Compositions comprising truncated casein polypeptides

Abstract

The invention relates to a truncated casein polypeptide (TCP), said truncated casein polypeptide being preferably selected from alpha S1-casein (αS1-casein), alpha S2-casein (αS2-casein), beta-casein (β-casein), or kappa-casein (κ-casein) polypeptide or a mixture thereof, and compositions comprising thereof. The invention further relates to a dairy product or dairy-like product comprising said truncated casein polypeptide (TCP). Such composition can also be used for the preparation of nutritional product. Finally, the invention further relates to methods of making the truncated casein polypeptide (TCP) of the invention and methods of making the dairy product or dairy-like product of the invention.

Description

COMPOSITIONS COMPRISING TRUNCATED CASEIN POLYPEPTIDESFIELD OF THE INVENTION

[0001] The invention relates to a truncated casein polypeptide (TCP), said truncated casein polypeptide being preferably selected from alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof, and particularly to truncated alpha Sl-casein (aS 1 -casein) and truncated kappa-casein polypeptide (TKC), and compositions comprising thereof. The invention further relates to a dairy product or dairy-like product comprising said truncated casein polypeptide (TCP), and particularly comprising truncated alpha Sl-casein (aSl-casein) or truncated kappa-casein polypeptide (TKC). According to the invention, compositions may further comprise recombinant beta-lactoglobulin (rBLG). Such composition can be used for the preparation of nutritional product. Finally, the invention further relates to methods of making the truncated casein polypeptide (TCP) of the invention and methods of making the dairy product or dairy- like product of the invention.BACKGROUND

[0002] Animal milk is composed largely of water, fat, protein, and lactose, e.g., cow milk is composed of 87% water, 5% lactose, 4% fat, 3% proteins and some minerals. The proteins in milk, e.g., casein and beta-lactoglobulin, account for the majority of the nutrition, taste, and texture of animal milk. Producing milk in animals has a substantial environmental cost. Further, the increasing human population combined with declining milk production has led researchers to seek to develop methods of making milk alternatives and milk from non-animal sources.

[0003] One alternative to make dairy product and dairy-like product with desired properties in terms of nutritional values and texture properties, is the use of beta-lactoglobulin (BLG) or recombinant beta-lactoglobulin (rBLG), a whey protein currently commercially produced and available in the market.

[0004] Enriched beta-lactoglobulin products and the use of recombinant beta-lactoglobulin allow to increase the concentration of beta-lactoglobulin in said products. In addition, recombinant beta-lactoglobulin (rBLG) can be used as protein complement in dairy-products or as source of milk proteins in non-animal dairy analogue products. Although BLG and rBLG provide interesting properties to some dairy products and dairy-like products in terms of nutritional value and food texture, for some applications, these properties are insufficient.

[0005] Beta-lactoglobulin (BLG) and recombinant beta-lactoglobulin (rBLG) gels tend to be elastic and can be very hard at high protein concentrations. Upon shearing, BLG gels yield a texture which is sandy / grainy instead of the desired smooth / creamy texture. Similar observations are drawn for rBLG gels. This inability to build optimal texture at relevant conditions limits the use of BLG and rBLG protein for several applications, in particular in dairy, such as stirred yogurts, processed cheese, cheese or desserts.

[0006] Therefore, it would be advantageous to develop methods to make dairy products and dairy-like products having texture properties similar or close to commercially available dairy products, and in the same time having similar or improved nutritional values. In parallel, it would be also advantageous if such products can be obtained from non-animal sources.SUMMARY OF THE INVENTION

[0007] The invention relates to a composition comprising a truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof.

[0008] In one embodiment, the casein polypeptide is truncated relative to SEQ ID NOs: 1 and 51 to 61.

[0009] In one embodiment, the casein polypeptide is truncated at the C-terminus, the N- terminus, or a combination thereof.

[0010] In one embodiment, the casein polypeptide is truncated by 10 to 150 amino acids relative to a full length alpha S 1-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein).

[0011] In one embodiment, the truncated casein polypeptide (TCP) comprises at least one cysteine, the cysteine preferably being a free or a modified cysteine.

[0012] In one embodiment, the truncated casein polypeptide (TCP) comprises an amino acid sequence between 28 and 199 amino acids.

[0013] In one embodiment, the truncated casein polypeptide (TCP) is a kappa-casein polypeptide (TKC) comprising between 56 and 161 amino acids or a truncated alpha Sl-casein (aS 1-casein) polypeptide comprising between 49 and 180 amino acids or a truncated alpha S2- casein (aS2-casein) polypeptide comprising between 39 and 199 amino acids or a truncated beta casein polypeptide comprising between 28 and 188 amino acids.

[0014] In one embodiment, the truncated casein polypeptide (TCP) comprises at least one additional modification, said modification being selected from substitution, deletion, truncation, insertion, phosphorylation, glycosylation, or a combination thereof.

[0015] In one embodiment, the truncated casein polypeptide (TCP) is non-glycosylated or is less than about 20% glycosylated, preferably less than about 10% glycosylated, and more preferably less than 5% glycosylated.

[0016] In one embodiment, the truncated casein polypeptide (TCP) is truncated alpha Sl- casein (aS 1-casein) polypeptide and comprises SEQ ID NO: 62 or SEQ ID NO: 63 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 62 or to SEQ ID NO: 63.

[0017] In one embodiment, the truncated casein polypeptide (TCP) does not comprise a phenylalanine amino acid residue.

[0018] In one embodiment, the truncated casein polypeptide (TCP) is a truncated kappa-casein (K-casein) polypeptide (TKC) and comprises between 56 and 161 amino acids, preferably the TKC does not comprise SEQ ID NO: 17.

[0019] In one embodiment, the truncated casein polypeptide (TCP) is a truncated kappa-casein (K-casein) polypeptide (TKC) and comprises SEQ ID NO: 5, SEQ ID NO: 27, or SEQ ID NO: 29 or an amino acid sequence with at least 90% identity to SEQ ID NO: 5, SEQ ID NO: 27, or SEQ ID NO: 29.

[0020] In one embodiment, the truncated casein polypeptide (TCP) or the truncated kappa- casein (K-casein) polypeptide (TKC) comprises an amino acid sequence according to any one of SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118 or comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118.

[0021] In one embodiment, the composition comprises at least one additional polypeptide, wherein the at least one additional polypeptide is selected from a beta-casein (β-casein), an alpha lactalbumin, a kappa casein (K-casein), an alpha S 1-casein (aS 1-casein), an alpha S2- casein (aS2-casein), a lactoferrin, a transferrin, a beta-lactoglobulin (BLG), and a serum albumin.

[0022] In one embodiment, the additional polypeptide of the composition according to the invention is a truncated polypeptide, preferably truncated at the C-terminus, the N-terminus, orboth, relative to a full-length beta-casein (β-casein), alpha lactalbumin, kappa casein (K-casein), alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), lactoferrin, transferrin, beta- lactoglobulin (BLG), or serum albumin.

[0023] In one embodiment, the alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide of which the casein polypeptide is truncated comprises a cow, human, sheep, goat, buffalo, bison, horse, yak, reindeer, donkey, moose, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or camel casein polypeptide, preferably a cow, buffalo, goat, sheep, human, camel, donkey, or horse casein polypeptide.

[0024] Another object of the invention is a composition comprising a truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2- casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof, and at least one recombinant beta-lactoglobulin (rBLG) polypeptide.

[0025] In one embodiment, the truncated casein polypeptide (TCP) comprises at least one cysteine involved in a disulfide bond with at least one of the recombinant beta-lactoglobulin polypeptide (rBLG).

[0026] A further object of the invention is a dairy product or dairy-like product comprising the composition as defined above, and in particular a composition comprising a truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2- casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof, and optionally at least one recombinant beta-lactoglobulin (rBLG) polypeptide.

[0027] In one embodiment, the dairy product of dairy-like product is milk, yogurt, curd, cheese, cream, cream cheese, butter, or ice cream.

[0028] In one embodiment, the dairy product or dairy -like product according to the invention further comprises one or more additional components selected from proteins, texture agents, lipids, flavor compounds, sweetening agents, color balancing agents, ashes, vitamins, and any combination thereof.

[0029] In one embodiment, the one or more additional components is animal-derived, non- animal derived, or a combination thereof.

[0030] In one embodiment, the composition of the dairy product or dairy-like product comprises from 1 to 10%, preferably from about 2% to about 5% by weight of a truncated casein polypeptide (TCP) and a plurality of aggregates.

[0031] In one embodiment, the aggregates comprise a diameter ranging from about 20 to 200 nm, preferably from about 40 to 100 nm as measured by dynamic light scattering.

[0032] In one embodiment, the composition of the dairy product or dairy-like product comprises from about 2% to about 5% by weight of a truncated casein polypeptide (TCP), and the TCP comprises an elastic modulus (G’) of about 0,1 mPa to about 2,000 Pa, preferably of about 100 Pa to about 1,500 Pa when the TCP is measured using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

[0033] In one embodiment, the composition of the dairy product or dairy-like product comprises about 2% to about 5% by weight of a truncated casein polypeptide (TCP), and the TCP comprises a viscosity modulus (G”) of about 1 mPa to about 500 Pa, preferably of about 10 Pa to about 300 Pa when the TCP is measured using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

[0034] In one embodiment, the composition of the dairy product or dairy-like product comprises about 2% to about 5% by weight of a truncated casein polypeptide (TCP) and the composition has a ratio of elasticity to viscosity for the TCP (tanD) of about 0.05 to about 100, preferably of about 0.05 to about 0.5, and more preferably of about 0.1 to about 0.4, when the TCP is measured using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

[0035] In one embodiment, the composition of the dairy product or dairy-like product comprises SEQ ID NO:62 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO:62, SEQ ID NO:29 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO:29, SEQ ID NO:63 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO:63 or SEQ ID NO:27 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 27.

[0036] In one embodiment, the dairy product or dairy-like product is yogurt or cheese, and the composition comprises: (i) a truncated alpha SI -casein (aS 1 -casein) polypeptide comprising SEQ ID NO:62 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:62; or (ii) a truncated kappa casein (K-casein) polypeptide(TKC) comprising SEQ ID NO:29 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:29.

[0037] In one embodiment, the dairy product or dairy-like product is an ultra-heat treatment beverage, and the composition comprises: (i) a truncated alpha Sl-casein (aSl-casein) polypeptide comprising SEQ ID NO:62 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:62; or (ii) a truncated kappa casein (K-casein) polypeptide (TKC)-comprising SEQ ID NO:27 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:27.

[0038] An additional object of the invention is a nutritional product comprising the composition as defined above, and in particular a composition comprising a truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2- casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof, and optionally at least one recombinant beta- lactoglobulin polypeptide.

[0039] In one embodiment, the composition of the nutritional product comprises a truncated casein polypeptide (TCP) with an amino acid score of at least 80%, preferably with an amino acid score of at least 94%.

[0040] In one embodiment, the composition of the nutritional product comprises a truncated casein polypeptide (TCP) with a protein digestibility corrected amino acid score (PDCAAS) of at least 0.94.

[0041] In one embodiment, the truncated casein polypeptide (TCP) of the nutritional product is an alpha Sl-casein (aS 1 -casein) polypeptide truncated at the N-terminus and C-terminus relative to a full length alpha Sl-casein (aSl-casein) polypeptide and comprising SEQ ID NO:62 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:62, preferably comprising SEQ ID NO:62.

[0042] In one embodiment, the truncated casein polypeptide (TCP) of the nutritional product, is an alpha Sl-casein (aS 1 -casein) polypeptide truncated at the N-terminus and C-terminus relative to a full length alpha Sl-casein (aSl-casein) polypeptide and comprising SEQ ID NO:63 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID 63, preferably comprising SEQ ID NO:63.

[0043] In one embodiment, the composition of the nutritional product comprises: (i) a truncated casein polypeptide (TCP), truncated at the N-terminus, the C-terminus, or acombination of both relative to a full-length casein polypeptide, wherein the TCP comprises SEQ ID NOs: 116 or 117, or comprises an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 116 or 117; or (ii) a truncated kappa casein (K-casein) polypeptide (TKC) truncated at the N-terminus relative to a full-length kappa casein polypeptide, wherein the TKC comprises SEQ ID NO: 118, or comprises an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 118.

[0044] In one embodiment, the nutritional product is for treating a metabolic disorder in an individual.

[0045] In one embodiment, the metabolic disorder is phenylketonuria or hyperphenylalaninemia.

[0046] Another object of the invention is a method for manufacturing a truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2- casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide, wherein the method comprises:(i) expressing the truncated casein polypeptide in a recombinant host;(ii) culturing the recombinant host under conditions suitable for the production of the truncated casein polypeptide; and(iii) harvesting the truncated casein polypeptide.

[0047] In one embodiment, the truncated casein polypeptide (TCP) obtained from the method of the invention is truncated at the N-terminus, at the C-terminus, or a combination of both relative to a full-length alpha Sl-casein (aS 1 -casein) polypeptide or a full-length kappa-casein (K-casein) polypeptide.

[0048] In one embodiment, the truncated casein polypeptide (TCP) obtained from the method of the invention is a truncated kappa-casein (K-casein) polypeptide (TKP) at the N-terminus, the C-terminus, or a combination of both, relative to a full-length kappa-casein (K-casein) polypeptide, and wherein the TKC comprises an amino acid sequence between 56 and 161 amino acids; and wherein preferably the TKC does not comprise SEQ ID NO: 17.

[0049] In one embodiment, the truncated casein polypeptide (TCP) obtained from the method of the invention is a truncated alpha Sl-casein (aSl-casein) polypeptide at the N-terminus, the C-terminus, or a combination of both, relative to a full-length alpha Sl-casein polypeptide,wherein the truncated alpha SI -casein (aS 1 -casein) polypeptide comprises an amino acid sequence between 49 and 180 amino acids.

[0050] In one embodiment, the truncated casein polypeptide (TCP) obtained according to the method of the invention comprises at least one cysteine.

[0051] In one embodiment, the method according to the invention further comprises one or more of the steps of combining the truncated casein polypeptide (TCP) with a recombinant beta-lactoglobulin (rBLG), heating and / or agitating the combination, followed by optionally cooling, concentrating and / or drying the combination.

[0052] In one embodiment, the truncated casein polypeptide (TCP) obtained according to the method of the invention has an increased titer of casein compared to a recombinant full-length casein.

[0053] In one embodiment, the recombinant host is a microbial host, a plant host, or a mammalian host.

[0054] In one embodiment, the recombinant host is a microbial host, preferably a bacteria, and more preferably a bacteria selected from Lactococci sp., Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis, Corynebacterium glutamicum, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis sp. PCC6803, Escherichia coli, or a combination thereof, and preferably the bacteria is Bacillus subtilis.

[0055] In one embodiment, the microbial host is a fungus, and preferably a fungus selected from Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida guilliermondii, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Endothia parasitica, Eremothecium ashbyii, Fusarium moniliforme, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. Cooney et Emerson, Mucor pusillus Lindt, Penicillium roquefortii, Pichia pastoris (also named Komagataella phaffii), Rhizopus niveus, Saccharomyces cervisea, Saccharomyces fragilis, Trichoderma reesei, or a combination thereof, more preferably the fungus is Pichia pastoris or Aspergillus oryzae.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following drawings form part of the present specification and are included to further demonstrate exemplary embodiments of certain embodiments of the present disclosure.

[0057] FIGS. 1A-1H depict full-length and truncated casein polypeptide sequences from various species, including alignment of sequences that were experimentally tested for recombinant expression in Pichia pastoris. SEQ ID NOs: 1-4 were used as experimental controls, which includes the intact kappa cow casein sequence (SEQ ID NO: 1). Degree of N- terminal truncation was varied across SEQ ID NOs: 5-19. In SEQ ID NOs: 20-25, C-terminal truncation was varied at a fixed degree of N-truncation.

[0058] FIGS. 2A-2B depict expression levels of intact and truncated casein sequences as analyzed by Western blotting (FIG. 2A) of secreted fraction after 96 well plate cultivation of recombinant Pichia strains for SEQ ID NOs: 5 and 10 (left panel), SEQ ID NO: 12 (middle panel) and SEQ ID NO: 1 (right panel), and Coomassie staining (FIG. 2B).

[0059] FIGS. 3A-3C depict a time course analysis of recombinant TKC (truncated kappa- casein) production during cultivation of Pichia pastoris in IL bioreactor analyzed by Western blotting. In FIG. 3A, lanes 2- 5 and lanes 6-9 refer to 0, 30, 54 and 70 h timepoints of fermentation for SEQ ID NOs: 5 and 26, respectively. Lanes 2-7 in FIG. 3B and FIG. 3C refer to 0, 30, 54, 70 and 93h timepoints of fermentation for SEQ ID NOs: 12 and 17, respectively.

[0060] FIG. 4 depicts expression levels of intact and truncated casein sequences as analyzed by Western blotting of secreted fraction with anti-HIS tag antibody after shake flask cultivation of recombinant Bacillus strains. Strain transformed with shuttle vector (no insert) was used as negative control.

[0061] FIG. 5 depicts an embodiment of a method of production of rBLG and TKC.

[0062] FIG. 6 depicts vector pHTOl, used in embodiments to express truncated caseins in recombinant Bacillus subtilis.

[0063] FIGS. 7A-7B depicts transmission electron microscopy (TEM) images (FIG. 7A) and DLS size distribution (FIG. 7B) showing soluble aggregates of recombinant BLG with an average size of 82 nm produced with the following parameters: 5% initial protein concentration; heat treatment at 80°C for 15 min; pH 6.7.

[0064] FIGS. 8A-8B depict TEM images of aggregates at 5% initial protein concentrationafter 15 minutes of heat treatment at 80 °C. FIG. 8A depicts SEQ ID NO:5 alone; FIG. 8B depicts SEQ ID NO:5-rBLG aggregates.

[0065] FIGS. 9A-9B depict TEM images of aggregates at 3% initial protein solution after 15 minutes of heat treatment at 80 °C. FIG. 9A depicts SEQ ID NO:5-rBLG aggregates; FIG. 9B depicts KC (full-length kappa-casein)-rBLG aggregates.

[0066] FIGS 10A -10C depict TEM images for a whey protein isolate (WPI) control, aggregates of SEQ ID N0:5 / WPI and aggregates of KC:WPI produced after 15 minutes of heat treatment at 95°C at a 3% initial protein solution. The ratio of KC:WPI and SEQ ID N0:5:WPI is 1: 1.

[0067] FIGS. 11A-11C depict DLS data related to size distribution by mass of SEQ ID N0:5 / WPI aggregates solution formed at three different temperatures.

[0068] FIGS. 12A-12C depict DLS data showing the impact of SEQ ID N0:5:WPI ratio in the size distribution and size of the aggregates.

[0069] FIG. 13 A is a DLS graph showing size distribution by intensity (left panel) and size distribution by mass (right panel) for aggregates of SEQ ID NO:29-rBLG. The y-axes represent intensity percentage and weight percentage, and the x-axes represent size. Peak polydispersity is shown above the graphs.

[0070] FIG. 13B is a DLS graph showing size distribution by intensity (left panel) and size distribution by mass (right panel) for aggregates of KC-rBLG. The y-axes represent intensity percentage and weight percentage, and the x-axes represent size. Peak polydispersity is shown above the graphs.

[0071] FIG. 13C is a TEM image of the aggregates formed by 2.5% SEQ ID NO:29 and 5% rBLG. The scale bar shows 500 pm.

[0072] FIG. 14 depicts titer levels of truncated alpha SI casein polypeptides as analyzed by SDS-PAGE densitometry (left panel) using full-length cow alpha S1 / S2 casein as a standard (right panel). SEQ ID NO: 62 and SEQ ID NO: 63 were serially diluted as shown across the bottom of the SDS-PAGE gel.

[0073] FIG. 15A depicts the solubility versus pH for SEQ ID NO:62 (blue) and SEQ ID NO:63 (orange) compared to full-length cow alpha S1 / S2 casein (gray). The y-axis representsthe percent solubility of the polypeptide, and the x-axis represents units of pH.

