examples 48-53, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 17, 18 or 19.

[0175] 55. The method as described in any one of Examples 48-54, wherein: 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 having glycine at position 99 corresponding to 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 having an alanine at position 99 corresponding to 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 contains glycine at position 99 corresponding to SEQ ID NO: 9; or 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 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: 12 and having glycine at position 101 corresponding to SEQ ID NO: 12.

[0176] 56. A dairy composition comprising a first polypeptide and optionally a second polypeptide, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase, and wherein the second polypeptide is a propeptide derived from the same original form of a protein deamidase.

[0177] 57. A dairy composition comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase, and wherein the second polypeptide is a propeptide derived from the same original form of a protein deamidase.

[0178] 58. The dairy composition as described in Examples 56 or 57, wherein: 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 having glycine at position 99 corresponding to 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 having an alanine at position 99 corresponding to 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 contains glycine at position 99 corresponding to SEQ ID NO: 9; or 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 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 position 101 corresponding to SEQ ID NO: 12.

[0179] 59. The dairy composition of any one of Examples 56-58, wherein the dairy 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.

[0180] 60. The dairy composition of any one of Examples 56-59, wherein the dairy composition is processed to produce an aerated dairy product, such as ice cream, whipped cream, whipped yogurt, whipped cream cheese, frothy milk, or whipped cream.

[0181] 61. An aerated dairy product produced by the method described in any one of Examples 48 to 55.

[0182] 62. The aerated dairy product as described in Example 61, wherein the aerated dairy product is ice cream, whipped cream, whipped yogurt, whipped cream cheese, foamed milk, whipped cream, or whipped cream spread.

[0183] 63. Use of a first polypeptide and optionally a second polypeptide for the production of dairy product compositions having improved aeration or foaming properties, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase, and wherein the second polypeptide is a propeptide derived from the same original form of a protein deamidase.

[0184] 64. Use of a first polypeptide and a second polypeptide for the production of dairy product compositions having improved aeration or foaming properties, wherein the first polypeptide is a mature protein deamidase derived from the original form of a protein deamidase, and wherein the second polypeptide is a propeptide derived from the same original form of a protein deamidase.

[0185] 65. The use as described in Example 62 or 63, wherein: 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 having glycine at position 99 corresponding to 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 having an alanine at position 99 corresponding to 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 contains glycine at position 99 corresponding to SEQ ID NO: 9; or 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 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 position 101 corresponding to SEQ ID NO: 12.

[0186] The invention described and claimed herein is not limited to the specific embodiments disclosed herein, as these embodiments are intended to serve as illustrative examples of several aspects of the invention. Any equivalent embodiments, as well as combinations of one or more of these embodiments, are intended to be within the scope of the invention.

[0187] This document cites several references, the disclosures of which are incorporated herein by reference in their entirety. The invention is further described through the following examples, which should not be construed as limiting the scope of the invention. Example Materials and Methods Example 1: Activation and purification of protein deamidases Purification of the original form: The proto-protein deamidase was expressed in a heterologous expression system, and cell clumps were removed by centrifugation followed by microbial filtration. The proto-protein was purified using standard protein purification techniques known to those skilled in the art. The purity of the final samples was examined by SDS-PAGE. These samples were used for activation.

[0188] Activation and purification of mature forms:The specific gamma-glutamyl endopeptidase was added to the purified original form and incubated at room temperature with gentle shaking. The progress of activation (from approximately 30 kDa to approximately 20 kDa) was monitored using standard SDS-PAGE. Once activation was complete, to separate the mature enzyme from the propeptide and inactivate / remove the gamma-glutamyl endopeptidase, the sample was buffer-exchanged using a column packed with Sephadex G-25 resin, following a standard protocol. The eluted fractions were examined by SDS-PAGE, and fractions containing the mature activated molecule were pooled. The resulting sample was loaded onto a cation exchanger (Source15S), calibrated using the same buffer, and eluted using a linear gradient (0–100%, over 20 CV). Peak fractions were analyzed by SDS-PAGE, and the relevant fractions (mature deamidase) were pooled. Concentrations were determined using A280 measurements on a NanoDrop device combined with theoretical extinction coefficients. The sample was frozen at -18°C until further use.