[0074] FIG. 15B depicts the solubility changes due to calcium addition for SEQ ID NO:62 (blue) and SEQ ID NO:63 (orange) compared to full-length cow alpha S1 / S2 casein (gray). The y-axis represents the percent solubility of the polypeptide, and the x-axis represents concentration of calcium (mM).

[0075] FIG. 16 depicts the deconvoluted mass spectrometry chart for SEQ ID NO:62.

[0076] FIG. 17 is a photograph of a 1.5 mL microcentrifuge tube. The gel formation is identified by the bracket.

[0077] FIG. 18A is a DLS graph showing size distribution by intensity (left panel) and size distribution by mass (right panel) for SEQ ID NO: 62. The y-axes represent intensity percentage and weight percentage, and the x-axes represent size. Peak polydispersity is shown above the graphs.

[0078] FIG. 18B is a TEM image of the pseudomicelles in the acidic gel formed by the self- aggregation of SEQ ID NO: 62. The scale bar shows 200 nm.

[0079] FIG. 18C is a DLS graph showing size distribution by intensity (left panel) and size distribution by mass (right panel) for the full-length cow alpha casein S 1 / S2 polypeptide. The y-axes represent intensity percentage and weight percentage, and the x-axes represent size. Peak polydispersity is shown above the graphs.

[0080] FIG. 18D is a TEM image of the full-length alpha S1 / S2 casein mixture following acidification. No aggregates or micelles are visible. The scale bar represents 200 nm.

[0081] FIG. 19A is a mass spectrometer graph showing a mass shift of the SEQ ID NO: 5 polypeptide (produced in Pichia pastoris) relative to the predicted or theoretical mass. The y- axis represents the percent intensity, and the x-axis represents mass based on mass / charge ratio.

[0082] FIG. 19B is a mass spectrometer graph showing no mass shift of SEQ ID NO: 29 produced in Bacillus subtilis.

[0083] FIG. 19C depicts a TKC sequence (SEQ ID NO: 5). The amino acid residues that are prone to glycosylation are underlined and in bold text.

[0084] FIG. 20 depicts the solubility versus pH for SEQ ID NO: 5 (blue) and SEQ ID NO:29 (orange) compared to full-length cow kappa casein (gray). The y-axis represents the solubility of the polypeptide (percent), and the x-axis represents units of pH.

[0085] FIG. 21 A is a DLS graph showing size distribution by intensity (left panel) and size distribution by mass (right panel) for the SEQ ID NO: 5. The y-axes represent intensity percentage and weight percentage, and the x-axes represent size. Peak polydispersity is shown above the graphs.

[0086] FIG. 2 IB is a DLS graph showing size distribution by intensity (left panel) and size distribution by mass (right panel) for the SEQ ID NO: 29. The y-axes represent intensity percentage and weight percentage, respectively, and the x-axes represent size. Peak polydispersity is shown above the graphs.

[0087] FIG. 21C-21E depict TEM images of solutions of SEQ ID NO:29 (FIG. 20C), cow full length kappa casein (FIG. 20D), and SEQ ID NO: 5 (FIG. 20E). The scale bar is 100 nm in FIG. 20D and 200 nm in FIGS. 20C and E.

[0088] FIG. 22A is a photograph of SEQ ID NO:62 gel formation in a microcentrifuge tube. The gel is identified by the bracket.

[0089] FIG. 22B is a photograph of Full Alpha AO gel formation in a microcentrifuge tube. The gel is identified by the bracket.

[0090] FIG. 22C is a photograph of SEQ ID NO: 29 gel formation in a microcentrifuge tube. The gel is identified by the arrow.

[0091] FIG. 22D is a photograph of Full Kappa AO gel formation in a microcentrifuge tube. The gel is identified by the bracket.

[0092] FIG. 23 A is a graph showing the elastic modulus (G’) for acidic gels containing 3.25% by wt (black bars) or 5% by wt (white bars) skimmed milk, SEQ ID NO: 62, SEQ ID NO: 29, Full Alpha AO, or rBLG. The y-axis represents elastic modulus G’ in Pascals (Pa).

[0093] FIG. 23B is a graph showing the ratio of viscosity to elasticity (tanD) for acidic gels containing 3.25% by wt (black bars) or 5% by wt (white bars) skimmed milk, SEQ ID NO: 62, SEQ ID NO: 29, Full Alpha AO, or rBLG. The y-axis represents tanD ratio.

[0094] FIG. 24 is a graph showing the solubility of SEQ ID NO:62 (black, dashed line), Full Alpha AO (black, solid line), sodium caseinate (dark gray line), and micellar casein (light grayline) in media of increasing pH. The y-axis represents the percent solubility, and the x-axis represents the pH of the medium.

[0095] FIG. 25A is a TEM image of a SEQ ID NO:62-rBLG blend showing aggregate formation. The scale bar represents 200 nm.

[0096] FIG. 25B is a TEM image of a cow full-length alpha Sl / S2-rBLG blend showing aggregate formation. The scale bar represents 200 nm.

[0097] FIG. 25C is a TEM image of rBLG alone showing aggregate formation. The scale bar represents 200 nm.

[0098] FIG. 26 is a graph showing the interfacial load of alpha-casein, recombinant full Alpha AO and SEQ ID NO: 62 at different protein concentration and pH.

[0099] FIG. 27 is a photograph of SEQ ID NO: 62 yogurt formation at a concentration of 3.25 wt% and 2% fat (coconut cream)

[0100] FIG. 28 are photographs of yogurt formation from 4 wt% of SEQ ID NO: 62 and 5 wt% of microp articulated rBLG (FIG. 28A), from 4 wt% of cold-gellable rBLG and 5 wt% of microparticulated rBLG (FIG. 28B) and from 4 wt% of recombinant full Alpha AO and 5 wt% of microparticulated rBLG (FIG. 28C).DETAILED DESCRIPTION

[0101] Any embodiment described herein, including those described only in the examples, can be combined with any one or more other embodiments, unless such combination is expressly disclaimed or is improper. Thus, the term “embodiment”, as used herein, is not to be considered as excluding features recited in other embodiments.

[0102] Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have meanings that are commonly understood by one of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0103] Definitions

[0104] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0105] The use of the term “or” in the claims is used to mean “and / or,” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0106] The term "about" is used herein explicitly or not. Every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and / or measurement conditions for such given value.

[0107] As used herein, “about” can mean plus or minus 10% of the provided value. Where ranges are provided, they are inclusive of the boundary values. “About” can additionally or alternately mean either within 10% of the stated value, or within 5% of the stated value, or in some cases within 2.5% of the stated value; or “about” can mean rounded to the nearest significant digit.

[0108] The terms “from ... to. . .” and “between . . . and ...” or “between” as used herein must be understood as including the boundaries mentioned. For example, a number between x and y explicitly includes the numbers x and y and any numbers that fall within x and y.

[0109] On the contrary, the expressions “greater than ... ”, “more than. . . ”, “higher than . . . ”, “less than ...” and “lower than ...” as used herein do not include the boundaries mentioned.

[0110] The abbreviation “% w / w” means weight / weight percent. Other abbreviations can be used to define mass percentage, such as “wt%” (weight percent).

[0111] As used herein, the terms “comprising” (and any variant or form of comprising, such as “comprise” and “comprises”), “having” (and any variant or form of having, such as “have” and “has”), “including” (and any variant or form of including, such as “includes” and “include”) or “containing” (and any variant or form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps.

[0112] The use of the term “for example” and its corresponding abbreviation “e.g.” means that the specific terms recited are representative examples and embodiments of the disclosure that are not intended to be limited to the specific examples referenced or cited unless explicitly stated otherwise.

[0113] The terms “optional” or “optionally” as used herein refer to a feature or structure being present or not, or an event or circumstance occurring or not, and that the description includes instances in which a particular feature or structure is present and instances in which the feature or structure is absent, or instances in which the event or circumstance occurs and instances in which the event or circumstance does not occur.

[0114] The terms “protein” and “polypeptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0115] The term “amino acid composition” is used herein to refer to the amino acid makeup of a polypeptide, which is typically calculated as the milligram amino acid per gram polypeptide. “Amino acid composition” may be calculated for a particular amino acid of interest, such as for example the amount of leucine in a given polypeptide, or for a group of amino acids, such as the amount of branched chain amino acids in a given polypeptide. Calculation may be based on the known sequence of the polypeptide, for example by the total molecular weight of the amino acid residues of interest divided by the total molecular weight of the polypeptide. The total molecular weight of the amino acid residues of interest is determined by the number of said residues in the polypeptide multiplied by the molecular weight of the residue in the context of the polypeptide (as opposed to the molecular weight of the free amino acid).

[0116] The term “amino acid score” and “AAS” are used interchangeably herein to refer to a score based on essential amino acid requirements published by the Food and Agriculture Organization (“FAO”) for different age groups (“Dietary protein quality evaluation in human nutrition: Report of an FAO Expert Consultation”, FAO Food Nutr. Paper, 92: 1-66, 2013). For instance, scoring patterns for protein quality evaluation are determined by calculating the ratio of an essential amino acid to protein requirement, expressed as mg amino acid per gram of protein. Using the scoring pattern determined for the protein requirements for a child (6 months to 3 years; see Table 5 of the referenced FAO report), the AAS can be defined. To calculate the AAS for a given polypeptide, for each essential amino acid, the ratio of the amount of an essential amino acid present in the polypeptide compared to the amount of that essential amino acid as recommended in the FAO report is determined. The AAS of that polypeptide is the lowest ratio determined for any of the essential amino acids. A polypeptide with an AAS of greater than 0.94 (94%) satisfies the essential amino acid requirements for all essential aminoacids and is generally considered a complete protein source.

[0117] The term “essential amino acid” is used herein to refer to an amino acid selected from histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

[0118] The term “recombinant” is a common term and known and understood in the art. When referring to a nucleic acid (e.g., a gene), the recombinant can be used, for example, to describe a nucleic acid that has been removed from its naturally occurring environment, a nucleic acid that is not associated with all or a portion of a nucleic acid abutting or proximal to the nucleic acid when it is found in nature, a nucleic acid that is operatively linked to a nucleic acid that it is not linked to in nature, or a nucleic acid that does not occur in nature. The term “recombinant” can be used, e.g., to describe cloned DNA isolates, or a nucleic acid including a chemically-synthesized nucleotide analog. A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion, or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome. When “recombinant” is used to describe a protein, it can refer to, for example, a protein that is produced in a cell of a different species or type as compared to the species or type of cell that produces the protein in nature. The term “recombinant host cell” as used herein refers to a cell into which a recombinant nucleic acid has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0119] The term “vector” as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double- stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectorsare capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell and are thereby replicated along with the host genome.

[0120] The term “host cell” as used herein refers to a cell into which a recombinant nucleotide sequence has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0121] The term “native protein” as used herein refers to protein in its natural state with intact structure, i.e., the structure is not altered by chemical or physical treatments. The natural state of the protein is its properly folded and assembled structure conferring stability and allowing functional activity.

[0122] The term “functionalized protein” as used herein refers to protein that has undergone a treatment to modify its properties via chemical or physical treatment. Methods to functionalize proteins include chemical treatments, such as, but not limited to, salt washing, chemical hydrolysis, and precipitation, and physical treatments, such as, but not limited to, heat treatment, high / low pressure application, sonication, and shearing.

[0123] The term “variant” when talking about microorganisms refers to a subtype of microorganism that is genetically distinct from a main strain of said microorganism.

[0124] The term “variant” when talking about protein refers to a modified form of a protein that may exhibit altered characteristics and / or properties.

[0125] Protein variants may contain one or more conservative substitutions and / or one or more tolerated substitutions. Tolerated substitutions can be substitutions which do not fall under the definition of conservative as provided below but are nonetheless phenotypically silent. The skilled person is aware that various amino acids have similar properties and thus are ‘conservative’. One or more such amino acids of a protein, polypeptide or peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that protein, polypeptide or peptide.

[0126] The term “phenotypically silent variants” is understood to refer to a variant whichincorporates one or more amino acid changes, including substitutions, insertions and deletions, in addition to those set out above, which variant has a similar phenotype to the corresponding polypeptide without said change(s). It is understood that “phenotypically silent variants” can be variants that do not have altered characteristics and / or properties in relation to native proteins thereof. For the purposes of this application, phenotype comprises attributes relevant to dairy applications such as nutrition, gelation, taste, etc., characterized as food functionalities.

[0127] Throughout the disclosure, the full name of an amino acids may be used interchangeably with the standard three letter and one letter abbreviations for each. For the avoidance of doubt, these include Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamic Acid (Glu, E), Glutamine (Gin, Q), Glycine (Gly, G), Histidine (His, H), Isoleucine (IIe, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0128] The amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulfur containing side chains). It should be appreciated that amino acid substitutions within the scope of the present disclosure can be made using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on an alanine may be replaced with an ethyl group, and / or that minor changes may be made to the peptide backbone. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present.

[0129] Substitutions of the nature described in the immediately preceding paragraph are often referred to as “conservative” or “semi-conservative” amino acid substitutions. The present disclosure therefore extends to use of a polypeptide comprising any of the amino acid sequences described above but with one or more conservative substitutions and or one or more tolerated substitutions in the sequence, such that the amino acid sequence of the truncatedcasein polypeptide (TCP) has at least 90% identity, such as 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to the TCP sequences disclosed herein. Since the truncated casein polypeptide (TCP) can be truncated alpha Sl-casein (asl-casein), alpha S2- casein (as2-casein), beta-casein (β-casein), or kappa-casein (K-casein), the amino acid sequence can have at least 90% identity, such as 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to the truncated alpha Sl-casein (asl-casein), alpha S2-casein (as2-casein), beta-casein (β-casein), or kappa-casein (K-casein) sequences disclosed herein.

[0130] “Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case can be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0131] One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present disclosure.

[0132] The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The “best alignment” is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions x 100).

[0133] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid polypeptides. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein polypeptides for use in the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSLBlast can be used to perform an iterated search which detects distant relationships between polypeptides (Id.). When utilizing BLAST, Gapped BLAST, and PSLBlast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0134] Mutations, including conservation and tolerated substitutions, insertions, and deletions, can be introduced into the sequences provided using any appropriate method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation independent cloning (LIC) procedures. These methods are detailed in many of the standard molecular biology texts. For further details regarding polymerase chain reaction (PCR) and restriction enzyme -based cloning, see Sambrook & Russell, (2001) Molecular Cloning - A Laboratory Manual (3rdEd.) CSHL Press. Further information on ligation independent cloning (LIC) procedures can be found in Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6. The truncated casein polypeptide (TCP) sequences provided by the disclosure can be obtained from solid state synthesis, or any other appropriate method known in the art.

[0135] The term “nutritional composition” and “nutritional product” are used interchangeably herein to refer to a composition that contains a recombinant polypeptide of the disclosure and which contains a desirable amount of amino acids, e.g., as dictated by U.S. government recommended daily allowance. The “nutritional composition” may also include any number of optional additional ingredients, including conventional food additives (synthetic or natural), for example one or more acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipients, flavorants, minerals, osmotic agents, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, minerals, and / or vitamins. The optional ingredients can be added in any suitable amount. The “nutritional compositions” may be a source of complete nutrition or may be a source of incomplete nutrition. The “nutritional composition” may provide daily dietary requirements for protein or for essential amino acids. In some embodiments, a “nutritional composition” or “nutritional product” can be a “dairy product” or dairy -like product”.

[0136] The term “complete nutrition” is used herein to refer to nutritional products and compositions that contain sufficient types and levels of macronutrients (protein, fats and carbohydrates) and micronutrients to be considered a sole source of nutrition for the animal to which it is being administered to. Patients can receive 100 percent of their nutritional requirements from such complete “nutritional compositions.”

[0137] The term “food product” as used herein refers to a composition that can be ingested by humans or animals, including domesticated animals (e.g., dogs, cats), farm animals (e.g., cows, pigs, horses), and wild animals (e.g., non-domesticated predatory animals). In various embodiments, the food products provided herein meet standards for food safety required by the U.S. Food and Drug Administration (FDA), the U.S. Department of Agriculture, the European Food Safety Authority, and / or other state or region food regulatory agencies. The term includes compositions that can be combined with or added to other ingredients to make compositions that can be ingested by humans or animals. In some embodiments, dairy products include food products.

[0138] The term “dairy product” as used herein refers to milk (e.g., whole milk [at least 3.25% milk fat], partly skimmed milk [from 1% to 2% milk fat], skim milk [less than 0.2% milk fat], cooking milk, condensed milk, flavored milk, goat milk, sheep milk, dried milk, evaporated milk, milk foam), and products derived from milk, including but not limited to yogurt (e.g., whole milk yogurt [at least 6 grams of fat per cup], low-fat yogurt [between 2 and 5 grams of fat per cup], nonfat yogurt [less than 0.5 percent milk fat by weight], Greek yogurt [strainedyogurt with whey removed], whipped yogurt, goat milk yogurt, Labneh [labne], sheep milk yogurt, yogurt drinks [e.g., whole milk Kefir, low-fat milk Kefir], stirred yogurt, Lassi), cheese (e.g., whey cheese such as ricotta and mozzarella, semi-soft cheese such as Havarti and Munster, medium-hard cheese such as Swiss and Jarlsberg, hard cheese such as Cheddar and Gouda, soft ripened cheese such as Brie and Camembert, cottage cheese, cream cheese, curd, powdered cheese, cheese flavor), cream (e.g., whipping cream, coffee whitener, coffee creamer, sour cream, creme fraiche), frozen confections (e.g., ice cream, smoothie, milk shake, frozen yogurt, sundae), butter, infant formula, weight loss beverages, nutritional beverages, pudding, buttermilk, milk protein concentrate, whey protein concentrate, whey protein isolate, casein concentrate, casein isolate, skim milk powder, whole milk powder, nutritional supplements, texturizing blends, flavoring blends, or coloring blends. In some embodiments, “dairy products” may or may not be enriched with proteins and used for products that are part of sport nutrition (e.g., high protein, low calorie diet, etc.), for products with high nutritional value (e.g., growth of children, elderly people, etc.), and for products used in the health field (e.g., mental health, well-being, gut microbia, treatment of diseases such as diabetes, osteoporosis, liver diseases, obesity, gastrointestinal disorders, allergy, blood pressure, eczema, digestion problems, amino acid disorders, etc.). The term “dairy product” may also encompass animal dairy products and animal products that include components of animal origin or include components of both animal original and non-animal origin.

[0139] The term “high protein dairy product” as used herein refers to a protein enriched product for which the total of protein concentration exceeds 5%, or for which the protein concentration is higher than that of standard products sold on the market and not considered protein rich. Such high protein dairy products are for example, but not limited to, high protein yogurts, high protein UHT (ultra heat treatment) drinks, high protein fruit preparations, high protein cream cheeses, high protein snacks, high protein bites, high protein cereal bars, high protein ready to drink milk, high protein milk beverages or high protein beverages.

[0140] The term “non-animal-derived” refers to a component (e.g., protein, lipid, carbohydrate) that is not native to an animal cell, like a recombinant protein produced by a microbial host or by a plant cell for instance. In some embodiments, the term “non-animal- derived” refers to components derived from naturally occurring or modified plants, algae, fungi, or microbes. The terms “non-animal product”, “non-animal dairy product”, “non-animal dairy analogs” refer to a product or dairy product that does not contain any components of animal origin, including milk or milk proteins of animal origin. Such products may include, for example, milk of vegetal origin (e.g., plant-based milk, for instance, from soy, oat, almond,coconut) or proteins extracted from plants, algae, fungi, or microbes. Similarly, the term “dairy- like product” refers to a product that does not contain any components of animal origin, including milk or milk proteins of animal origin, but may include, for example, milk of vegetal origin (e.g., plant-based milk, for instance, from soy, oat, almond, coconut) or proteins extracted from plants, algae, fungi, or microbes.

[0141] The term “animal-derived”, as used herein, refers to component, such as a milk protein, produced by an animal, for instance, a cow. In some embodiments, the term “animal- derived” refers to a mammal-produced milk or a mammal-produced dairy product.

[0142] The term “milk protein” as used herein refers to a protein that is found in a mammal- produced milk or a protein having a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the sequence of a protein that is found in a mammal -produced milk. Examples of milk proteins include, but are not limited to, β-casein, K-casein, a-Sl-casein, a-S2-casein, a-lactalbumin, P- lactoglobulin, lactoferrin, transferrin, and serum albumin.

[0143] The term “lipids” as used herein refers to one or more molecules (e.g., biomolecules) that include a fatty acyl group (e.g., saturated or unsaturated acyl chains). The term “lipids” includes oils, phospholipids, free fatty acids, phospholipids, monoglycerides, diglycerides, and triglycerides.

[0144] The term “plant-derived lipid” as used herein refers to a lipid obtained from and / or produced by a plant (e.g., monocot or dicot).

[0145] The terms “Protein Digestibility Corrected Amino Acid Score” and “PDCAAS” are used interchangeably herein to refer to the determination of the effectiveness with which absorbed dietary amino acids meet the indispensable amino acid requirement at safe levels of protein intake. The method to compute PDCAAS has been outlined in detail with associated caveats in the 2007 protein requirements (see Report of the Joint FAO / WHO / UNU Expert Consultation, 2007). The term “Chemical Amino Acid Score” refers to the ratio for each amino acid (mg / g protein) in the food ingredient or formulation on a reference pattern of amino acids (mg / g protein) according to the following equation 1 :Chemical amino acid score % = 100 x [(mg of amino acid in 1 g test protein) / (mg of amino acid in reference pattern)] Equation 1(World Health Organization, 1991).The “PDCAAS” is computed by correcting the Chemical Amino Acid Score of the indispensable amino acids by the protein fecal or ileal digestibility according to the following equation 2:PDCAAS % = weighted protein digestibility for the food formulation * chemical amino acid score. Equation 2The recommendations are to use appropriate and available fecal or ileal digestibility values and the reference requirements and scoring patterns for a reference adult of FAO / WHO (2013) report on dietary protein quality.

[0146] The term “sweetening agent” as used herein refers to a saccharide (e.g., a monosaccharide, a disaccharide, or a polysaccharide) or an artificial sweetener (e.g., a small molecule artificial sweetener or a protein artificial sweetener) that, when added to a composition, makes the composition taste sweet when ingested by a mammal, such as a human. Non-limiting examples of sweetening agents are described elsewhere herein. Additional examples of sweetening agents are known in the art.

[0147] The term “ash” is a common term known and understood in the art and represents one or more ions, elements, minerals, and / or compounds that can be found in a mammal-produced milk. Non-limiting ions, elements, minerals, and compounds that are found in mammal- produced milk are described elsewhere herein and also known in the art.

[0148] The term “color balancing agent” or “coloring agent” as used herein refers to an agent added to a composition to modulate the color of the composition, e.g., to make the color of the composition appear more similar to a mammal-produced milk. Non-limiting examples of color balancing agents or coloring agents include P-carotene and annatto. Other examples of coloring balancing agents are known in the art. A color balancing agent or a coloring agent can be produced by or obtained from a plant.

[0149] The term “flavor” as used herein refers to the taste and / or the aroma of a food or drink.

[0150] The term “texture agent” as used herein refers to any substance added to food products to modify their physical properties, particularly their texture and mouthfeel, without significantly affecting their flavor or nutritional value. Texture agents are widely used in the food industry to achieve desired textures, stabilize formulations, thicken liquids, and form gels in a variety of food products, contributing to the overall sensory experience of the food. It can be derived from numerous sources, such as for example plants, animals, and seaweeds.

[0151] The term “vitamin” as used herein refers to organic molecules (or a set of closely related molecules called vitamers) that are essential to an organism in small quantities for proper metabolic function.

[0152] The term “predicted molecular weight” or “theoretical molecular weight” are used interchangeably herein and refer to the molecular weight of a polypeptide that is calculated based on the atomic weight of each of the atoms in the molecule.

[0153] The term “substantially unglycosylated” as used herein refers to a polypeptide in which less than 10% of the glycosylation sites are glycosylated, i.e. covalently linked to a glycan molecule. Glycosylation adds additional molecular weight to a glycosylated protein, which is detectable using art standard techniques, such as, but not limited to, mass spectrometry. Accordingly, “substantially unglycosylated” may refer to an actual or measured molecular weight of a polypeptide that exhibits a mass shift that is less than about 1 kDa as compared to the predicted or theoretical molecular weight. For instance, a “substantially unglycosylated” polypeptide may have an actual molecular weight that is within about 0.05 to about 1 kDa compared to the predicted or theoretical molecular weight.

[0154] The term “micelle” as used herein refers to a generally (or roughly) spherical supramolecular structure that exists as a dispersion within a composition. In some embodiments, a “micelle” may have, e.g., a surface that is composed of a charged outer layer. In other embodiments, a “micelle” can encapsulate one or more biomolecules. For example, a “micelle” can encapsulate two or more proteins (e.g., an a-casein protein and a K-casein protein). In some embodiments, a “micelle” has a diameter of between about 10 nm and about 350 nm.

[0155] Pichia pastoris as used herein is also known as Komagataella phaffii. The use of Pichia pastoris herein is understood to include / synonymous with the name and organism Komagataella phaffii.Introduction

[0156] Beta-Lactoglobulin (BLG) is a whey protein present in many mammalians milk, and in particular in cow and sheep’s milk. Whey proteins have been recognized as a protein source and contain biologically active components potentially beneficial to the human health.

[0157] Beta-Lactoglobulin (BLG) is the major whey protein, accounting for more than 40% of the total whey protein. Bovine P -lactoglobulin (BLG) is a protein of 162 amino acid residueswith a molecular weight of 18.4 kDa. Because of its beneficial effects on human health, but also because of its physical properties and intrinsic characteristics, BLG is of direct interest in the food industry. Indeed, the amino acid content in BLG exceeds the Food and Agriculture nutritional intake recommendations both for children aged 2 to 5 and adults.

[0158] An alternative to the native whey proteins are recombinant whey proteins, which are currently used in dairy industry. In particular, recombinant beta-lactoglobulin (rBLG) is used in dairy products to increase the concentration of said beta-lactoglobulin (US 5,795,611, US 2019 / 0216106, US 9,924,728, WO2022 / 251263 and WG2022 / 239000). In addition, recombinant beta-lactoglobulin (rBLG) can be prepared from non-animal source and are consequently used as the sole source of milk proteins in clean-label non-animal dairy analogue products.

[0159] Optimal texture is required in animal-free dairy alternative such as animal-free yoghurts, fresh cheese, cheese, etc. However, BLG and recombinant BLG gels tend be elastic and can be very hard at high protein concentrations. Upon shearing, these gels yield a texture that is sandy / grainy instead of the desired smooth / creamy texture. Texture obtained with formulations containing no or reduced fat is watery. This inability to build optimal texture at conditions compatible with finish products processes limits the use of BLG and rBLG for several applications such as stirred yogurts, processed cheese, cheese or desserts, and in particular in non-animal dairy analogue products.

[0160] BLG gel texture can be tuned by addition of kappa casein (unmodified) or caseinomacropeptide (CMP, a bioactive peptide derived from K-casein by the action of chymosin during cheese manufacturing) to some extent. However, CMP is not able to modify BLG gel hardness by disrupting its thiol network. In case of intact kappa casein, two cysteines are present and a graft polymer thiol network of BLG and kappa casein is expected. Therefore, significant termination of the intermolecular BLG thiol network does not occur.

[0161] To the best of the inventors’ knowledge, there is no commercial product currently available with recombinant casein, e.g., recombinant K-casein, due to the low titers achieved during recombinant overexpression. Intact kappa caseins have low stability under microbial fermentation conditions due to high susceptibility to proteases and low solubility limit.

[0162] The inventors have identified truncated casein polypeptides (TCP) that gives improved properties in terms of nutritional values and food texture characteristics. Thus, truncated casein polypeptides (TCP) according to the invention provides interesting propertiesto dairy-products or dairy-analogue products made therefrom.

[0163] Further, without to be bound to any theory, it is believed that truncated casein polypeptide act as efficient BLG thiol polymerization terminators and chaperones to modulate BLG aggregation driven by non-covalent forces when their amino acid sequence comprises at least one cysteine.

[0164] The invention also provides methods to generate TCP (truncated casein polypeptide)- rBLG complex, e.g., truncated alpha Sl-casein polypeptide and rBLG complex or truncated kappa-casein polypeptide and rBLG (TKC-rBLG) complex, under appropriate conditions (protein concentrations, time / temperature for heat treatment, salt and pH) to enable formulations containing BLG with optimal texture for several applications such as dairy products, e.g., stirred yogurts, processed cheese and cheese, at the relevant product conditions such as >10% protein concentration and in the pH range of 4.6-7.0. The TCPs, e.g., TKCs, provided herein also show high levels, or high titer, of recombinant production when compared to intact casein, e.g., kappa-casein, due to high stability and solubility under microbial fermentation conditions. In some embodiments, TCP-rBLG, e.g., TKC-rBLG, formation occurs during product formulation without any heat treatment and provides the texture benefits. In some cases, TCPs, such as truncated alpha Sl-casein polypeptide or TKC, can provide optimal texture for the dairy application even in the absence of any BLG. For instance, in some embodiments, a truncated alpha Sl-casein polypeptide having the amino acid sequence represented by SEQ ID NO:62 or SEQ ID NO: 63 exhibits optimal texture even in the absence of BLG (i.e., a truncated alpha Sl-casein alone). In some embodiments, a TKC having the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:29 exhibits optimal texture even in the absence of BLG. In some embodiments, an unglycosylated or substantially unglycosylated TCP or TKC exhibits optimal texture even in the absence of BLG (e.g., SEQ ID NO:29).

[0165] Apart from texture, truncated casein polypeptides (TCP) of the invention, provided herein contribute to favorable taste and flavor profile in dairy product and dairy analogue product applications, e.g., cheese applications.

[0166] According to the invention, truncated casein polypeptides (TCP) are preferably selected from truncated alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta- casein (β-casein), or kappa-casein (K-casein) polypeptides. In other words, the casein polypeptide according to the invention is truncated relative to a full length alpha Sl-casein(aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide.

[0167] From a nutritional standpoint, casein, and in particular alpha Sl-casein (aSl-casein) and kappa-casein (K-casein), has a complete essential amino acid profile that is different from whey proteins, high digestibility and contains precursors for bioactive peptides such as casoparan and casoxicidin. Thus, in some embodiments, the truncated casein polypeptide (TCP) is preferably a truncated alpha Sl-casein (aS 1 -casein) or a truncated kappa-casein (K- casein) polypeptide (TCK).

[0168] In embodiments, the invention provides a composition comprising (i) a truncated casein polypeptide (TCP) relative to a full length alpha S 1-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide; and optionally (ii) a recombinant beta-lactoglobulin (rBLG) polypeptide.

[0169] It may be desirable for a human diet to comprise a dietary protein source which is of high quality. The human body typically is not able to synthesize some amino acids necessary for health and growth, such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Dietary proteins that provide all essential amino acids are referred to as high quality proteins, or complete proteins. Foods that have higher protein quality are considered more beneficial in a mammalian diet compared to proteins that do not. Complete proteins promote maintenance of muscle mass, a healthy body mass index, and glycemic balance. Additionally, by increasing the amount of complete protein in the diet, the total amount of protein consumption may be decreased compared to a diet in which low quality proteins are primarily consumed. Therefore, in some embodiments, the truncated casein polypeptides, and in particular truncated alpha Sl-casein (aS 1-casein) and truncated kappa-casein (K-casein) polypeptide (TCK) according to the invention are selected for use in the production of food products, dairy products, or dairy -like products that are high in protein and have amino acid compositions that meet daily requirements for essential amino acids, for general nutrition.

[0170] In some embodiments, the truncated casein polypeptides (TCP) and the truncated kappa-casein polypeptides (TKC) of the disclosure are selected for use in the production of food products, dairy products, or dairy-like products which satisfy the protein needs for those requiring specialized nutrition, including, but not limited to, the nutritional needs of ill or elderly individuals or individuals with certain conditions such as pregnancy,short-term medical needs such as hospitalization, or long-term medical needs such as chronic conditions like diabetes or metabolic disorders.

[0171] Dietary products that are enriched with protein, either for general nutrition or specialized nutrition, are typically enriched with proteins that are relatively easy to access and low-cost to purify. Such proteins include whey proteins, casein, and albumin. Additionally, individuals with metabolic disorders frequently require the exclusion of at least one essential amino acid from their diets and high enrichment of other amino acids. Some dietary products comprise free amino acids which are added at amounts to provide a complete nutritional amino acid profile. In some embodiments, free amino acids may have an extremely bitter taste which is not completely masked by the addition of other ingredients, such as sugars and / or flavorants. Therefore, in some embodiments, these dietary products are not desirable to those who must consume them. Accordingly, in embodiments, the truncated casein polypeptides (TCP) and the truncated kappa-casein polypeptides (TKC) of the disclosure are selected for producing food products, dairy products, or dairy-like products that are tailored to provide a desired nutritional profile and / or that also are provided in a cost-effective manner.Truncated Casein Polypeptide (TCP)

[0172] The invention concerns a composition comprising a truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof.

[0173] It is understood that truncation of amino acid sequence corresponds to an irreversible modification which consists in shortening the sequence by removing a portion of said sequence.

[0174] In one preferred embodiment, the truncated casein polypeptide (TCP) according to the invention is a truncated alpha (a) casein, preferably selected from alpha S 1-casein (aS 1-casein) and alpha S2-casein (aS2-casein), a truncated beta-casein (β-casein), or a truncated kappa- casein (K-casein).

[0175] In another preferred embodiment, the truncated casein polypeptide (TCP) is truncated relative to SEQ ID NOs: 1 and 51 to 61 as defined hereafter, i.e. the truncated casein polypeptide (TCP) is truncated relative to sequences selected from SEQ ID NOs: 1, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 and 61. Therefore, in some preferred embodiments, the invention provides a casein polypeptide truncated relative to a full-length alpha Sl-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein), andpreferably relative to a protein comprising SEQ ID NOs: 1 or 51-61. Preferably, the truncated casein polypeptide (TCP) is truncated relative to sequences selected from SEQ ID NOs: 1, 52, 53 and 54.

[0176] Preferably, the truncated casein polypeptide (TCP) is truncated at the C-terminus, the N-terminus, or a combination of both. For instance, in some preferred embodiments, the casein polypeptide is truncated at the N-terminus. In other preferred embodiments, the casein polypeptide is truncated at the C-terminus. Still, in other preferred embodiments, the casein polypeptide is truncated at both the N-terminus and the C-terminus. Preferably, the truncated casein polypeptide (TCP) is a truncated alpha Sl-casein (aSl-casein) polypeptide, which is truncated at the N-terminus, the C-terminus, or a combination of both, or a truncated kappa- casein (K-casein) polypeptide, which is truncated at the N-terminus, the C-terminus, or a combination of both.

[0177] The truncated casein polypeptide (TCP) is preferably truncated by 10 to 150 amino acids and more preferably by 10 to 100 amino acids relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide. In other preferred embodiments, the truncated casein polypeptide (TCP) is truncated by 10 to 90 amino acids, or by 10 to 80 amino acids, or by 10 to 70 amino acids, or by 10 to 60 amino acids or even by 10 to 50 amino acids relative to a full length alpha Sl- casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K- casein) polypeptide. Still in other embodiments, the truncated casein polypeptide (TCP) is truncated by 15 to 45 amino acids, preferably by 20 to40 amino acids, more preferably by 25 to 35 amino acids, or by 25 to 30 amino acids relative to a full length alpha Sl-casein (aSl- casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide.

[0178] In some preferred embodiments, the truncated casein polypeptide (TCP) comprises an amino acid sequence comprising between 28 to 199 amino acids. Preferably, the truncated casein polypeptide (TCP) comprises between 56 and 161 amino acids or between 49 and 180 amino acids.

[0179] In one particular and preferred embodiment, the truncated casein polypeptide (TCP) is: a truncated kappa-casein polypeptide (TKC) comprising an amino acid sequence having between 56 and 161 amino acids ora truncated alpha SI -casein polypeptide comprising an amino acid sequence having between 49 and 180 amino acids, or a truncated alpha S2-casein polypeptide comprising an amino acid sequence having between 39 and 199 amino acids or a truncated beta casein polypeptide comprising an amino acid sequence having between 28 and 188 amino acids.

[0180] Preferably, the truncated casein polypeptide (TCP) comprises: a truncated kappa-casein polypeptide (TKC) comprising between 56 and 161 amino acids, and preferably selected from SEQ ID NOs: 5, 27, 29 and 118; or a truncated alpha SI -casein polypeptide comprising between 49 and 180 amino acids, and preferably selected from SEQ ID NOs: 62, 63, 116 and 117, or a truncated beta casein polypeptide comprising between 28 and 188 amino acids, and preferably selected from SEQ ID NOs: 88 and 95, or a truncated alpha S2-casein polypeptide comprising between 39 and 199 amino acids, and preferably selected from SEQ ID NOs: 84 and 111, or a truncated casein polypeptide (TCP) comprising between 64 and 160 amino acids, preferably between 64 and 140 amino acids, preferably between 64 and 115 amino acids, preferably between 64 and 110 amino acids, or preferably between 70 and 115 amino acids, or preferably between 80 and 160 amino acids, preferably between 80 and 115 amino acids, preferably between 80 and 110 amino acids, preferably between 80 and 100 amino acids, or preferably between 100 and 140 amino acids, and wherein the truncated casein polypeptide is preferably selected from SEQ ID NOs : 5, 27, 29, 62, 63, 116, 117 and 118. .

[0181] Still, in other prefered embodiments, the truncated casein polypeptide (TCP) comprises 104 amino acids, 115 amino acids, 125 amino acids, 130 amino acids, 137 amino acids, 139 amino acids, 93 amino acids, 120 amino acids, 122 amino acids, 70 amino acids, or 106 amino acids.

[0182] In some preferred embodiments, the truncated casein polypeptide (TCP) contains at least one cysteine. It is believed that cysteine can bound another cysteine, thus leading to the formation of graft polymer thiol network. When present, the cysteine can be a free or modified cysteine. In one preferred embodiment, the truncated casein polypeptide (TCP) comprising at least one cysteine is a truncated kappa-casein (K-casein) polypeptide. In another preferred embodiment, the truncated casein polypeptide (TCP) comprising at least one cysteine is atruncated alpha SI -casein (aS 1 -casein) polypeptide. Still in other preferred embodiments, the truncated casein polypeptide (TCP) according to the invention, and preferably selected from truncated alpha Sl-casein (aSl-casein) polypeptide and kappa-casein (K-casein) polypeptide, comprises at least one additional modification resulting in the insertion of one more cysteine into the truncated polypeptide.

[0183] According to the invention, the truncated casein polypeptides (TCP) of the invention; e.g. comprising the sequences disclosed herein, can further comprise at least one additional modification, said modification being selected from substitution, deletion, truncation, insertion, phosphorylation, glycosylation, or a combination thereof. Examples of additional modifications are variants and substitutions such as phenotypically silent variants, tolerated substitutions, conservative substitutions, semi-conservative substitutions, and the like. Substitutions can be natural or artificial substitutions. Preferably, substitutions increase nutrition score, improve texture potential, or any combination thereof.

[0184] In some embodiments and when additional modifications exist, the truncated casein polypeptide (TCP) comprises about one to about 20 additional modifications, preferably about one to about ten additional modifications. It is understood that the additional modifications are relative to the full-length casein polypeptide from which it was derived, such as through recombinant DNA technologies.