[0189] Purification of propeptide: The propeptide of the protein deamidase was cloned and expressed in a heterologous expression system. At the end of fermentation, cell clumps were removed by centrifugation followed by microbial filtration. The resulting samples were buffer-exchanged using a column packed with Sephadex G-25 resin, following standard protocol. The peak fractions were combined and loaded onto an anion exchange column (Source 15Q) equilibrated with the same buffer. The elution and wash fractions were combined and loaded onto a Source 15Q column. The elution and wash fractions were combined and concentrated using a pressurized UF cell equipped with a 5 MWCO membrane. The concentration of the resulting osmotic residue was determined using measurements on an A280 device on a NanoDrop instrument, combined with the theoretical extinction coefficient. Example 2: Skim milk treated and foamed with deamidase Enzyme treatment: Weigh 4 × 250 g of pasteurized skim milk (Egelykke, Arla Foods, Vibi, Denmark; 3.5 g protein / 100 g; 0.1 g fat / 100 g; 4.7 g carbohydrates / 100 g) into a blue-capped bottle and heat to 55°C in a water bath. Add a mature protein deamidase (derived from *Cryptospira viscera*; 350 IPA(U) / g) containing the amino acid sequence of SEQ ID NO: 2 at a concentration of 0, 1.4, 3.4, or 6.8 IPA(U) / g of milk protein and incubate for 30 min. After enzyme treatment, transfer the bottle to a 75°C water bath and hold at 72°C for 15 seconds. Then cool the bottle on ice and store at 5°C for at least 90 minutes.

[0190] Foaming in a milk frother:Weigh 120 g (+ / - 1 g) of cold (5°C) enzyme-treated milk. Heat 2 × 4 120 g samples, each covering dose responses of 0, 1.4, 3.4, and 6.8 IPA (U) / g milk protein, to 50°C or 60°C. Add the heated samples to a milk frother (Severin SM3587 Spuma 700 plus; Severin Elektrogerate GmbH, Sunden, Germany) and stir for 60 seconds without further heating. Transfer the frothed milk to a 500 ml measuring cup and measure the total volume, foam volume, and liquid volume separately. After 10 minutes, measure the total volume, foam volume, and liquid volume again at room temperature to assess foam stability. The sample treated with 0 IPA (U) / g protein serves as a control.

[0191] Table 1: Foaming results of skim milk treated with protein deamidase of SEQ ID NO: 2 The addition of the protein deamidase of SEQ ID NO: 2 significantly increased foam volume at both 50°C and 60°C.

[0192] In addition to volume, the quality of the foam was also evaluated. The airiness of the foam (ranging from large, clearly defined, individually visible bubbles to uniform microbubbles with a silky appearance) and cohesion were analyzed. Foam cohesion was assessed when transferring the frothed milk from the frother to the measuring cup. The foam cohesion was rated in the quality assessment as follows: None (foam separates from the liquid milk, and there is no airiness in the liquid phase, making it impossible to create latte art); Poor (foam separates from the liquid milk, and there is some airiness in the liquid phase, offering some possibility of creating latte art, but with the possibility of foam clumps); Good (foam partially blends with the liquid milk, and there is good airiness in the liquid phase, offering a good possibility of creating latte art); and Very Good (foam completely blends with the liquid milk, and there is a high degree of airiness in the liquid phase, offering an excellent possibility of creating latte art).

[0193] Table 2: Quality Assessment of Foamed Skim Milk The addition of the protein deamidase of SEQ ID NO: 2 improved foam volume and quality at both 50°C and 60°C. A positive correlation was observed between enzyme dosage and foam volume and the formation of microfoams with a silky appearance and good foam cohesion. Example 3: Semi-skimmed milk and whole milk treated and foamed with deamidase Enzyme treatment:Four × 500 g of pasteurized semi-skimmed milk (Allerle Company, Vibi, Denmark; 3.5 g protein / 100 g; 1.5 g fat / 100 g; 4.6 g carbohydrate / 100 g) and four × 500 g of pasteurized whole milk (Allerle Company; 3.4 g protein / 100 g; 3.5 g fat / 100 g; 4.6 g carbohydrate / 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 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. The bottles were then cooled on ice and stored at 5°C for at least 90 minutes.

[0194] Foaming in a milk frother: Weigh 120 g (+ / - 1 g) of cold (5°C) enzyme-treated semi-skimmed or whole milk. Heat 3 × 4 120 g samples, representing dose responses of 0, 1.4, 3.4, and 6.8 IPA (U) / g milk protein, to 40°C, 50°C, or 60°C. Add the heated samples to a milk frother (Severin SM3587 Spuma 700 plus) and stir for 60 seconds without further heating. Transfer the frothed milk to a 500 ml measuring cup and measure the total volume, foam volume, and liquid volume separately. After 10 minutes, measure the total volume, foam volume, and liquid volume again at room temperature to assess foam stability.