[0185] In some preferred embodiments, truncated casein polypeptides (TCP) may be non- glycosylated or substantially non-glycosylated. Glycosylated amino acid residues are covalently linked to a N-glycan or O-glycan molecules, such as, but not limited to N- acetylglucosamine, galactose, galactosamine, glucose, glucosamine, N-acetylglucosamine, mannose, N-acetylmannosamine, mannosamine, xylose, N-acetylneuraminic acid, N- glycolylneuraminic acid, 2-keto-3deosynononic acid, fucose, glucuronic acid, iduronic acid, galacturonic acid, and mannuronic acid.

[0186] As one having ordinary skill in the art will readily appreciate, the amount of glycosylation of a polypeptide can be measured using techniques and equipment readily available in the art, such as deconvoluted mass spectrometry. In some embodiments, glycosylation adds between about 2 kDa to about 4 kDa to a polypeptide. Further, the amino acid sequence of a particular polypeptide can be used to provide a predicted or theoretical molecular weight of that polypeptide. Therefore, the skilled artisan can compare the deconvoluted mass spectrometry data to determine if the actual molecular weight of thepolypeptide is greater than the predicted or theoretical molecular weight to determine the amount of glycosylation.

[0187] The amino acid residues themselves can be used to make predictions and / or identify likely glycosylation sites. According to some embodiments of the invention, O-linked glycans are attached to the hydroxyl oxygen of serine, threonine, hydroxylysine, or hydroxyproline side chains, and / or N-linked glycans are attached to the nitrogen of asparagine or arginine side chains, e.g., at Asn-X-Ser / Thr sequons or at Ans-X-Cys sequons.

[0188] In some embodiments, the truncated casein polypeptide (TCP) is preferably non- glycosylated, or substantially non-glycosylated. When referring to “substantially non- glycosylated” TCP, it is understood that the TCP is less than about 20% glycosylated. Preferably, the non-glycosylated TCP is less than about 15% glycosylated, more preferably less than about 10% glycosylated, and even more preferably less than about 5% glycosylated.

[0189] According to some preferred embodiments the non-glycosylated or substantially non- glycosylated truncated casein polypeptide (TCP) is selected from truncated alpha Sl-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein casein (β-casein), and kappa-casein (K- casein) polypeptide. More preferably, the non-glycosylated or substantially non-glycosylated truncated casein polypeptide (TCP) is selected from truncated alpha Sl-casein (aS 1-casein) and truncated kappa-casein (K-casein) polypeptide. According to one preferred embodiment, the substantially non-glycosylated truncated casein polypeptide (TCP) is a truncated kappa- casein (K-casein) polypeptide corresponding to SEQ ID NO: 29, said sequence being preferably produced from Bacillus subtilis.

[0190] According to the invention, and in preferred embodiments, the difference between the actual molecular weight and the theoretical molecular weight indicates that the majority of glycosylation sites are not glycosylated (i.e., not covalently linked to a glycan molecule). Such “substantially non-glycosylated” or “substantially unglycosylated” polypeptide may have an actual molecular weight that is within about 1 kDa compared to the predicted or theoretical molecular weight, preferably an actual molecular weight that is within about 0.5 kDa compared to the predicted or theoretical molecular weight, or an actual molecular weight that is the same as the predicted or theoretical molecular weight.

[0191] In other preferred embodiments, the truncated casein polypeptide (TCP), said truncated casein polypeptide being preferably selected from truncated alpha Sl-casein (aSl- casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein), is notphosphorylated, or comprises less than about 10% of phosphorylation sites, or even less than about 5% of phosphorylation sites. Polypeptide phosphorylation is a post-translational modification wherein phosphate groups are covalently linked to certain amino acid residues, such as serine, threonine, tyrosine and / or histidine amino acid residues. The phosphorylation of a polypeptide can be characterized using a variety of art-standard techniques, including, but not limited to antibody detection (e.g., P-Serine antibodies), 2-dimensional gel analysis, SDS page gels, mass spectrometry, and the like. According to the invention, and in some preferred embodiments, the truncated casein polypeptide (TCP) does not include any P-Serine (Phosphorylated serine) sites. According to some preferred embodiments, the truncated casein polypeptide (TCP) is not phosphorylated, and is selected from truncated alpha S 1 -casein (aS 1- casein) polypeptide. More preferably, the non-phosphorylated truncated casein polypeptide (TCP) is selected from SEQ ID NOs: 62 and 63, and said sequence being preferably produced from Aspergillus Oryzae.

[0192] Still in other preferred embodiments, the truncated casein polypeptide (TCP) does not comprise a phenylalanine amino acid residue in its polypeptide. According to some preferred embodiments, the truncated casein polypeptides (TCP) that do not comprise phenylalanine residue are truncated alpha SI -casein (aSl-casein) polypeptides. Examples of such sequences, i.e. that do not comprise phenylalanine residue are SEQ ID NOs: 63, 116, 117, or 118.

[0193] According to the invention, the truncated casein polypeptide (TCP), i.e. the truncated alpha Sl-casein (aSl-casein), the alpha 2-casein (aS2-casein), the beta-casein (β-casein), or the kappa-casein (K-casein) is preferably produced from a casein polypeptide found in milk of cow, human, sheep, goat, buffalo, bison, horse, yak, reindeer, donkey, moose, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, rabbit, whale, baboon, gibbons, orangutang, mandrill, pig, wolf, fox, lion, tiger, echidna, or camel. Preferably, the TCP is truncated relative to a casein produced in milk from cow, buffalo, goat, sheep, human, camel, donkey, or horse. In other words, the SEQ ID NOs: 1 and 51 to 61 from which the truncated casein polypeptide (TCP) according to the invention is preferably produced, is a casein produced in milk from cow, buffalo, goat, sheep, human, camel, donkey, or horse.

[0194] In some preferred embodiments, the truncated casein polypeptide (TCP) is a truncated alpha Sl-casein (aSl-casein) polypeptide, which is truncated at the N-terminus, C-terminus or both and the polypeptide comprises 49 to 180 amino acids. In some preferred embodiments, the truncated casein polypeptide (TCP) is a truncated alpha S 1 -casein (aS 1 -casein) polypeptidecomprising at least 90% sequence identity to SEQ ID NOs: 62, 63, 116, or 117. Preferably, the truncated alpha Sl-casein (aSl-casein) polypeptide comprises at least 95%, 96%, 97%, 98% or even 99% sequence identity to SEQ ID NOs: 62, 63, 116, or 117. Yet, in preferred embodiment, the truncated alpha Sl-casein (aS 1 -casein) polypeptide is selected from SEQ ID NOs: 62, 63, 116, and 117, preferably from SEQ ID NOs: 62 and 63.

[0195] In some preferred embodiments, the truncated casein polypeptide (TCP) is a truncated alpha S2-casein (aS2-casein) polypeptide, which is truncated at the N-terminus, C-terminus or both and the polypeptide comprises 39 to 199 amino acids. Preferably, the truncated alpha S2- casein polypeptide is selected among SEQ ID NOs: 84 and 111.

[0196] In some preferred embodiments, the truncated casein polypeptide (TCP) is a truncated beta-casein comprising 28 to 188 amino acids. Preferably the truncated beta-casein polypeptide is selected among SEQ ID NOs: 88 and 95.

[0197] In other preferred embodiments, the truncated casein polypeptide (TCP) is a truncated kappa-casein polypeptide (TKC) and comprises 56 to 161 amino acids, or 64 to 160 amino acids but preferably does not comprise SEQ ID NO: 17. Preferably, the truncated kappa-casein polypeptide (TKC) is selected from SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 and 118, more preferably from SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33 and 118, even more preferably from 5, 27, 29 and 118.

[0198] In preferred embodiments, the truncated casein polypeptide (TCP), preferably selected from truncated alpha S 1-casein (aS 1-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein casein (β-casein), and truncated kappa-casein (K-casein), corresponds to polypeptides comprising at least 90% sequence identity to SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, and 118. Preferably, the truncated casein polypeptide (TCP), preferably selected from truncated alpha Sl-casein (aS 1-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein), and truncated kappa-casein (K-casein), comprises at least 95%, 96%, 97%, 98% or even 99% sequence identity to SEQ ID NOs :5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, and 118. Yet, in preferred embodiment, the truncated casein polypeptide (TCP), preferably selected from truncated alpha Sl-casein (aSl- casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein) and truncated kappa-casein (K-casein), is selected from SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, and 118, and more preferably from SEQ ID NOs: 5, 27, 29, 62, 63, 116, 117 and 118.

[0199] Still in other preferred embodiments, the truncated casein polypeptide (TCP) according to the invention has the amino acid sequence represented by SEQ ID NO:5, SEQ ID NO:27, SEQ ID NO:29, (cow truncated kappa casein polypeptide), SEQ ID NO: 62, SEQ ID NO: 63 (cow truncated alpha Sl-casein polypeptide), SEQ ID NOs: 116 (cow truncated alpha S 1-casein polypeptide), SEQ ID NO: 117 (cow truncated alpha S 1-casein polypeptide), or SEQ ID NO: 118 (cow truncated kappa casein polypeptide).

[0200] In other preferred embodiments, the truncated casein polypeptide (TCP) according to the invention has an amino acid sequence with at least 90% sequence identity to SEQ ID NOs: 5, 27, 29, 62, or 63, and more preferably at least 95% sequence identity to SEQ ID NOs: 5, 27, 29, 62, or 63.

[0201] Overall and preferably, the truncated casein polypeptide (TCP) according to the invention, and preferably selected from truncated alpha S 1-casein (aS 1-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein), and truncated kappa-casein (K- casein), includes the amino acid sequence represented by SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118. In some preferred embodiments, the TCP or truncated kappa-casein (K-casein) (TKC) of the disclosure includes an amino acid sequence with at least 90% sequence identity to SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118, and preferably at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118.

[0202] Preferably, the truncated casein polypeptides (TCP) of the invention, e.g., comprising the sequences disclosed herein, include variants and substitutions such as phenotypically silent variants, tolerated substitutions, conservative substitutions, semi-conservative substitutions, and the like.

[0203] In some embodiments, the TCP of the invention is non-naturally occurring. For example, the TCP is additionally modified relative to a full-length casein, such as through recombinant DNA technologies. In embodiments, the non-naturally occurring TCP of the invention have markedly different properties than casein polypeptides found in nature. In some embodiments, the modification occurs naturally.

[0204] In some embodiments, the TCP of the invention is naturally occurring.

[0205] In embodiments, the TCP of the invention comprises an amino acid substitution, deletion, insertion, phosphorylation, glycosylation, or a combination thereof or relative to awild-type or native casein protein. The truncation may be a truncation found in nature or an engineered truncation.

[0206] In some embodiments, the TCP according to the disclosure, and preferably selected from truncated alpha S 1-casein (aS 1-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein), and truncated kappa-casein (K-casein) comprises an amino acid sequence of any one of SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118 or a fragment or variant thereof with at least 90% sequence identity to SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33,40, 62, 63, 116, 117, or 118.

[0207] Further to the casein polypeptide truncated (TCP) relative to a full length alpha Sl- casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K- casein) as defined herein, the composition according to the invention can comprise at least one additional polypeptide, wherein the at least one additional polypeptide is selected from a beta- casein, an alpha lactalbumin, a kappa casein, an alpha S 1-casein, an alpha S2-casein, a lactoferrin, a transferrin, a beta-lactoglobulin, and a serum albumin.

[0208] Preferably, the additional polypeptide is a truncated polypeptide, said truncated polypeptide being preferably truncated at the C-terminus, the N-terminus, or both, relative to a full-length beta-casein, alpha lactalbumin, kappa casein, alpha S 1-casein, alpha S2-casein, lactoferrin, transferrin, beta-lactoglobulin, or serum albumin.

[0209] According to the invention, it is understood that the composition comprising a casein polypeptide truncated (TCP) relative to a full length alpha S 1-casein (aS 1-casein), alpha S2- casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) as defined herein, can comprise one or more truncated casein polypeptide, which can be named “additional polypeptide” and as defined above. Accordingly, the composition can comprise a blend of different TCP with similar or different sequences (mixture of alpha casein S I, alpha S2 casein, beta casein, kappa casein, or any combination thereof), or a blend of sequences coding for different types of truncated caseins or for the same type of truncated casein, or a blend of sequences from different species, or any combination thereof.

[0210] In some preferred embodiments, the composition may include truncated caseins and animal-derived or non-animal-derived caseins, said caseins being different from truncated casein polypeptide according to the invention. The animal-derived or non-animal derived caseins can be full-length caseins, or modified / truncated / trimmed, etc. relative to full-length caseins.

[0211] In other preferred embodiments, truncated casein polypeptide (TCP) may have undergone particular treatment (e.g., physical or chemical) which allows them to be functionalized, for example. It is also understood that the composition according the invention can comprise functionalized TCP or non-functionalized TCP or a mixture thereof. Thus, according to the disclosure, an object of the invention is a composition comprising a truncated casein polypeptide (TCP) relative to a full length alpha S 1-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof, said truncated casein polypeptide (TCP) being as defined herein.

[0212] According to some preferred embodiments, the composition comprises a truncated casein polypeptide (TCP), wherein the truncated casein polypeptide is truncated relative to a protein comprising SEQ ID NOs: 1 or 51-61, and preferably relative to protein sequences selected from SEQ ID NOs: 1, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 and 61.

[0213] According to some preferred embodiments, the composition comprises a truncated casein polypeptide (TCP), said truncated casein polypeptide being preferably selected from truncated alpha S 1-casein (aS 1-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein), and truncated kappa-casein (K-casein) polypeptides.

[0214] Preferably, the composition comprises a truncated casein polypeptide (TCP), said truncated casein polypeptide comprising: a truncated kappa-casein polypeptide (TKC) comprising between 56 and 161 amino acids, and preferably selected from SEQ ID NOs: 5, 27, 29 and 118; or a truncated alpha SI -casein polypeptide comprising between 49 and 180 amino acids, and preferably selected from SEQ ID NOs: 62, 63, 116 and 117, or a truncated beta casein polypeptide comprising between 28 and 188 amino acids, and preferably selected from SEQ ID NOs: 88 and 95, or a truncated alpha S2-casein polypeptide comprising between 39 and 199 amino acids, and preferably selected from SEQ ID NOs: 84 and 111, or a truncated casein polypeptide (TCP) comprising between 64 and 160 amino acids, preferably between 64 and 140 amino acids, preferably between 64 and 115 amino acids, preferably between 64 and 110 amino acids, or preferably between 70 and 115 amino acids, or preferably between 80 and 160 amino acids, preferably between 80 and 115 amino acids, preferably between 80 and 110 amino acids, preferably between 80 and 100 amino acids, or preferably between 100 and 140 amino acids, and wherein the truncated casein polypeptide is preferably selected from SEQ ID NOs : 5, 27, 29, 62,63, 116, 117 and 118.

[0215] In some preferred embodiments, the composition comprises a truncated casein polypeptide (TCP), preferably selected from a truncated alpha Sl-casein (asl-casein), a truncated alpha S2-casein (as2-casein), a truncated beta-casein (β-casein), and a truncated kappa-casein (K-casein) polypeptide, and said truncated casein polypeptide comprises at least one cysteine. In this case, the truncated casein polypeptide (TCP) is preferably a truncated alpha Sl-casein (asl-casein) or a a truncated kappa-casein (K-casein)

[0216] In some preferred embodiments, the composition comprises a truncated casein polypeptide (TCP) which is not glycosylated or which is not substantially glycosylated. Preferably, the composition comprises a non-glycosylated or substantially non glycosylated truncated alpha Sl-casein (aSl-casein) or truncated kappa-casein (K-casein) polypeptide, and more preferably, the substantially non-glycosylated truncated casein polypeptide (TCP) is a truncated kappa-casein (K-casein) polypeptide, preferably corresponding to SEQ ID NO: 29, and said sequence being preferably produced from Bacillus subtilis.

[0217] In other preferred embodiments, the composition comprises a truncated casein polypeptide (TCP), preferably selected from truncated alpha S 1-casein (aS 1-casein), alpha S2- casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein), and the truncated casein polypeptide (TCP) is not phosphorylated, or comprises less than about 10% of phosphorylation sites, or even less than about 5% of phosphorylation sites. Preferably, the composition comprises a non-phosphorylated truncated casein polypeptide (TCP) which is preferably a truncated alpha Sl-casein (aS 1-casein) polypeptide. More preferably, the composition comprises a non-phosphorylated truncated casein polypeptide (TCP) selected from SEQ ID NOs: 62 and 63, and said sequence being preferably produced from Aspergillus Oryzae.

[0218] Still in other preferred embodiments, the composition comprises a truncated casein polypeptide (TCP), preferably selected from truncated alpha S 1-casein (aS 1-casein), alpha S2- casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein), said truncated casein polypeptide does not comprise a phenylalanine amino acid residue in its polypeptide. According to some preferred embodiments, the truncated casein polypeptides (TCP) that do not comprise phenylalanine residue are truncated alpha Sl-casein (aS 1-casein) polypeptides, such as SEQ ID NOs: 63, 116, 117, or 118.Recombinant beta-lactoglobulin (rBLG)

[0219] Another object of the invention is a composition comprising a truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (asl-casein), alpha S2-casein (as2- casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof, and at least one recombinant beta-lactoglobulin (rBLG) polypeptide.

[0220] Recombinant beta-lactoglobulin (rBLG) can be obtained commercially, or can be prepared by methods known in the art, such as through fermentation, including precision fermentation. (See, e.g., Nielsen et al, Annual Review of Food Science and Technology 2024 15: 1, Geistlinger et al., Chapter 21 - Case study on whey protein from fermentation, Cellular Agriculture, Academic Press, 2024, pp 323-342.) For purposes of providing a dairy-free composition comprising rBLG, a non-animal source can be used.

[0221] Recombinant beta-lactoglobulin (rBLG) can be native rBLG (bovine sequence expressed in a recombinant host for instance), or it can be functional, meaning that a specific treatment has been applied to the native rBLG, thereby bringing specific functions to the recombinant protein. Examples of functionalized rBLG are disclosed in PCT / EP2024 / 077629 and PCT / EP2024 / 077614, wherein mention is made of microp articulated rBLG and cold- gellable rBLG respectively.

[0222] In some preferred embodiments, the composition comprises a truncated casein polypeptide (TCP), preferably selected from alpha Sl-casein (aSl-casein), alpha 2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein), as defined above and one or more recombinant beta-lactoglobulin (rBLG). In preferred embodiment, said rBLG is functionalized rBLG, preferably it is a cold-gellable rBLG or a microparticulated rBLG. However, rBLG can also be native rBLG in compositions according to the invention. According to preferred embodiments, the truncated casein polypeptide (TCP) is a truncated alpha Sl-casein (asl-casein) polypeptide or a truncated kappa-casein (K-casein) polypeptide (TKC).

[0223] Preferably, the composition comprising a truncated casein polypeptide (TCP), preferably a truncated kappa-casein polypeptide (TKC), comprises a blend of recombinant beta-lactoglobulin (rBLG). It is understood that the blend of rBLG can comprise similar or different rBLG sequences. In some preferred embodiments, the blend of rBLG comprises native rBLG and functionalized rBLG. In other preferred embodiments, the blend comprises modified, truncated, trimmed, etc. rBLG. rBLG sequences in the blend can be obtained from same or different species.

[0224] In some preferred embodiments, the compositions according to the invention comprises a truncated kappa-casein polypeptide (TKC) comprising between 56 and 161 amino acids in length and a recombinant beta-lactoglobulin (rBLG). Preferably, the TKC is selected from SEQ ID NO:5, SEQ ID NO:27, SEQ ID NO: 29 and SEQ ID NO: 118, and more preferably, the TKC is selected from SEQ ID NO: 5, SEQ ID NO: 29 and SEQ ID NO: 118.

[0225] In other preferred embodiments, the compositions according to the invention comprises a truncated alpha SI -casein (aS 1 -casein) polypeptide comprising between 49 and 180 amino acids in length and a recombinant beta-lactoglobulin (rBLG). Preferably, the truncated alpha SI -casein (aSl-casein) polypeptide is selected from SEQ ID NO: 62 and SEQ ID NO: 63.