[0195] The volume results for foaming from 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 airiness and cohesion is described in Tables 4 and 6, respectively. The sample processed with 0 IPA (U) / g protein is the control sample.

[0196] Table 3: Foaming results of semi-skimmed milk treated with protein deamidase of SEQ ID NO: 2 Table 4: Quality Assessment of Foamed Semi-skimmed Milk Table 5: Foaming results of whole milk treated with protein deamidase of SEQ ID NO: 2 Table 6: Quality Assessment of Foamed Whole Milk The addition of the protein deamidase in SEQ ID NO: 2 unexpectedly improved the foaming ability of both semi-skimmed and whole milk. The milk quality was improved in terms of airiness (resulting in the generation of more microfoams with a silky appearance) and excellent cohesion. Example 4: Treatment and foaming of skim milk with deamidase, purified deamidase, and commercial deamidase The protein deamidase of the present invention is produced from an inactive polypeptide in its original form. This original form is cleaved to release a propeptide from the mature protein deamidase. In this example, samples containing the mature protein deamidase (with or without the propeptide) were measured. The molar concentration of the added propeptide was within twice the molar concentration of the mature protein deamidase.

[0197] Enzyme treatment: 8 × 120 g (+ / - 1 g) of skim milk (Egelykke, Allergy; 3.5 g protein / 100 g; 0.1 g fat / 100 g; 4.7 g carbohydrate / 100 g) was weighed into a blue-capped bottle and heated to 55°C in a water bath. No protein deamidase or propeptide was added to the control sample. Mature protein deamidase containing the amino acid sequence of SEQ ID NO: 2 (with or without the propeptide containing SEQ ID NO: 16) 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 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. A 200 μl subsample of the inactivated sample was retained for ammonium analysis. Then cool the bottle on ice and store it at 5°C for at least 90 minutes.

[0198] 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) while stirring for 80 seconds. The frothed milk was then transferred to 250 ml measuring cups, and the total volume, foam volume, and liquid volume were measured for each sample. After 10 minutes, the total volume, foam volume, and liquid volume were measured again at room temperature to assess the stability of the foam.

[0199] Ammonium determination: The activity assay consists of two separate parts: 1) an enzymatic step in which ammonia is formed by the catalysis of a protein deamidase; and 2) a non-enzymatic detection step in which the ammonia formed in step (1) is derivatized into an indophenol blue compound with maximum absorption at 630 nm.

[0200] In step (1), ammonia is produced by deamidation of a protein deamidase. In step (2), the generated ammonia reacts with phenol under alkaline conditions to form dioxane. This reaction is catalyzed by sodium pentacyanonitroferro(III) ferric(III) nitrosyl ferrate (sodium nitroprusside). "Colorimetric reagent solution A" contains phenol and sodium nitroprusside. "Colorimetric reagent solution B" provides the alkaline reaction conditions. The intermediate is then oxidized by adding sodium hypochlorite ("Colorimetric reagent solution C") to form indophenol blue. This compound absorbs visible light at 630 nm. The enzyme activity is then calculated using a standard curve.

[0201] Measurement procedure: Step (1) Enzymatic step of ammonia formation Reagents: The assay dilution solution was: 0.2 M sodium phosphate buffer, 0.01% Triton X-100, pH 6.5. Assay buffer: Same as above. Used for preparing stock solutions and diluting samples for protein deamidases (hereinafter referred to as "enzymes").

[0202] Substrate solution: 30 mM Z-Gln-Gly (Merck C6154-1G), in the assay dilution (check pH after dissolution).

[0203] Termination solution: 0.4 M TCA Standard: NH4Cl diluted in the assay dilution solution (ammonium standard for IC, Merck 59755-100ML, 1000 mg / L NH4Cl). + (in water) (see also the "Standard Curve" section).

[0204] The enzyme product is dissolved / diluted in the assay buffer and a suitable dilution is prepared to produce a linear assay response.

[0205] Incubation: 1. Add 10 μL of diluted enzyme sample in triplicate to the wells of a 96-well microtiter plate (MTP).

[0206] 2. Add 100 μL of substrate solution to each well.

[0207] 3. For blank samples, add 100 μL of 0.4 M TCA solution.

[0208] 4. Seal the board with a transparent sealing film.

[0209] 5. Incubate the plate at 37°C and 500 rpm for 10 minutes on a thermostatic mixer equipped with a heated lid.

[0210] 6. To terminate the reaction, carefully add 100 μL of 0.4 M TCA solution (except for the blank sample, which already contains TCA).

[0211] Total reaction volume: 210 μL Step (2) Ammonia Detection Step Reagents: Colorimetric reagent A: 4% (w / v) phenol, 0.015% (w / v) sodium pentacyanonitrosylferro(III) ferrate dihydrate (sodium nitroprusside) (Na2[Fe(CN)5NO]∙2H2O).

[0212] Colorimetric reagent B: 5% (w / v) potassium hydroxide.

[0213] Colorimetric reagent C: 28% (w / v) potassium carbonate, 6% (v / v) sodium hypochlorite (Sigma-Aldrich 239305-25ml, <5% can be replaced with Cl2).

[0214] Incubation: 1. Transfer 15 μL from each well in step (1) to a new 96-well MTP.

[0215] 2. Transfer 45 μL of Milli-Q water into each well.

[0216] 3. Add 30 μL of colorimetric reagent B to each well (on the lab bench, gently shake to mix).

[0217] 4. Add 60 μL of colorimetric reagent A to each well (shake gently by hand on the lab bench to mix).

[0218] 5. Add 60 μL of colorimetric reagent C to each well (shake gently by hand on the lab bench to mix).

[0219] 6. Color development: Carefully seal the plate and place it on the lab bench for 30 minutes.

[0220] 7. Carefully transfer the MTP to the microplate reader and measure the absorbance at 630 nm.

[0221] Total reaction volume: 210 μL Standard curve: Standard stock solution: 1000 mg NH4 + / L.

[0222] In the ammonia detection procedure, a standard curve is prepared by adding ammonium standards at different dilutions to the determination dilution buffer. Specifically, 15 μL of diluted ammonia standard is mixed with 45 μL of water, and then the colorimetric reagents B, A, and C are added in the order given above.

[0223] The amount of enzyme that produces 1 μmol of ammonia per minute at 37°C is defined as 1 unit (indophenol assay unit; IPA (U)): in It can be abbreviated as in • C NH4+ It is the ammonia concentration in the reaction solution obtained from the ammonium standard curve (i.e., taking into account the dilution of the pre-diluted ammonium standard solution in the ammonia derivatization step).

[0224] • 18.04 is the molecular weight of ammonium used in the standard solution.

[0225] • V 反应 It is the reaction volume in the pore when ammonia is generated (210 μL).

[0226] • V 酶 This is the volume (10 μL) of enzyme solution added to the well when ammonia is generated.

[0227] • V NH3检测 This is the reaction volume in the well when detecting ammonia (210 μL).

[0228] The volume results and ammonium determination results for all tested protein deamidases are shown in Table 7. The precursor added to the sample contained the amino acid sequence of SEQ ID NO: 16. The quality of the generated foam in terms of airiness and cohesion is described in Table 8. No protein deamidase was added to the milk in the control sample.

[0229] Table 7: Foaming results of skim milk treated with protein deamidase When transferring the foam from the frother to the measuring cup, the quality of the foam was evaluated in terms of airiness (from clearly visible bubble size to uniform microfoam with a silky appearance) and cohesion (the ability to create latte art and avoid clumps of foam).

[0230] Table 8: Quality assessment of skim milk treated with protein deamidase In terms of higher foam, higher ammonium release, and improved foam quality, the formulation containing both the mature protein deamidase and the propeptide unexpectedly outperformed the purified mature form and the commercial product Amano PG500. Example 5: Skim milk treated with deamidase, purified deamidase, and foamed using FoamScan.

[0231] Protein deamidase purification Purify the pro-deamidase form of a protein containing the amino acid sequence SEQ ID NO: 1, 21, 23, or 24 according to Example 1. The pro-deamidase polypeptide is enzymatically cleaved to produce a mature protein deamidase, as described in Example 1. In samples containing the propeptide, no further purification step to remove the propeptide is performed. In samples where the propeptide is absent, the propeptide is removed as described in Example 1.

[0232] Enzyme treatment: 80 g of skim milk (Egelykke, Arla Foods Group, Denmark; 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. A protein deamidase containing the original amino acid sequence of SEQ ID NO: 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.

[0233] Foaming in FOAMSCAN™: Following the manufacturer's instructions, 60 ml of enzyme-treated milk and enzyme-free control milk were applied to tubes in a FOAMSCAN™ (Teclis Scientific, Lyon, France) using the following settings: initial liquid volume = 60 ml, nitrogen 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 (t½) of the foam volume after the nitrogen flow stopped. The final foam volume (in mL) and the t½ of the foam volume (in seconds) were recorded, and the results are shown in Table 9.