[0226] Still in other preferred embodiments, the compositions according to the invention comprises a truncated alpha S2-casein (aS2-casein) polypeptide comprising between 39 and 199 amino acids in length and rBLG. Preferably, the truncated alpha S2-casein (aS2-casein) polypeptide is selected from SEQ ID NO: 84 and SEQ ID NO: 111.

[0227] In other preferred embodiments, the compositions according to the invention comprises a truncated beta casein (β-casein) polypeptide comprising between 28 and 188 amino acids in length and rBLG. Preferably, the truncated beta-casein (β-casein) polypeptide is selected from SEQ ID NO: 88 and SEQ ID NO: 95.

[0228] Preferably, the truncated casein polypeptide (TCP) according to the compositions of the present invention, and comprising recombinant beta-lactoglobulin (rBLG), are selected from amino acid sequence SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 84, 88, 95, 111, 116, 117, and 118. More preferably, the TCP are selected from amino acid sequence SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, and 118, and even more preferably from SEQ ID NOs: 5, 27, 29, 62 and 63.

[0229] According to other preferred embodiments, the composition according to the invention comprises: (i) a non-naturally occurring kappa-casein (K-casein) polypeptide truncated at the N-terminus, the C-terminus, or a combination of both, (“TKC”) relative to a full-length kappa-casein polypeptide; and (ii) a recombinant beta-lactoglobulin polypeptide, wherein the TKC comprises an amino acid sequence between 64 and 160 amino acids; and wherein the TKC preferably does not comprise SEQ ID NO: 17. Preferably, the TKC is truncated at the N-terminus relative to a full-length kappa-casein (K-casein) polypeptide, or preferably, the TKC is truncated at the C-terminus relative to a full-length kappa-caseinpolypeptide, or preferably the TKC is truncated at both the N-terminus and the C-terminus relative to a full-length kappa-casein polypeptide.

[0230] Still according to other preferred embodiments, the composition according to the invention comprises: (i) a kappa-casein (K-casein) polypeptide truncated at the N-terminus, the C-terminus, or a combination of both, (TKC) relative to a full-length kappa-casein polypeptide; and (ii) a recombinant beta-lactoglobulin polypeptide, wherein the TKC comprises an amino acid sequence between 56 and 161 amino acids; and wherein the TKC preferably does not comprise SEQ ID NO: 17.

[0231] In some preferred embodiments, the composition according to the invention and comprising a truncated casein polypeptide (TCP) relative to a full length alpha SI -casein (asl- casein), alpha S2-casein (as2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide or a mixture thereof, and at least one recombinant beta-lactoglobulin (rBLG) polypeptide, comprises from 1 to 10 wt% of TCP and rBLG, wt% being expressed in relation to the total weight of the composition. Preferably, the composition comprises TCP, as defined herein, and rBLG at a content ranging from 2 to 8 wt%, preferably from 2.5 to 7.5 wt%, and more preferably from 3 to 5 wt%. Alternatively, the content of truncated casein polypeptide (TCP), preferably selected from alpha Sl-casein (asl-casein), alpha S2-casein (as2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide, and recombinant beta- lactoglobulin (rBLG) can be expressed in weight ratio. In this case, the TCP / rBLG weigh ratio ranges from 1 / 10 to 5 / 1, preferably from 1 / 5 to 3 / 1, preferably from 1 / 2 to 2 / 1, or from 3 / 2 to 1.Dairy products and Dairy-like products

[0232] The present invention also relates to dairy products and dairy-like products that have the texture and sensation of dairy. In some embodiments, the dairy products and dairy-like products are free from milk produced by an animal, and overall free of animal -derived components. These products may also be termed non-animal dairy analogs. In other embodiments, the dairy products and dairy-like products can contain animal-derived components and may be referred to herein as “dairy products”. It is also worth to note that dairy-products and dairy-like products can contain both non-animal derived and animal- derived components.

[0233] Dairy-products and dairy-like products according to the invention comprise a casein polypeptide truncated (TCP) relative to a full length aSl-casein (alpha Sl-casein), aS2-casein(alpha S2-casein), β-casein (beta-casein), or K-casein (kappa-casein) polypeptide as defined above or a mixture thereof.

[0234] In some embodiments, dairy-products and dairy-like products according to the invention comprises a truncated casein polypeptide (TCP) relative to a full length alpha Sl- casein (asl-casein), alpha S2-casein (as2-casein), beta-casein (β-casein), or kappa-casein (K- casein) polypeptide as defined above or a mixture thereof and one or more recombinant beta- lactoglobulin (rBLG) as defined above.

[0235] It is understood that the dairy products and dairy-like products according to the invention can comprise a composition as defined herein, i.e. a composition according to the invention and comprising one or more truncated casein polypeptide (TCP) relative to a full length alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein). It is also understood that additional components such as recombinant beta-lactoglobulin (rBLG), or additional polypeptides as defined herein can be present into the composition, and therefore into the dairy -products or dairy-like products.

[0236] Advantageously, dairy products or dairy-like products according to the invention have one or more characteristics of a classic dairy food product, such as taste, aroma, appearance, handling, mouthfeel, density, structure, texture, elasticity, springiness, coagulation, binding, leavening, aeration, foaming, creaminess and emulsification. Advantageously, the dairy product or dairy-like products according to the invention have improved or modified properties as compared to a classic dairy product, the properties being preferably selected from the viscosity, the foaming effect, the buffering effect, storage time, opacity, and smell. According to the invention, “classic dairy food products” refer to dairy products produced from animal milk and preferably commercially available.

[0237] Dairy products and dairy-like products of the invention, i.e. comprising a truncated casein polypeptide (TCP) relative to a full length alpha S 1-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide as defined above or a mixture thereof and optionally one or more recombinant BLG (rBLG) as defined above may include additional components comprising a protein, texture agent, lipid, flavor compound, sweetening agent, color balancing agent, an ash, a vitamin, or any combination thereof. The additional components can be animal-derived or non-animal-derived. In some preferred embodiments, the dairy-products or dairy like-products according to the invention comprise at least one additional component, said at least one additional component being ofanimal-derived or non-animal derived. In other preferred embodiments, the dairy -products or dairy like-products according to the invention comprise one or more additional components, said additional components being of animal-derived or non-animal derived or a combination thereof.

[0238] The texture agent includes one or more of Maltodextrin, native starches, modified starches, cellulose derivatives (e.g., microcrystalline cellulose), carrageenans, xanthan gum, guar gum, locust bean gum, acacia gum, agar-agar, gelatin, pectin, alginate, plant proteins (e.g., soy proteins, pea proteins).

[0239] When present, lipids are selected from the group consisting of sunflower oil, coconut oil, tributyrin, mono- and di-glycerides, free fatty acids, and phospholipids. According to the invention, the free fatty acids comprise at least one fatty acid selected from the group of butyric acid, caproic acid, caprylic acid, and capric acid, and the phospholipids are selected from soy lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, or rapeseed lecithin phospholipids. Preferably, the monoglycerides and diglycerides are plant- derived monoglycerides and diglycerides, or are bacteria-derived monoglycerides and diglycerides.

[0240] In some embodiments, the compositions according to the invention include ash. In these embodiments, the ash may include one or more of calcium, phosphorus, potassium, sodium, citrate, and chloride.

[0241] Ash can further include minerals, which are selected from sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc, chloride, manganese, selenium, iodine, retinol, carotene, vitamins, vitamin D, vitamin E, vitamin B 12, thiamin and riboflavin. In some embodiments, the ash includes anions, such as phosphate, citrate, sulfate, carbonate, and chloride.

[0242] As used in the composition according to the invention, the flavor compounds include at least one flavor compound selected from the group consisting of: 5-decalactone, ethyl butyrate, 2-furyl methyl ketone, 2,3-pentanedione, y-undecalactone, and 5-undecalactone.

[0243] The composition as described herein may include one or more sweetening agents. Preferably, the sweeting agent is a saccharide, such as a saccharide selected from the group consisting of glucose, mannose, maltose, fructose, galactose, lactose, sucrose, monatin, and tagatose. The one or more sweetening agents can be an artificial sweetener, and preferablyselected from the group of stevia, aspartame, cyclamate, saccharin, sucralose, mogrosides, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lacititol, mabinlin, malititol, mannitol, miraculin, monatin, monelin, osladin, pentadin, sorbitol, thaumatin, xylitol, acesulfame potassium, advantame, alitame, aspartame-acesulfame, sodium cyclamate, dulcin, glucin, neohesperidin dihyrdochalcone, neotame, and P-4000.

[0244] When present in the composition according to the invention and as described herein, the color balancing agents are preferably P-carotene or annatto.

[0245] The composition according to the invention can further include a vitamin. The vitamin is preferably selected from one or more of lipid soluble vitamins, water soluble vitamins, thiamin [vitamin B l], riboflavin [vitamin B2], niacin 0 [vitamin B3], pantothenic acid [vitamin B5], vitamin B6 [pyridoxine], vitamin B 12 [cobalamin], vitamin C, folate, vitamins A, vitamin D, vitamin E, or vitamin K.

[0246] In some embodiments, the dairy-like product is a vegan dairy-like product. In some embodiments, the dairy-like product further comprises one or more of (a) a plant-derived oil; (b) a plant-derived starch: (c) a sugar; and (d) a salt. In some embodiments, the dairy-like product further comprises a flavoring selected from cheddar flavor, parmesan flavor or mozzarella flavor.

[0247] In some embodiments, the fats of the dairy products or dairy-like products include triglycerides. In other embodiments, the fats comprise high-oleic oil. In some embodiments, the high-oleic oil further includes one or more of mono unsaturates, oleic, linoleic, linolenic and saturates. In some embodiments, the fats comprise short chain fatty acids. In some embodiments, the short chain fatty acids include butanoic, hexanoic, octanoic, and decanoic acids. In some embodiments, one or more of the fats comprised trans -esterified fatty acids. In some embodiments, one or more of the fats are isolated from plants. In some embodiments, the plant is selected from one or more of sunflower, com, olive, soy, peanut, walnut, almond, sesame, cottonseed, canola, safflower, flax seed, palm, palm kernel, palm fruit, coconut, babassu, shea butter, mango butter, cocoa butter, wheat germ and rice bran oil.

[0248] In some embodiments, the dairy products or dairy-like products comprise sugars, such as, but not limited to, galactose, sucrose, glucose, fructose and maltose. In some embodiments, the dairy substitute food product is essentially free of lactose.

[0249] In some embodiments, the dairy products or dairy-like products include an acidifier,such as gluconolactone (or glucono-delta-lactone), which mimics lactic ferments. Other suitable acids used in the production of dairy-like products include, but are not limited to, lactic acid, citric acid, and the like.

[0250] In some embodiments, the dairy-product or dairy-like product as defined herein further includes one or more of animal-derived or non-animal derived beta-lactoglobulin, serum albumin, lactoferrin, alpha lactalbumin and transferrin. Preferably, the beta- lactoglobulin is a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth beta-lactoglobulin. The serum albumin is preferably a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth serum albumin. In some embodiments, the lactoferrin is a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth lactoferrin. In some embodiments, the transferrin is a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth transferrin protein. The alpha lactalbumin is preferably a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth alpha lactalbumin.

[0251] In some embodiments, the dairy product or dairy-like product according to the invention and as defined herein, further includes one or more of animal-derived or non-animal derived kappa casein, beta casein, alpha S 1 casein, and alpha S2 casein, preferably, the kappa- , beta-, alpha SI- or alpha S2-casein being a cow, human, sheep, goat, buffalo, camel, horse, donkey, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, opossum, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or woolly mammoth kappa-, beta-, alpha SI- or alpha S2-casein protein.

[0252] According to the invention and in some embodiments, a non-animal component, and in particular a non-animal protein, can be a native or recombinant non-animal protein, or hydrolyzed native or recombinant non-animal protein, or combinations thereof. In other embodiments, non-animal components, and in particular non-animal proteins, can derive from non-animal sources including naturally occurring or modified plants, algae, fungi, or microbes.

[0253] Examples of suitable plants include, but are not limited to, spermatophytes (spermatophyta), acrogymnospermae, angiosperms (magnoliophyta), ginkgoidae, pinidae, mesangiospermae, cycads, Ginkgo, conifers, gnetophytes, Ginkgo biloba, cypress, junipers, thuja, cedarwood, pines, angelica, caraway, coriander, cumin, fennel, parsley, dill, dandelion, helichrysum, marigold, mugwort, safflower, camomile, lettuce, wormwood, calendula, citronella, sages, thyme, chia seed, mustard, olive, coffee, capsicum, eggplant, paprika, cranberry, kiwi, vegetable plants (e.g., carrot, celery), tagetes, tansy, tarragon, sunflower, wintergreen, basil, hyssop, lavender, lemon verbena, marjoram, melissa, patchouli, pennyroyal, peppermint, rosemary, sesame, spearmint, primroses, samara, pepper, pimento, potato, sweet potato, tomato, blueberry, nightshades, petunia, morning glory, lilac, jasmin, honeysuckle, snapdragon, psyllium, wormseed, buckwheat, amaranth, chard, quinoa, spinach, rhubarb, jojoba, cypselea, chlorella, manila, hazelnut, canola, kale, bok choy, rutabaga, frankincense, myrrh, elemi, hemp, pumpkin, squash, curcurbit, manioc, dalbergia, legume plants (e.g., alfalfa, lentils, beans, clovers, peas, fava coceira, frijole bola roja, frijole negro, lespedeza, licorice, lupin, mesquite, carob, soybean, peanut, tamarind, wisteria, cassia, chickpea, garbanzo, fenugreek, green pea, yellow pea, snow pea, lima bean, fava bean), geranium, flax, pomegranate, cotton, okra, neem, fig, mulberry, clove, eucalyptus, tea tree, niaouli, fruiting plants (e.g., apple, apricot, peach, plum, pear, nectarine), strawberry, blackberry, raspberry, cherry, prune, rose, tangerine, citrus (e.g., grapefruit, lemon, lime, orange, bitter orange, mandarin), mango, citrus bergamot, buchu, grape, broccoli, brussels, sprout, camelina, cauliflower, rape, rapeseed (canola), turnip, cabbage, cucumber, watermelon, honeydew melon, zucchini, birch, walnut, cassava, baobab, allspice, almond, breadfruit, sandalwood, macadamia, taro, tuberose, aloe vera, garlic, onion, shallot, vanilla, yucca, vetiver, galangal, barley, corn, curcuma aromatica, ginger, lemon grass, oat, palm, pineapple, rice, rye, sorghum, triticale, turmeric, yam, bamboo, barley, cajuput, canna, cardamom, maize, oat, wheat, cinnamon, sassafras, lindera benzoin, bay laurel, avocado, ylang-ylang, mace, nutmeg, moringa, horsetail, oregano, cilantro, chervil, chive, aggregate fruits, grain plants, herbal plants, leafy vegetables, non-grain legume plants, nut plants, succulent plants, land plants, water plants, delbergia, millets, drupes, schizocarps, flowering plants, non-flowering plants,cultured plants, wild plants, trees, shrubs, flowers, grasses, herbaceous plants, brushes, lianas, cacti, green algae, tropical plants, subtropical plants, temperate plants, and derivatives and crosses thereof.

[0254] Examples of suitable algae include, but are not limited to, green algae (e.g., Chlorella), brown algae (e.g., Alaria marginata, Analipus japonicus, Ascophyllum nodosum, Ecklonia sp, Eisenia bicyclis, Hizikia fusiforme, Kjellmaniella gyrata, Laminaria angustata, Laminaria longirruris, Laminaria Longissima, Laminaria ochotensis, Laminaria claustonia, Laminaria saccharina, Laminaria digitata, Laminaria japonica, Macrocystis pyrifera, Petalonia fascia, Scytosiphon lome), red algae (e.g., Gigartinaceae, Soliericeae, Chondrus crispus, Chondrus ocellatus, Eucheuma cottonii, Eucheuma spinosum, Furcellaria fastigiata, Gracilaria bursa- pastoris, Gracilaria lichenoides, Gloiopeltis furcata, Gigartina acicularis, Gigartina bursa- pastoris, Gigartina pistillata, Gigartina radula, Gigartina skottsbergii, Gigartina stellata, Palmaria palmata, Porphyra columbina, Porphyra crispata, Porhyra deutata, Porhyra perforata, Porhyra suborbiculata, Porphyra tenera, Porphyridium cruentum, Porphyridium purpureum, Porphyridium aerugineum, Rhodella maculate, Rhodella reticulata, Rhodella violacea, Rhodophyceae, Rhodymenia palmata), and derivatives and crosses thereof.

[0255] Examples of suitable fungi include, but are not limited to, Aspergillus sp., Aspergillus nidulans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida albicans, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Candida versatilis, Chrysosporium lucknowense, Debaryomyces hansenii, Endothia parasitica, Eremothecium ashbyii, Fusarium sp., Fusarium gramineum, Fusarium moniliforme, Fusarium venenatum, Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces marxianus var. lactis, Kluyveromyces thermotolerans, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. Cooney et Emerson, Mucor pusillus Lindt Myceliophthora thermophile, Neurospora crassa, Penicillium roquefortii, Physcomitrella patens, Pichia sp., Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Rhizopus niveus, Rhodotorula sp., Saccharomyces sp., Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum, Sporidiobolus johnsonii, Sporidiobolus salmonicolor,Sporobolomyces roseus, Trichoderma, Trichoderma reesei, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, Zygosaccharomyces rouxii, and derivatives and crosses thereof.

[0256] Examples of suitable microbes include, but are not limited to, firmicutes, cyanobacteria (blue-green algae), oscillatoriophcideae, bacillales, lactobacillales, oscillatoriales, bacillaceae, lactobacillaceae, Acetobacter suboxydans, Acetobacter xylinum, Actinoplane missouriensis, Arthrospira platensis, Arthrospira maxima, Bacillus cereus, Bacillus coagulans, Bacillus subtilus, Bacillus cerus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactococcus lactis, Lactococcus lactis Lancefield Group N, Lactobacillus reuteri, Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides strain NRRL B-512(F), Micrococcus lysodeikticus, Spirulina, Streptococcus cremoris, Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus, Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes, Streptomyces rubiginosus, Tetrahymena thermophile, Tetrahymena hegewischi, Tetrahymena hyperangularis, Tetrahymena malaccensis, Tetrahymena pigmentosa, Tetrahymena pyriformis, and Tetrahymena vorax, and Xanthomonas campestris, and derivatives and crosses thereof.

[0257] In some embodiments, dairy-like products are non- animal-derived food products, except that such products may include truncated casein polypeptides, such as alpha SI, alpha S2, beta or kappa casein, or other proteins and / or recombinant beta-lactoglobulin (rBLG) polypeptides that have high sequence identity to animal polypeptides. In other words, while the dairy like products may include, e.g., truncated bovine casein polypeptide, and preferably truncated alpha SI, alpha S2, beta or kappa casein polypeptide, the dairy like product itself is not produced by an animal. For instance, the dairy-like product is not derived from and does not contain milk produced by a mammal.

[0258] As defined above, the dairy products and dairy-like products according to the invention have the texture and sensation of dairy, and can have one or more characteristics of a classic dairy food product, such as taste, aroma, appearance, handling, mouthfeel, density, structure, texture, elasticity, springiness, coagulation, binding, leavening, aeration, foaming, creaminess and emulsification. Also, advantageously, the dairy products or dairy-like products according to the invention have improved or modified properties as compared to a classic dairy product, the properties being preferably selected from limitation, the viscosity, the foaming effect, the buffering effect, storage time, opacity, and smell. Examples of dairy-products anddairy-like products are milk, yogurts, curd, cheese, cream, cream cheese, butter and ice cream.

[0259] Dairy products and dairy like-products according to the invention are advantageously selected from milk, yogurt, curd, cheese, cream, cream cheese, butter or ice cream.