[0234] Ammonium determination: Perform as described in Example 4.

[0235] The volume results for all protein deamidase tests and the ammonium determination results are shown in Table 9. No protein deamidase was added to the milk in the control sample. The generated ammonium is shown after subtracting the blank.

[0236] Table 9: Foaming results of skim milk treated with protein deamidase As shown in Table 9, protein deamidases derived from various *Cyperobacter* species improved the foaming of skim milk compared to samples without added enzymes. Surprisingly, the final foam volume was higher in the samples with peptides present compared to enzyme treatment that removed peptides. The half-life of the foam volume was also prolonged in the peptide-containing enzyme treatment. Furthermore, the measured ammonia production was correlated with the final foam volume. In conclusion, the presence of peptides has a significant impact on the generated milk foam, increasing enzyme efficiency.

Claims

1. A method for producing an improved aerated dairy product, the method comprising: a) obtaining a dairy product composition; b) adding a first polypeptide to the dairy product composition, wherein the first polypeptide is a mature protein deamidase derived from a pro-form protein deamidase; c) optionally adding a second polypeptide to the dairy product composition, wherein the second polypeptide is a pro-peptide derived from the same pro-form protein deamidase; and d) aerating the dairy product composition to produce an aerated dairy product; wherein the aerated dairy product is improved compared to a similar method in which the first polypeptide and optionally the second polypeptide are not added to the dairy product composition.

2. The method of claim 1, wherein the dairy product composition comprises whipped cream, milk, cream, condensed milk, evaporated milk, milk serum, reconstituted milk, fermented milk, milk protein concentrate, whey protein concentrate, or a combination thereof.

3. The method of 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 to SEQ ID NO: 2, 5, 8, or 11.

4. The method of any one of the preceding claims, wherein the second polypeptide is a pro-peptide comprising a mutation that reduces the binding affinity of the pro-peptide to a deamidase domain compared to a parent pro-peptide.

5. The method of any one of the preceding claims, wherein the second polypeptide is a pro-peptide comprising: 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 to SEQ ID NO: 3, and a glycine at a 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 to SEQ ID NO: 6, and an alanine at a 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 to SEQ ID NO: 9, and having a glycine at a position corresponding to position 99 of SEQ ID NO: 9; or 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 to SEQ ID NO: 12, and having a glycine at a position corresponding to position 101 of SEQ ID NO:

12.

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

7. The method of any one of the preceding claims, wherein: 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 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 to SEQ ID NO: 3 and having a glycine at a 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 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 to SEQ ID NO: 6 and having an alanine at a 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 to 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 to SEQ ID NO: 9 and has a glycine at a position corresponding to position 99 of SEQ ID NO: 9; or 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 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 to SEQ ID NO: 12 and has a glycine at a position corresponding to position 101 of SEQ ID NO:

12.

8. A dairy composition comprising a first polypeptide and optionally a second polypeptide, wherein the first polypeptide is a mature protein deamidase derived from a pro-form protein deamidase, wherein the second polypeptide is a pro-peptide derived from the same pro-form protein deamidase.

9. The dairy composition of claim 8, wherein: 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 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 to SEQ ID NO: 3 and has a glycine at a 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 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 to SEQ ID NO: 6 and has an alanine at a 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 to 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 to SEQ ID NO: 9 and has a glycine at a position corresponding to position 99 of SEQ ID NO: 9; or 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 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 to SEQ ID NO: 12 and has a glycine at a position corresponding to position 101 of SEQ ID NO:

12.

10. The dairy composition of claim 8 or 9, wherein the dairy composition comprises a whipped cream, a milk, a cream, a condensed milk, a light milk, a condensed milk, a reconstituted milk, a fermented milk, a milk protein concentrate, a whey protein concentrate, or a combination thereof.

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

12. Aerated dairy product produced by the method according to any one of claims 1 to 7.

13. 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 cream or whipped cream topping.

14. Use of a first polypeptide and optionally a second polypeptide for the production of a dairy composition having improved aeration or foaming properties, wherein the first polypeptide is a mature protein deamidase derived from a pro-form protein deamidase and wherein the second polypeptide is a pro-peptide derived from the same pro-form protein deamidase.

15. Use according to claim 14, wherein: 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 a glycine at a 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 an alanine at a 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 to 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 to SEQ ID NO: 9 and a glycine at a position corresponding to position 99 of SEQ ID NO: 9; or 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 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 to SEQ ID NO: 12 and a glycine at a position corresponding to position 101 of SEQ ID NO: 12.

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