[0260] In some embodiments, the dairy product or dairy-like product according to the invention as defined herein and comprising the composition of the disclosure comprises one or more truncated casein polypeptide (TCP) of the disclosure at about 1% to about 10% by weight of the composition, or about 2% to about 9%, or about 2% to about 8%, or about 2% to about 7%, or about 2% to about 6%, or about 2% to about 5%, or about 3% to about 10%, or about 3% to about 9%, or about 3% to about 8%, or about 3% to about 7%, or about 3% to about 6%, or about 3% to about 5%, or about 4% to about 5%, or about 2%, or about 2.5%, or about 3%, or about 3.5%, or about 4%, or about 5% by weight of the composition. According to this embodiment, the dairy product or dairy like product is preferably yogurt or cheese. More preferably, the composition for use in the yogurt or cheese of the disclosure contains about 2% to about 5% of truncated casein polypeptide (TCP).

[0261] In some embodiments, truncated casein polypeptide (TCP) of the disclosure suitable for use in a composition for producing dairy product and dairy like -product, and preferably cheese or yogurt, may form aggregates or micelles. It is within the purview of the skilled artisan to measure aggregate or micelle diameter using art standard techniques, such as, but not limited to dynamic light scattering (DLS), which measures Brownian motion of particles to calculate aggregate or micelle diameter. In some embodiments, the TCP in the composition for producing dairy product and dairy like products, and preferably cheese or yogurt, comprises a plurality of aggregates having a diameter of between about 5 to about 1000 nm, or about 5 nm to about 800 nm, or about 5 nm to about 500 nm, or about 10 nm to about 200 nm, or about 20 nm and about 200 nm, or about 20 nm to about 150 nm, or about 30 nm to about 125 nm, or about 40 nm to about 100 nm as measured by DLS. Preferably, the TCP aggregates have a diameter of about 40 nm to about 100 nm as measured by DLS.

[0262] In some preferred embodiments, the dairy product or dairy-like product according to the invention and as defined herein comprising truncated casein polypeptide (TCP) of the disclosure, comprise from about 1% to about 10% by weight of TCP, preferably from about 2% to about 5% by weigh of TCP, and a plurality of aggregates. Preferably, the aggregates have a diameter of between about 5 to about 1000 nm, or about 5 nm to about 800 nm, or about 5 nm to about 500 nm, or about 10 nm to about 200 nm, or about 20 nm to about 200 nm, or ofabout 20 to 150 nm, or preferably of about 30 nm to about 125 nm, or about 40 nm to about 100 nm as measured by DLS.

[0263] Acidic gelling capacity is a desired trait for application in dairy products and dairy like-products, and preferably for yogurt products of the disclosure. In some embodiments, compositions for use in producing the yogurt products may contain one or more truncated casein polypeptide (TCP), preferably selected from truncated alpha Sl-casein (aSl-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein) or truncated kappa- casein (K-casein) polypeptide (TKC), of the disclosure that exhibit acidic gelling capacity at concentrations of between about 1% and about 10% by weight of the composition, or about 2% to about 8%, or about 3% to about 6%, or about 2% to about 5%, or about 2%, or about 2.5%, or about 3%, or about 3.5%, or about 4%, or about 4.5%, or about 5% by weight of the composition. The compositions for use in dairy products and dairy like -products, as for example yogurt applications, may include one or more TCPs preferably selected from truncated alpha Sl-casein (aSl-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein) or truncated kappa-casein (K-casein) (TKC) polypeptides capable of forming acidic gels with desired elasticity and viscosity traits, which traits are measurable using art standard techniques. For instance, the elasticity modulus (G’) and viscosity modulus (G”) can be measured using a rheometer equipped with cylindrical geometry. In some embodiments, the elasticity modulus and / or the viscosity modulus for an acidic gel containing one or more TCP, preferably selected from truncated alpha Sl-casein (aSl-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein) or truncated kappa-casein (K-casein) (TKC) polypeptides of the disclosure may be determined using a rheometer equipped with, e.g., a coaxial geometry and set to oscillate at 1 Hz.

[0264] In preferred embodiments, the TCP, preferably selected from truncated alpha Sl- casein (aSl-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein) or truncated kappa-casein (K-casein) (TKC) polypeptides in the composition at a concentration of about 2% to about 5% by weight has an elasticity modulus (G’) of about 0.1 to 2000 Pascals (Pa), preferably of about 1 to 2000 Pa, more preferably of about 10 to 2000 Pa, more preferably of about 20 to 2000 Pa, more preferably of about 50 to about 2,000 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. In other preferred embodiments, the TCP preferably selected from truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides in the composition at a concentration of about 2% to about 5% by weight has an elasticity modulus (G’) of about 100 Pa to about 1,500 Pa when measuring using a rheometer at 25 °C to 30 °Cusing coaxial cylinder geometry oscillating at 1 Hz or preferably the elasticity modulus (G’) is about 150 Pa to about 1,500 Pa, or about 200 Pa to about 1,500 Pa, or about 200 Pa to about 1,500 Pa, or about 250 Pa to about 1,500 Pa, or about 300 Pa to about 1,500 Pa, or about 350 Pa to about 1,500 Pa, or about 400 Pa to about 1,500 Pa, or about 450 Pa to about 1,500 Pa, or about 500 Pa to about 1,500 Pa, or about 100 Pa to about 600 Pa, or about 100 Pa to about 500 Pa.

[0265] In some preferred embodiments, the elasticity modulus (G’) of the TCP, preferably selected from truncated alpha SI -casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, at a concentration of about 5% by weight of the composition is about 1,000 Pa to about 1,500 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. In other preferred embodiments, the elasticity modulus (G’) of the TCP, preferably selected from truncated alpha SI -casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, at a concentration of about 3% by weight of the composition is about 100 Pa to about 600 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. In other preferred embodiments, the elasticity modulus (G’) of the TCP, preferably selected from truncated alpha SI -casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides at a concentration of about 5% by weight of the composition is about 100 Pa to about 300 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

[0266] According to one preferred embodiment, the truncated casein polypeptide (TCP) in the composition is a truncated alpha SI -casein polypeptide, and the elasticity modulus (G’) is comprised between 100 and 700 Pa, preferably between 200 and 700 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. According to this embodiment, the truncated alpha Sl-casein polypeptide is selected from SEQ ID NOs: 62 and 63.

[0267] According to another preferred embodiment, the truncated casein polypeptide (TCP) in the composition is a truncated kappa-casein polypeptide, and the elasticity modulus (G’) is comprised between 100 and 500 Pa, preferably between 100 and 400 Pa, and more preferably between 100 and 300 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. According to this embodiment, the truncated kappa- casein polypeptide is selected from SEQ ID NOs: 27 and 29.

[0268] In some embodiments, the TCP, preferably selected from truncated alpha S l-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, in the composition at a concentration of about 2% to about 5% by weight has a viscosity modulus (G”) of about 1 mPa to about 10 Pascals (Pa) when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. Preferably, the viscosity modulus (G”) of the composition is of about 10 mPa to about 500 Pa, preferably from about 100 mPa to about 500 Pa, or more preferably from about 1 to 500 Pa, or even more preferably from about 10 to about 500 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

[0269] Still in other preferred embodiments, the TCP, preferably selected truncated alpha S 1- casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, in the composition at a concentration of about 2% to about 5% by weight has a viscosity modulus (G”) of about 10 Pa to about 300 Pa, preferably of about 50 Pa to about 300 Pa, or about 60 Pa to about 300 Pa, or about 70 Pa to about 300 Pa, or about 80 Pa to about 300 Pa, or about 90 Pa to about 300 Pa, or about 100 Pa to about 300 Pa, or about 200 Pa to about 300 Pa when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

[0270] In some embodiments, the ratio of elasticity to viscosity for the TCP, preferably selected from truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, in the composition is measured (tanD). Preferably, according to the invention, the TCP, preferably selected truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, in the composition at a concentration of about 2% to about 5% by weight of the composition has a tanD of about 0.001 to about 200, or about 0.05 to about 200, more preferably of about 0.05 to 150, and more preferably of about 0.05 to 100, when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. In other preferred embodiments, the composition comprising TCP according to the disclosure, has a ratio of elasticity to viscosity for the TCP (tanD) of about 0.001 to about 75, or about 0.05 to about 75, preferably of about 0.05 to about 50, more preferably of about 0.05 to 25, and even more preferably of about 0.05 to about 10 when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. Still in other preferred embodiments, the tanD of the TCP, preferably selected from truncated alpha Sl-casein, truncated alpha S2 casein, truncated beta- casein or truncated kappa-casein (TKC) polypeptides, in the composition is of about 0.001 to about 10, or about 0.05 to about 8, of about 0.05 to about 5, of about 0.05 to about 3, of about0.05 to about 2, of about 0.05 to about 1, or preferably of about 0.05 to 0.5, or more preferably of about 0.05 to about 0.4, or even more preferably of about 0.1 to about 0.4, or about 0.1 to about 0.2 when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

[0271] According to some preferred embodiments, the truncated casein polypeptide (TCP) in the composition is a truncated alpha SI -casein polypeptide, and the ratio of elasticity to viscosity (tanD) is comprised between 0.05 and 5, preferably between 0.05 and 3, more preferably between 0.05 and 2, even more preferably between 0.05 and 1. Advantageously, the ratio of elasticity to viscosity (tanD) is comprised between 0.05 and 0.5 when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. According to this embodiment, the truncated alpha Sl-casein polypeptide is selected from SEQ ID NOs: 62 and 63.

[0272] According to other preferred embodiments, the truncated casein polypeptide (TCP) in the composition is a truncated kappa-casein polypeptide, and the ratio of elasticity to viscosity (tanD) is comprised between 0.05 and 5, preferably between 0.1 and 3, more preferably between 0.1 and 2, even more preferably between 0.1 and 1 when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. According to this embodiment, the truncated kappa-casein polypeptide is selected from SEQ ID NOs: 27 and 29. Advantageously, the ratio of elasticity to viscosity (tanD) is comprised between 0.15 and 0.8, preferably between 0.15 and 0.6 when measuring using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz. According to this embodiment, the truncated kappa-casein polypeptide is selected from SEQ ID NOs: 27 and 29.

[0273] Advantageously, dairy products or dairy-like products according to the invention comprise the composition as defined herein, and wherein the truncated casein polypeptide (TCP) comprise SEQ ID NO: 62, or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 62, or SEQ ID NO : 29, or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 29, or SEQ ID NO: 63, or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 63, or SEQ ID NO: 27, or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 27.

[0274] In some embodiments, compositions containing one or more of the truncated casein polypeptides (TCP), preferably selected from truncated alpha S 1-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, and asdescribed above may be used to make yogurt dairy products or dairy-like products. Advantageously, TCP, preferably selected from truncated alpha SI -casein, truncated alpha S2- casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides , has an amino acid sequence represented by SEQ ID NO: 62 or SEQ ID NO: 29 or an amino acid sequence 90%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NO: 62 or to SEQ ID NO: 29. Preferably, the compositions containing SEQ ID NO: 62 or SEQ ID NO: 29 exhibit the acidic gelling capacity, elasticity, and viscosity traits described above and may be used to make for example spoonable yogurt.

[0275] In some preferred embodiments, the dairy product or dairy like -product is yogurt or cheese, and the composition comprises (i) a truncated alpha SI -casein polypeptide comprising SEQ ID NOs: 62 or an amino acid sequence with at least 90%, or 95%, or 96%, or 97%, or 98% or 99% sequence identity to SEQ ID NO: 62, or (ii) a truncated kappa-casein polypeptide (TKC) comprising SEQ ID NOs: 29 or an amino acid sequence with at least 90%, or 95%, or 96%, or 97%, or 98% or 99% sequence identity to SEQ ID NO: 29.

[0276] In some embodiments, a yogurt product is provided herein that includes a composition with one or more TCP of the disclosure, TCP being preferably selected from truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, such as SEQ ID NO:62 or SEQ ID NO:29, and one or more additional components. For non-animal derived yogurt products of the disclosure, the one or more additional components include, but are not limited to, plant-based proteins, plant-based fat, texturizer(s), salt, flavoring agents, colorants, minerals, and sugar(s). Suitable texturizers include starch, gums, carraghenans, xanthan, and pectins. For animal derived yogurt products of the disclosure, the additional components included, but are not limited to, milk, milk protein, cream, sodium chloride, calcium chloride, vitamins, minerals, sugar(s), flavoring agents, and colorants.

[0277] In some embodiments, a cheese product is provided herein that includes a composition with one or more TCP, TCP being preferably selected from truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, of the disclosure, such as SEQ ID NO: 62 or SEQ ID NO: 29, and one or more additional components. For non-animal derived cheese products of the disclosure, the one or more additional components include, but are not limited to, plant-based proteins, plant-based fat, texturizer(s), salt, flavoring agents, colorants, and cultures. Suitable texturizers include starch, gums, carraghenans, xanthan, and pectins. For animal derived cheese products of thedisclosure, the additional components included, but are not limited to, milk, milk protein, cream, sodium chloride, calcium chloride, Rennet, and cheese cultures.

[0278] In some embodiments, curd capacity is a desired trait for application in cheese products of the disclosure. As one having ordinary skill in the art would understand, curds have much less water holding capacity as compared to acidic gels. Water holding capacity is the amount of water that a sample, e.g., gel or curd, can absorb per unit of weight and can be determined by weighing the object before and after centrifugation according to the following formula:% water holding capacity = 100 x (Wl-W2) / W 1 Equation 3 where W1 is the weight of the object pre-centrifugation and W2 is the weight of the object post-centrifugation. In some embodiments, the TCP, preferably selected from truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, of the composition exhibits curd formation in conditions conducive to curd formation, e.g., “rennet” conditions or in the presence of 1% lactic acid (see Example 6B). In some embodiments, the TCP, preferably selected from truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-casein or truncated kappa-casein (TKC) polypeptides, in the composition for use in making cheese products is present in the composition at a concentration of about 2% to about 5% by weight and forms a curd in rennet conditions or in the presence of lactic acid. In some preferred embodiments, the composition includes an alpha Sl-casein polypeptide, and preferably a truncated alpha Sl-casein polypeptide, with an amino acid sequence represented by SEQ ID NO: 62 or a truncated kappa-casein polypeptide (TKC) with an amino acid sequence represented by SEQ ID NO: 29, or an amino acid sequence with at least 90%, or 95%, or 96%, or 97%, or 98% sequence identity to either SEQ ID NO: 62 or SEQ ID NO: 29.

[0279] In some embodiments, heat stability is also a desired trait for application in ultra-high temperature (“UHT”) dairy products and dairy-like products of the invention, such as beverages of the disclosure. Heat stability of a composition or polypeptide can be determined using any suitable art-standard technique, such as, but not limited to differential scanning calorimetry, differential scanning fluorimetry, circular dichroism spectroscopy, fluorescence spectroscopy, and the like.

[0280] In some embodiments, the truncated casein polypeptides (TCP), and preferably TCP selected from truncated alpha Sl-casein, truncated alpha S2-casein, truncated beta-caseinor truncated kappa-casein (TKC) polypeptides, of the composition for use in UHT products of the disclosure is heat stable for at least 5 minutes at 80 °C or at least 5 minutes at 85°C, or at least 5 minutes at 90 °C.

[0281] In some preferred embodiments, the composition for use in UHT products comprises a TCP having an amino acid sequence represented by SEQ ID NO: 62, or an amino acid sequence with at least 90%, or 95%, or 96%, or 97%, or 98% sequence identity to SEQ ID NO: 62.

[0282] In other preferred embodiments, the composition for use in UHT products comprises a truncated kappa-casein polypeptide (TKC) having an amino acid sequence represented by SEQ ID NO: 27, or an amino acid sequence with at least 90%, or 95%, or 96%, or 97%, or 98% sequence identity to SEQ ID NO: 27.

[0283] In other preferred embodiments, a UHT product is provided herein that includes a composition with one or more TCPs or TKCs of the disclosure, such as SEQ ID NO: 62 or SEQ ID NO: 27, and one or more additional components, as defined herein. For non-animal derived UHT products of the disclosure, the one or more additional components include, but are not limited to, plant-based proteins, plant-based fat, texturizer(s), salt, flavoring agents, colorants, minerals, sugar(s), and sweeteners. Suitable texturizers include starch, gums, carraghenans, xanthan, and pectins. For animal derived UHT products of the disclosure, the additional components included, but are not limited to, milk, milk protein, cream, sodium chloride, calcium chloride, vitamins, minerals, sugar(s), sweeteners, flavoring agents, and colorants.

[0284] In some preferred embodiments, the dairy product or dairy like-product is an ultra- heat treatment beverage, and the composition comprises (i) a truncated alpha SI -casein polypeptide comprising SEQ ID NOs: 62 or an amino acid sequence with at least 90%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NO:62, or (ii) a truncated kappa- casein polypeptide (TKC) comprising SEQ ID NOs: 27 or an amino acid sequence with at least 90%, or 95%, or 96%, or 97%, or 98% or 99% sequence identity to SEQ ID NO: 27.

[0285] In some embodiments, the dairy-like product is preferably non-animal dairy analog of milk, yogurt, curd, cheese, cream, cream cheese, butter, or ice cream. In other embodiments, the dairy-like product comprises both animal and non-animal components, and is a dairy analog of milk, yogurt, curd, cheese, cream, cream cheese, butter, or ice cream. The dairy-like products can be used as nutritional products, for sports nutrition, as infant formula, or for health products(e.g., treatment for specific disorders).

[0286] According to the invention and in preferred embodiments, the truncated casein polypeptide (TCP), and preferably the truncated alpha SI -casein, the truncated alpha S2-casein, the truncated beta-casein or the truncated kappa-casein (TKC) polypeptides and more preferably the truncated alpha-S 1 casein polypeptide or the truncated kappa-casein polypeptide (TKC) of the disclosure has an amino acid score of at least 0.070 (70%), preferably at least 0.80 (80%), preferably at least 0.90 (90%), and more preferably equal to or greater than 0.94 (94%) without any further constraints on the amount of any amino acid, e.g., for use in a product considering generally a nutritional composition. This nutritional composition may be used as a protein source by an individual who does not have a metabolic disorder which restricts protein intake. Such a nutritional composition may be useful to an individual with a reduced appetite or a reduced ability to eat.

[0287] In some embodiments, a truncated casein polypeptide (TCP), preferably a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, of the disclosure has an amino acid sequence that includes certain amino acids at amounts above the amount needed for an amino acid score of equal to or greater than 0.94 (94%). For example, the amino acid leucine is an important factor in stimulating muscle protein synthesis, and more generally branched-chain amino acids (BCAA; includes valine, leucine, and isoleucine) are plentiful in muscle proteins, stimulate muscle growth in the body, and provide energy during exercise. A nutritional composition high in leucine and / or BCAA may be needed and / or desired for individuals interested in increasing and / or preserving muscle mass, such as professional athletes (which may be referred to as “sports nutrition”) and the elderly, who tend to lose muscle mass as part of the aging process, or hospitalized people with limited appetite (which may be referred to as “medical nutrition”). Leucine also may play a role in managing blood sugar levels and help control appetite. Therefore, in some embodiments, a nutritional composition high in leucine may be needed and / or desired by individuals interested in better maintenance of their blood sugar levels and / or weight, such as diabetics, individuals who are pre-diabetic, and individuals struggling with weight control. In one embodiment, the TCP and preferably the truncated alpha Sl-casein polypeptide with an amino acid score of equal to or greater than 0.94 (94%) is SEQ ID NO: 62. In another embodiment, the truncated casein polypeptide (TCP), and preferably the truncated alpha Sl-casein, the truncated alpha S2-casein, the truncated beta-casein or the truncated kappa-casein (TKC) polypeptide, have a theoretical amino acid scores of equal to or greater than 0.94 (94%). As one having ordinary skill in the art will appreciate, a theoreticalamino acid score is one that has been calculated but not confirmed experimentally. For instance, in an embodiment, the truncated casein polypeptide (TCP), and preferably the truncated alpha SI -casein with a theoretical amino acid score of equal to or greater than 0.94 (94%) is SEQ ID NO:63.

[0288] In some embodiments, dairy products or dairy -like products with high protein or essential amino acid content, or that are optimized for other dietary considerations may be referred to herein as a “nutritional product.” In some embodiments, a dairy product, dairy -like product, or nutritional product of the disclosure include a TCP, and preferably a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, with an amino acid score of 0.8 (or 80%) or higher, e.g., 0.8 (80%), 0.82 (82%), 0.84 (84%), 0.86 (86%), 0.88 (88%), 0.90 (90%), 0.92 (92%), 0.94 (94%), 0.96 (96%), 0.98 (98%), 1.0 (100%), or higher. In some embodiments, a dairy product, dairy-like product, or nutritional product of the disclosure may include a TCP, preferably a truncated alpha Sl- casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, with an amino acid score of 0.90 (90%) or higher, or 0.94 (94%) or higher, or 1.0 (100%) or higher. In some embodiments, the amino acid score is between 0.9 (90%) and 1.5 (150%), e.g., 0.94 (94%) and 1.2 (120%), or 0.94 (94%) and 1.0 (100%). As one having ordinary skill in the art would appreciate, a dairy product, dairy-like product, or nutritional product containing a TCP, and preferably a truncated alpha Sl-casein, a truncated alpha S2- casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, with an amino acid score at 0.94 (94%) or higher is considered a nutritional product.

[0289] The amino acid score can be corrected to account for protein digestability (i.e., protein digestability corrected amino acid score or PDCAAS). The PDCAAS is determined by measuring the protein digestability of the TCP, and preferably according to the invention of the truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, using techniques available in the art, such as, but not limited to the INFOGEST in vitro method, Near Real Digestive Tract (NERDT) dynamic biomimetic in vitro digestion system, and others. The percent digestibility is then factored into the amino acid score. In some embodiments, the PDCAAS is capped at 1.0. In some embodiments, the capped PDCAAS of the TCP, and preferably of the truncated alpha Sl- casein, the truncated alpha S2-casein, the truncated beta-casein or the truncated kappa-casein (TKC) polypeptide, is 0.85 or higher, e.g., 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97. 0.98, 0.99 or higher. In some embodiments, a dairy product, dairy-like product, or nutritional product of the disclosure may include a TCP and preferably a truncated alpha S 1-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide with a capped PDCAAS of between 0.9 and 1.0, or between 0.92 and 1.0, or between 0.94 and 1.0.

[0290] In some embodiments, the TCP, and preferably the truncated alpha Sl-casein, the truncated alpha S2-casein, the truncated beta-casein or the truncated kappa-casein (TKC) polypeptide of the disclosure has an AAS of at least about 94%. In some preferred embodiments, the TCP, and preferably the truncated alpha Sl-casein, the truncated alpha S2- casein, the truncated beta-casein or the truncated kappa-casein (TKC) polypeptide of the disclosure has a capped PDCAAS of between 0.94 and 1.0. Advantageously, the TCP with a capped PDCAAS between 0.94 and 1.0 is a TCP, and preferably a truncated alpha Sl-casein polypeptide with an amino acid sequence represented by SEQ ID NO: 62 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 62, or an amino acid sequence with at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NO: 62.

[0291] In some embodiments, a TCP according to the invention, and preferably selected from a truncated alpha S 1 -casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, suitable for use in products for an individual (e.g., a patient) with a metabolic disorder associated with abnormal and / or deficient processing of one or more amino acids may be selected for an amino acid composition based on the needs of the individual. For instance, Phenylketonuria (PKU) and Hyperphenylalaninemia (HPA) are inherited metabolic disorders in which phenylalanine (Phe) cannot be properly processed. Therefore, phenylalanine in the diet needs to be avoided as much as possible. In an embodiment, a TCP of the disclosure that does not contain any phenylalanine amino acid residues in its polypeptide, such as a TCP, and preferably selected from a truncated alpha Sl- casein polypeptide or a truncated kappa-casein polypeptide, with the amino acid sequence set forth in any one of SEQ ID NOs: 63, 116, 117, or 118, is suitable as a complete protein source for individuals with PKU or HPA. In an embodiment, the TCP, preferably selected from a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, is suitable for as a complete protein source for PKU or HPA patients has no phenylalanine residues, and other than phenylalanine, has an amino acid score equal to or greater than 0.94. Other metabolic disorders may lead to abnormal and / or deficient processing of, for example, tyrosine (e.g., tyrosinemia), cysteine (e.g., sulfur oxidase deficiency), methionine (e.g., homocystinuria, sulfur oxidase deficiency), leucine (e.g., isovaleric acidemia), tryptophan (e.g., glutaric acidemia), and / or lysine (e.g., glutaric acidemia).

[0292] In an embodiment, a TCP, preferably selected from a truncated alpha SI -casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, of the disclosure is selected for use in products for individuals with a metabolic disease or disorder, including, but not limited to, Tyrosinaemia, Maple Syrup Urine Disease, Methylmalonic acidaemia, Homocystinurea, Glutaric aciduria, Isovaleric acidaemia, and Hyper- lysinaemia. For example, a nutritional composition suitable as a complete protein source for Tyrosinaemia patients may comprise a TCP of the disclosure, preferably selected from a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptides, which has low to no tyrosine with an amino acid score equal to or greater than 0.94.

[0293] In some embodiments, a TCP of the disclosure, preferably selected from a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, may be further modified by substituting 1-10 amino acids to reduce or reach zero phenylalanine content for use in a product suitable for consumption by an individual with PKU or HPA. In some embodiments, a TCP of the disclosure, preferably selected from a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, may be further modified by substituting 1-10 amino acids to reduce or reach zero tyrosine content for use in a product suitable for consumption by an individual with tyrosinemia. In some embodiments, a TCP of the disclosure, preferably selected from a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, may be further modified by substituting 1-10 amino acids to reduce or reach zero cysteine content for use in a product suitable for consumption by an individual with sulfur oxide deficiency. In some embodiments, a TCP of the disclosure, preferably selected from a truncated alpha S 1 -casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, may be further modified by substituting 1-10 amino acids to reduce or reach zero methionine content for use in a product suitable for consumption by an individual with homocystinuria or sulfur oxide deficiency. In some embodiments, a TCP of the disclosure, preferably selected from a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, may be further modified by substituting 1-10 amino acids to reduce or reach zero leucine content for use in a product suitable for consumption by an individual with isovaleric acidemia. In some embodiments, a TCP of the disclosure, preferably selected from a truncated alpha Sl-casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, may be further modified by substituting 1-10amino acids to reduce or reach zero lysine and / or tryptophan content for use in a product suitable for consumption by an individual with glutaric acidemia.

[0294] In some embodiments, provided herein is a dairy-like product, dairy product, or nutritional product with zero phenylalanine content that contains a TCP of the disclosure, said TCP being preferably selected from a truncated alpha SI -casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide. In some embodiments, the TCP, and preferably the truncated alpha SI -casein, the truncated alpha S2-casein, the truncated beta-casein or the truncated kappa-casein (TKC) polypeptide, has a phenylalanine content of zero. In some preferred embodiments, the TCP is an alpha SI -casein polypeptide that is truncated at the N-terminus and C-terminus relative to a full length alpha SI -casein polypeptide and has an amino acid sequence represented by SEQ ID NO: 63 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 63. In other preferred embodiments, the amino acid sequence of the TCP has at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NO: 63. In other preferred embodiments, the TCP is an alpha Sl-casein protein that is truncated at the N-terminus, the C-terminus, or both relative to a full length alpha Sl-casein polypeptide and has an amino acid sequence represented by SEQ ID NOs: 116 or 117 or has an amino acid sequence with at least 90% sequence identity to SEQ ID NOs: 116 or 117. Still in other preferred embodiments, the amino acid sequence of the TCP has at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NOs: 116 or 117. In some embodiments, the nutritional product for use in a nutritional product with zero phenylalanine content contains a truncated kappa-casein (TKC) that is truncated at the N- terminus, the C-terminus, or both. In some preferred embodiments, the TKC has an amino acid sequence represented by SEQ ID NO: 118 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 118. In other preferred embodiments, the amino acid sequence of the TKC has at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NO: 118.

[0295] In some embodiments, provided herein is a method of treating an individual with a metabolic disorder, by administering to the individual a nutritional product of the disclosure. In some embodiments, the metabolic disorder is PKU or HPA and the nutritional product contains a TCP of the disclosure, preferably selected from a truncated alpha S 1 -casein, a truncated alpha S2-casein, a truncated beta-casein or a truncated kappa-casein (TKC) polypeptide, having a phenylalanine content of less than 3, or less than 2, or less than 1, or zero. In some embodiments, the TCP, preferably selected from the truncated alpha Sl-casein, the truncated alpha S2-casein, the truncated beta-casein or the truncated kappa-casein (TKC)polypeptide, has a phenylalanine content of zero. In some embodiments, the TCP is an alpha SI -casein polypeptide that is truncated at the N-terminus and C-terminus relative to a full length alpha SI -casein polypeptide and has an amino acid sequence represented by SEQ ID NO: 63 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 63. In some preferred embodiments, the amino acid sequence of the TCP has at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NO:63. In some embodiments, the TCP is an alpha S 1-casein protein that is truncated at the N-terminus, the C-terminus, or both relative to a full length alpha S 1-casein polypeptide and has an amino acid sequence represented by SEQ ID NOs: 116 or 117 or has an amino acid sequence with at least 90% sequence identity to SEQ ID NOs: 116 or 117. In some preferred embodiments, the amino acid sequence of the TCP has at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NOs: 116 or 117. In some embodiments, the nutritional product for use in a nutritional product with zero phenylalanine content contains a TKC that is truncated at the N-terminus, the C-terminus, or both. In some preferred embodiments, the TKC has an amino acid sequence represented by SEQ ID NO: 118 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 118. In some preferred embodiments, the amino acid sequence of the TKC has at least 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID NO: 118.

[0296] In some embodiments, the metabolic disorder is PKU or HPA.

[0297] In some embodiments, the dairy products or dairy-like products may be used as nutritional products, sports nutritional products, infant formula, health products (e.g., to be consumed by individuals which specific disorders, such as metabolic disorders). Exemplary products are shown below:Method of manufacturing the TCP

[0298] Another object of the invention concerns a method for manufacturing a truncated casein polypeptide (TCP) relative to a full length alpha S 1-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide, wherein the method comprises: (i) expressing the truncated casein polypeptide in a recombinant host; (ii)culturing the recombinant host under conditions suitable for the production of the truncated casein polypeptide; and (iii) harvesting the truncated casein polypeptide. It is understood that the method for manufacturing truncated casein polypeptide (TCP) comprises a method for manufacturing a truncated alpha Sl-casein (aSl-casein), alpha S2-casein (aS2-casein), beta- casein (β-casein), or kappa-casein (K-casein) polypeptide (TKC). Preferably, the method relates to a method for manufacturing truncated alpha S 1 -casein (aS 1 -casein) polypeptide or truncated kappa-casein polypeptide (TKC).

[0299] As defined above, the truncated casein polypeptide, and preferably the truncated alpha Sl-casein (aSl-casein) or the truncated kappa casein (TKC) polypeptide is truncated at the N- terminus, C-terminus or a combination of both relative to the full length alpha Sl-casein (aSl- casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide, or preferably to a full length of alpha Sl-casein (aS 1 -casein), or kappa-casein (K- casein) polypeptide.

[0300] In some preferred embodiments, the truncated casein polypeptide (TCP) is an alpha- S1 casein (aS 1 -casein) polypeptide truncated at the N-terminus, C-terminus or combination of both relative to a full length of alpha Sl-casein (aS 1 -casein) polypeptide, and the truncated alpha Sl-casein (aS 1 -casein) polypeptide comprises an amino acid sequence comprising between 49 and 180 amino acids. Preferably, the amino acid sequences are selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 116 and SEQ ID NO: 117.

[0301] In some preferred embodiments, the truncated casein polypeptide (TCP) is a kappa- casein polypeptide (TKC) truncated at the N-terminus, C-terminus or combination of both relative to a full length of kappa-casein (K-casein) polypeptide, and the TKC comprises an amino acid sequence comprising between 56 and 161 amino acids, and the TKC does not comprises SEQ ID NO: 17. Preferably, the amino acid sequences are selected from SEQ ID NO: 5, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 118.

[0302] Yet in some preferred embodiments, the truncated casein polypeptide (TCP), the TCP being selected from truncated alpha Sl-casein (asl-casein), alpha S2-casein (as2-casein), beta- casein (β-casein), or kappa-casein (K-casein) polypeptide, comprises at least one cysteine. In this case, the TCP is more preferably selected from truncated alpha Sl-casein (aS 1 -casein) or kappa-casein (K-casein) polypeptide, as defined above. In preferred embodiments, the truncated alpha Sl-casein (aSl-casein) or kappa-casein (K-casein) polypeptide are selected from SEQ ID NO: 5, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 62, SEQ ID NO: 63, SEQID NO: 116, SEQ ID NO: 117 and SEQ ID NO: 118. Still in more preferred embodiments, TCP is truncated alpha Sl-casein (asl-casein) polypeptide and is selected from SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 116 and SEQ ID NO: 117, and more preferably from SEQ ID NO: 62 and SEQ ID NO: 63. In other more preferred embodiments, TCP is truncated kappa- casein (K-casein) polypeptide (TCK) and is selected from SEQ ID NO: 5, SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 118, and more preferably from SEQ ID NO: 5, SEQ ID NO: 27 and SEQ ID NO: 29.

[0303] In embodiments, the disclosure provides a nucleic acid, and / or a cell comprising a nucleic acid encoding a truncated casein polypeptide (TCP), and in particular encoding a truncated alpha Sl-casein (aSl-casein) polypeptide or a truncated kappa-casein (TKC) polypeptide, as disclosed herein. The first step of the method comprises expressing the truncated casein polypeptide (TCP) in a recombinant host. In some embodiments, the nucleic acid is DNA or RNA. The nucleic acid may be non-naturally occurring and / or purified and / or engineered. In other embodiments, the nucleic acid is naturally occurring. The nucleic acid sequence may be codon optimized, in accordance with the expression system utilized.

[0304] Methods of producing recombinant proteins such as truncated casein polypeptide (TCP) and in particular, truncated alpha Sl-casein (aS 1 -casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein), and truncated kappa-casein (K-casein) polypeptide are well known in the art. Nucleic acids encoding the protein can be cloned into expression constructs or vectors, which are then transfected into host cells, such as a microbial host cell. Exemplary host cells include, but are not limited to bacteria cells, yeast cells, insect cells, algae such as microalgae, plant cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the protein. Thus, in some preferred embodiments according to the invention, the recombinant host is a microbial host, a plant host or a mammalian host.

[0305] In some embodiments, the recombinant host is a microbial host, and preferably a bacteria. Bacterial hosts include, but are not limited to, Lactococci sp., Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glutamicum, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis sp. 6803 and E.coli or a combination thereof. In preferred embodiments, the bacterial host cell is Bacillus subtilis. In some embodiments, the strain of Bacillus subtilis exhibits reduced glycosylation of the expressed truncated casein polypeptide (TCP).

[0306] In some embodiments, the microbial host is a fungus. Fungal host cells include, but are not limited to Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida guilliermondii, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Endothia parasitica, Eremothecium ashbyii, Fusarium moniliforme, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, M ucor miehei var. Cooney et Emerson, Mucor pusillus Lindt, Penicillium roquefortii, Pichia pastoris, also named Komagataella phaffii, Rhizopus niveus, Saccharomyces cervisea, Saccharomyces fragilis, and Trichoderma reesei or a combination thereof. In preferred embodiments, the fungal host is Pichia pastoris or Aspergillus oryzae.

[0307] In some embodiments, the host cell is a plant cell. Plant hosts include, but are not limited to Nicotiana bethamiana, Nicotiana tabacum, Arabadopsis thaliana, and more generally Arabidopsis, carrots, tomato, tobacco, rice, soybean lettuce, and potato.

[0308] In some embodiments, the host cell is an algae. Algae hosts include, but are not limited to Chlamydomonas reinhardtii, Volvox carteri, Chlorella sp., Phaeodactylum tricomutum (diatom), Synechococcus, Schizochy trium, and D. salina.

[0309] Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present) or Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Methods of producing recombinant antibodies are also known in the art, see, e.g., US4816567 or US5530101.

[0310] In some embodiments, the methods disclosed herein purify the TCP, e.g., TKC, duringthe manufacturing process. In some embodiments, the TCP polypeptide is secreted into a host cell culture supernatant before isolating the supernatant of the host cell culture comprising the TCP polypeptide. In embodiments, the host cell for expressing TCP is a bacterial, fungal, plant, or mammalian cell.

[0311] The present disclosure also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one nucleic acid as described above. The present disclosure also provides a recombinant host cell which comprises one or more constructs as above. As mentioned, a nucleic acid encoding a specific polypeptide of the disclosure forms an aspect of the present disclosure, as does a method of production of the specific polypeptide comprising expression from a nucleic acid encoding a specific polypeptide of the disclosure. Expression may conveniently be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, a specific polypeptide may be isolated and / or purified using any suitable technique, then used as appropriate.

[0312] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be any suitable vectors known in the art, including plasmids or viral vectors (e.g. 'phage, or phagemid), as appropriate. For further details see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual: 2ndEdition, Cold Spring Harbor Laboratory Press (1989). Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. eds., Short Protocols in Molecular Biology, 2nd Edition, John Wiley & Sons (1992).

[0313] Further, the disclosure provides methods comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

[0314] In some embodiments, the nucleic acid of the disclosure may be integrated into the genome (e.g. chromosome) of the host cell. In embodiments, integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

[0315] In some embodiments, the nucleic acid encoding the TKC polypeptide may be inserted operably linked to a promoter in an expression construct or expression vector for further cloning (amplification of the DNA) or for expression in a cell-free system or in cells. As used herein, the term "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and / or external stimulus, or in a tissue specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial expression and / or temporal expression of said nucleic acid. As used herein, the term "operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0316] Many vectors for expression of a protein in cells, e.g., the truncated casein polypeptide (TCP), such as truncated alpha SI -casein (aSl-casein), truncated alpha S2-casein (aS2-casein), truncated beta-casein (β-casein), or truncated kappa-casein (K-casein) polypeptide, are commercially available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a protein (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled person will be aware of suitable sequences for expression of a protein. Exemplary signal sequences include prokaryotic secretion signals (e.g., pel B, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

[0317] Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S.pombe, include, but are not limited to, the ADH1 promoter, the GAL 1 promoter, the GALA promoter, the CUP1 promoter, the PHOS promoter, the nmt promoter, the RPR 1 promoter, or the TEF1 promoter.

[0318] The host cells used to produce the protein may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0319] Following the step of expressing the truncated casein polypeptide (TCP) in a recombinant host, the method for manufacturing a truncated casein polypeptide according to the invention comprises a step of (ii) culturing the recombinant host under conditions suitable for the production of said truncated casein polypeptide. Culturing recombinant host is known from the person of ordinary skill in the art, and this step includes one or more of the following steps: inoculation of sterilized media (Examples: LB and YPD media), growth of cells at a temperature comprised between 30 and 37 °C with stirring at approximatively 500 round per minute (rpm), addition of inducer such as IPTG or methanol if required to induce protein production. The culture of the recombinant host is stopped before high degree of lysis was observed, typically after 48 to 96 hours when the recombinant host is bacteria or yeast. It is understood that the skilled artisan will be able to determine the conditions for a suitable and efficient culture depending on the recombinant host and the sought truncated casein polypeptide.

[0320] Consequent to the expression of the truncated casein polypeptide in a recombinant host and the culture of said recombinant host, the truncated casein polypeptide is harvested. Methods to harvest said truncated casein polypeptide are known in the art and include one or more of the following steps: removing the biomass by centrifugation or microfiltration, filtration of the biomass through a membrane with appropriate MWCO, preferably having a MWCO in the range of 10-100 kDa.

[0321] The method according to the present invention, i.e. comprising the steps of (i) expressing the truncated casein polypeptide (TCP) in a recombinant host, (ii) culturing the recombinant host under conditions suitable for the production of TCP, and (iii) harvesting the TCP, can further comprise a step (iv) of combining the TCP with a recombinant beta- lactoglobulin (rBLG), and heating and / or agitating the combination, followed by optionalcooling, concentrating and / or drying the combination.

[0322] According to some preferred embodiments, the truncated casein polypeptide (TCP) is a truncated alpha S 1-casein (aS 1-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide, and more preferably a truncated alpha S 1-casein (aS 1- casein) or kappa-casein (K-casein) polypeptide.

[0323] In some embodiments, the recombinant host producing the truncated casein polypeptide (TCP) further comprises a nucleic acid sequence encoding for the rBLG. In some embodiments, the recombinant host producing the TCP is a different type of host cell (e.g., different type of bacteria cell, yeast cell, insect cell, algae cell, plant cell, or mammalian cell) than the recombinant host producing the rBLG. In some embodiments, the recombinant host producing the TCP and the recombinant host producing the rBLG can be in the same cell culture medium (co-culture). In some embodiments, the recombinant host producing the TCP and the recombinant host producing the rBLG can be in different culture media.

[0324] According to the method of the invention, and when comprising a step (iv) of combining the truncated casein polypeptide (TCP) with a recombinant beta-lactoglobulin (rBLG), the heating step is preferably performed at a temperature comprised between 65 °C and 95 °C, preferably between 70°C and 95 °C, and more preferably between 80°C and 95 °C. Advantageously, the temperature is 80 °C.

[0325] Agitation of the combination comprising the truncated casein polypeptide (TCP) and the recombinant beta-lactoglobulin (rBLG) is performed to make a suspension of the combination. The person of ordinary skill in the art will know to determine the appropriate rotational speed. Advantageously, the rotational speed is of 1 to 500 rpm (round per minutes).

[0326] Further optional steps can also be performed. These steps are preferably selected from cooling, concentrating and drying or a combination thereof.

[0327] It is understood that the cooling step corresponds of making the composition less warm. The cooling step can be performed by leaving the composition to cool down by itself, for example at room temperature or by transfer of thermal energy. Methods of cooling are known in the art, and the person of ordinary skill in the art will be able to choose and apply the appropriate method.

[0328] When needed, concentration of the aqueous solution is preferably performed according to the well-known methods of the technical field. Such methods includecentrifugation, dialysis, precipitation or salting out, filtration or chromatography. The person of ordinary skill in the art will be able to choose the appropriate method and to apply the appropriate conditions.

[0329] According to the invention, suitable drying methods are for examples freeze-drying, spray drying and supercritical drying. The person of ordinary skill in the art will know to choose the drying method of choice and suitable parameters.

[0330] Advantageously, the method for manufacturing the truncated casein polypeptide (TCP) according to the invention provides a truncated casein polypeptide (TCP) with an increase titer of casein compared to a full recombinant length casein. In preferred embodiments, titer of at least 10%, preferably at least 20%, and more preferably a least 30% are obtained. In preferred embodiments, titer of 10 to 200%, preferably of 10 to 100%, more preferably of 10 to 80%, or more preferably of 10 to 50%, or even more preferably of 10 to 30% are obtained. Such titers are preferably obtained for truncated casein polypeptide (TCP) preferably selected from truncated alpha SI -casein or truncated kappa-casein (TKC) polypeptides, and more preferably from polypeptides corresponding to amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 62, or SEQ ID NO: 63.

[0331] According to the invention, titer is measured by SDS-PAGE densitometry, as disclosed in Rehbein et al., «Integrated protocol for reliable and fast quantification and documentation of electrophoresis gels», 2015, or by high performance liquid chromatography (HPLC) or quantitate mass spectrometry.

[0332] In some preferred embodiments, the truncated casein polypeptide (TCP) is obtained according to the method described above, and the recombinant host is Pichia pastoris, Bacillus subtilis, or Aspergillus oryzae. Examples of such truncated casein polypeptides are SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 33 and SEQ ID NO: 40. Preferably, the truncated casein polypeptide (TCP) is a truncated kappa-casein polypeptide (TKC) or a truncated alpha Sl-casein, and the recombinant host is Pichia pastori, Bacillus subtilis, or Aspergillus oryzae.

[0333] In some preferred embodiments, the obtained truncated casein polypeptides are truncated alpha Sl-casein polypeptides, preferably selected from SEQ ID NO: 62 and SEQ ID NO: 63 and a titer of 10 to 200%, preferably of 10 to 100%, more preferably of 10 to 80%, or more preferably of 10 to 50%, or even more preferably of 10 to 30% is obtained. In otherpreferred embodiments, the obtained truncated casein polypeptides are truncated kappa-casein polypeptides, preferably selected from SEQ ID NO: 29 and SEQ ID NO: 30 and a titer of 10 to 200%, preferably of 10 to 100%, more preferably of 10 to 80%, or more preferably of 10 to 50%, or even more preferably of 10 to 30% is obtained.Description of the SequencesGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

[0347] SEQ ID NO: 14 Cow kappa casein with N-terminal truncation.SYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQP TTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASPEV IESPPEINTVQVTSTAV

[0348] SEQ ID NO: 15 Cow kappa casein with N-terminal truncation.YPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLEASP EVIESPPEINTVQVTSTAV

[0349] SEQ ID NO: 16 Cow kappa casein with N-terminal truncation.SRYPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLE ASPEVIESPPEINTVQVTSTAV

[0350] SEQ ID NO: 17 Cow kappa casein with N-terminal truncation.VLSRYPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTIEAVESTVATLE ASPEVIESPPEINTVQVTSTAV

[0351] SEQ ID NO: 18 Cow kappa casein with N-terminal truncation.IAKYIPIQYVLSRYPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINTIASGEPTSTPTIEA VESTVATLEASPEVIESPPEINTVQVTSTAV

[0352] SEQ ID NO: 19 Cow kappa casein with N-terminal truncation.IRCEKDERFFSDKIAKYIPIQYVLSRYPSYGLNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHPHPHLSFMAIPPKKNQDKTEIPTINT IASGEPTSTPTIEAVESTVATLEASPEVIESPPEINTVQVTSTAV

[0411] The present disclosure has been described with respect to representative examples that are to be considered illustrative embodiments that do not limit the scope of the disclosure which is defined solely by the claims. All references to publications, including scientific publications, treatises, textbooks, patent applications and issued patents are hereby incorporated by reference for all purposes.EXAMPLESExample 1. Sequence design of truncated kappa caseins and empirical testing of production potential in Pichia pastorisTiters achieved during recombinant overexpression of intact kappa caseins are very low possibly because of low stability under microbial fermentation conditions due to high susceptibility to proteases and low solubility limit. Our objective was to identify a casein fragment design with optimal degree of truncation that can be produced in Pichia pastoris at high titer. Sequence design also takes into account the requirements of a milk protein ingredient such as essential amino acid content, emulsification capacity and texture building at pH relevant for dairy applications.Mature bovine kappa casein is 169 AA long. There are 7140 unique truncation designs theoretically possible for fragments greater than 50 amino acids long. This does not include any insertions or deletions and only truncation from N- and C-termini. The theoretical library size will increase astronomically when considering the natural variations across multiple species and synthetic variations by protein engineering. Given this large sequence space, it is not obvious which particular truncated sequence or a subset of sequences would display desired functionality and production feasibility.Sequence candidates were prioritized for experimental testing after in-silico ranking of sequence properties (FIG. 1). Recombinant DNA construction using synthesized genes containing epitope (Strep II) tag, Pichia transformation, Pichia cultivation in 96 well plates, induction of recombinant protein production using methanol and analysis of secreted fraction by SDS-PAGE and Western blotting were performed as per standard protocols. Control sequences known in the prior art did not show detectable expression when analyzed by Coomassie staining or Western blotting with epitope tag antibody. In comparison, out of the 22 truncated sequences tested, eight sequences (SEQ ID NO: 5, 10, 12, 16, 17, 21, 26 and 33) showed higher expression. As representative examples, expression data for SEQ ID NO: 5, 10, 12 (truncated) in comparison to intact casein (SEQ ID NO: 1) is shown in FIG. 2.Example 2. Production of recombinant truncated kappa casein in Pichia pastoris under fermentation conditionsHit clones for SEQ ID NO: 5, 12, 17 and 26 that showed promising levels of expression in microtiter plate cultivation were tested in bioreactors under standard process conditions with methanol induction. The strongest expression was observed for SEQ ID NO: 5 as witnessed byCoomassie bands of increasing intensity with induction time (FIG. 3). Detectable expression was observed for SEQ ID NO: 26. However, for SEQ ID NO: 12 and 17, target bands were not detected possibly due to high proteolysis.By extension, it is likely that cow kappa casein sequences with identical or similar degree of N-terminal truncation as SEQ ID NO: 5 will also show good recombinant production titer. SEQ ID NO: 28 and 29 are shown as examples containing R118C and S 176R variations respectively. Of the many natural variations observed in cow kappa sequences, these two were shown as examples. Sequence of interest may also show combination of these natural variations.By extension, it is likely that cow kappa casein sequences with identical or similar degree of N-terminal truncation as SEQ ID NO: 5 and containing 1-10 non-natural or engineered variations will also show good recombinant production titer. SEQ ID NO: 45-48 are shown as examples containing non-natural or engineered variations to modify functional (gelation pH and hardness) and nutritive (essential amino acid content) properties.By extension, it is likely that kappa casein sequences from other species with identical or similar degree of N-terminal truncation as SEQ ID NO: 5 will show good recombinant production titer. SEQ ID NO: 38-44 are described elsewhere herein (see, for example, FIG. 1). Sequence of interest may also show combination of natural variations observed in each species and engineered variations as mentioned above.Based on the detectable expression observed for SEQ ID NO: 26, it is likely that kappa casein sequences with identical or similar degree of N-terminal truncation as SEQ ID NO: 26 will also show good recombinant production titer. Of the many natural variations observed in cow kappa sequences, SEQ ID NO: 27 and 31-37 are described elsewhere herein (see, for example, FIG. 1). Sequence of interest may also show combination of these natural variations.Example 3. Production of recombinant truncated kappa casein in Bacillus subtilis under fermentation conditionsBacillus subtilis WB800N strain and a standard genetic design of IPTG-inducible promoter and amyQ signal peptide were used to test recombinant production of intact (SEQ ID NO: 1 and 51) and truncated kappa casein sequences (SEQ ID NO: 5, 8, 17 and 40). The construct used for expression in this Example is shown in FIG 6. Expression levels were analyzed by Western blotting of secreted fraction with anti-HIS tag antibody after shake flask cultivation of recombinant Bacillus strains. As shown in FIG.4, SEQ ID NO: 5 and 40 showed strongexpression when compared to other sequences (truncated and intact). By alignment of SEQ ID NO: 5 and 40, it is clear that the degree of N-terminal truncation is the same in both sequences. Thus, a truncation design that works well for both cow and sheep kappa caseins in Bacillus and in Pichia was identified.It is likely that SEQ ID NO: 5 or a similar sequence will show high titer in other commonly used hosts for recombinant protein production. Specifically bacterial hosts could be Lactococci sp., Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis and Corynebacterium glutamicum, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis sp. 6803 and E. coli.Fungal hosts could be Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida guilliermondii, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Endothia parasitica, Eremothecium ashbyii, Fusarium moniliforme, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. Cooney et Emerson, Mucor pusillus Lindt, Penicillium roquefortii, Pichia pastoris, also named Komagatella phaffi, Rhizopus niveus, Saccharomyces cervisea, Saccharomyces fragilis, and Trichoderma reesei.Example 4. Method of production of rBLG and TKC complex and TKC-rBLG characterization (size, shape and charge)Upon heating above 60°C, the free thiol group that is typically buried in folded BLG is exposed and is expected to react with thiol groups available in TKC via disulfide bond (FIG. 5). In this example, the total protein is 3-7.5% w / w, and the pH is 6-8. In embodiments agitation is optionally included, at a stirring speed of about 0-500 rpm. The holding time ranges from 30 seconds to 90 minutes. In some embodiments, the treatment is followed by cooling, drying and concentrating. It is plausible that similar effects could be obtained with alpha S2 casein fragments containing cysteines. Examples of such fragments are shown in SEQ ID NO: 49 and 50. It is plausible to get similar effect with full or truncated alpha SI, alpha S2 and beta casein sequences containing free cysteine. In some cases, this would involve introducing free cysteine in alpha SI and beta sequences by natural or non-natural cysteine substitutions.A preassessment of TKC-rBLG aggregates was performed under miniaturized conditions (100 uL of sample) by using a Thermocycler system for heating and time control.To evaluate TKC-rBLG aggregates the following materials were used: TKC (SEQ ID NO: 5) powder used in the example 2 is obtained by precision fermentation, purification and lyophilization and has a TKC content of 80% on powder basis; purified full length kappa casein (KC) (Sigma Aldrich), WPI (Whey Protein Isolate) powder (Reference PRONATIV 95LL, Lactalis, France) contains 91% w / w of protein and was produced by filtration of bovine milk and spray-drying; and recombinant BLG (rBLG). rBLG powder used in the examples is rBLG obtained by precision fermentation, purification and lyophilization and has the following composition (table 1). BLG purity analysis was performed by HPLC and compared to WPI (bovine whey protein isolate) used as a reference. Different ratios of the proteins were evaluated after heat treatment at temperatures ranging from 70 to 95 °C at 15 minutes of heat treatment.Table 1: rBLG powder composition. Composition is expressed in weight % relative to total weight of the composition. The protein is rBLG (100%).TKC-rBLG aggregates were formed by mixing recombinant TKC powder (80% TKC on powder basis) and rBLG powder (73.2% rBLG on powder basis) in different proportions to set a defined TKC on rBLG ratio. Alternatively, rBLG powder can be replaced by WPI powder, and TKC powder can be replaced by full length kappa casein (KC). Demineralized water was added to the mix in order to obtain a defined total protein concentration (from 3% to 5% w / w total protein solution). The pH of this solution is set at a defined value between 6 and 8 using either sodium hydroxide or chloridric acid concentrated solutions (IM). The protein solution is then heated, under moderate agitation (150 rpm), at a defined temperature between 70 and 95°C during 15 min using a thermocycler system.Soluble aggregates of pure rBLG, pure TKC, TKC-rBLG, KC-rBLG, KC-WPI, TKC-WPI depicted in FIG.7 to 13 were obtained using protocol described above using the parameters of table 2.Table 2: Conditions of production of soluble aggregates of FIG. 7 to 13. The reaction time was 15 min, and pH of the solution was 6.7.Selected samples were submitted for TEM images. DLS was performed on all samples to evaluate poly dispersity and size of the aggregates at the different conditions. As shown in FIG. 7A, the particle size of soluble aggregates of rBLG formed under heat treatment at 80°C for 15 minutes varied from 40 nm to 100 nm and shape was characterized by branched fibril-like structures. FIG. 7B depicts size distribution of soluble aggregates after 15 minutes of heat treatment at 80 °C using DES, with an average particle size of 82 nm. FIG. 8 A depicts SEQ ID NO: 5 alone obtained from a 5% total protein concentration after 15 minutes of heat treatment at 80 °C. FIG. 8B depicts SEQ ID NO: 5-rBEG aggregates in a ratio of 2:3 SEQ ID NO: 5-rBLG. FIG. 8A shows that TKC alone does not aggregate at this concentration. By contrast SEQ ID NO: 5-rBLG (FIG. 8B) aggregates assume a different shape by aggregating in shorter, fragmented fibril structures when comparing to rBLG alone (FIG. 7A).FIG. 9A depicts a TEM image of SEQ ID NO: 5-rBLG aggregates at 3% initial protein solution after 15 minutes of heat treatment at 80 °C. FIG. 9B depicts a TEM image of full-length kappa casein (KC)-rBLG aggregates under these same conditions. The ratio of SEQ ID NO: 5-rBLG and KC-rBLG is 2: 1. FIGS. 9 A and 9B demonstrate that truncated kappa casein, TKC, has a different effect on aggregation under heat treatment when compared to full length kappa casein,KC, in the presence of rBLG. The SEQ ID N0:5-rBLG aggregates (FIG. 9A) present short fibrils, while KC-rBLG present spherical aggregates (FIG. 9B).FIGS. 10A-10C show TEM images for aggregates obtained after 15 minutes of heat treatment at 95 °C: A) control solution WPI, B) truncated kappa casein (TKC) (SEQ ID NO:5) aggregated with WPI; and C) full length kappa casein KC aggregated with WPI; all present in 1 : 1 ratio and 3% total protein concentration. SEQ ID NO:5 formed aggregates with WPI different in shape and size when compared to KC / WPI, as depicted in FIGS 10B and IOC. KC / WPI aggregates are a spherical / amorphous shape, though less spherical and homogeneous compared to the WPI control. In contrast, SEQ ID N0:5-WPI aggregates have a fibril-like shape, similar to those observed for rBLG aggregates (FIG.7A), though longer in size. FIGS 10B and IOC also demonstrate truncation effect on the aggregation process when comparing KC-WPI or WPI alone with SEQ ID N0:5-WPI.When decreasing the temperature from 95 to 70 °C for the formation of TKC-rBLG aggregates, a decrease in the average particle size was observed. FIGS. 11A-11C depict DLS data related to size distribution by mass of SEQ ID N0:5-WPI aggregates, in a 1: 1 ratio, formed at three different temperatures. No significant impact was observed at 95° C or 85° C, where the average particle size was between 100 nm to 1,000 nm. At 70° C the average particle size decreased down to 5 nm to 10 nm. A particle size in the range of 2 nm to 10 nm is characteristics of native non-aggregated proteins. At 70° C, the size distribution graph in FIG. 11C is composed of a unique population with an average at around 6 nm indicating no aggregation.The ratio SEQ ID N0:5:WPI was varied to evaluate the impact in the size of the aggregates. FIGS. 12A-12C depict the size distribution graphs obtained for the aggregate solutions under different SEQ ID NO :5: WPI ratios: 2:1 (FIG. 12A), 1: 1 (FIG. 12B), and 1:2 (FIG. 12C). The total protein and the temperature were held constant, 3% total protein solution, and 95 °C temperature. The size of aggregates varied from 100 nm to 1000 nm.In order to evaluate the influence of calcium on the aggregation of the SEQ ID NO:5-rBLG aggregates during heating, a solution of 2.5% SEQ ID NO: 5 produced from Pichia pastoris was added to a 5% rBLG solution and heated to 85 °C for 15 minutes in the presence and absence of 1 mM calcium.Summarized in Table 3 are the aggregate size distributions in the SEQ ID NO: 5-rBLG aggregate compared to rBLG alone and the impact of calcium on the size distributions of the aggregates. As shown in Table 3, the presence of calcium increases the peak distribution ofsize of the aggregates in the rBLG solutions (from 40 nm to 90 nm). However, when SEQ ID NO: 5 was added to rBLG, the complex exhibited a decrease in aggregate particle size (from 90 nm to 60 nm), which was followed by a decrease in turbidity. These observations are indicative of a chaperone effect that SEQ ID NO: 5 has on the aggregation of rBLG.Table 3: Production of soluble aggregates at 85 °C, pH 6.7 for 15 minutes in the presence or absence of calcium and aggregate size distributionThe above analysis was also carried out on another TKC, a cow TKC polypeptide (SEQ ID NO: 29) that was expressed in Bacillus subtilis. SEQ ID NO: 29 differs slightly from SEQ ID NO: 5 in that the former has a natural variation Serl76Arg (see FIG. 1). Particle size and shape of soluble were investigated for aggregates comprising 2.5% SEQ ID NO:29 and 5% rBLG that were formed under heat treatment at 85 °C for 15 minutes. As shown in FIG. 13 A, aggregates of SEQ ID NO:29-rBLG were determined to fall into two main populations ranging from 50 nm to 120 nm and from 120 nm to 800 nm. In contrast, aggregates of full-length bovine kappa casein KC-rBLG at a 1:2 ratio exhibiting a polydisperse peak at 100 nm and soluble aggregates ranging from 80 nm to 150 nm (see FIG. 13B).These results demonstrated a clear effect of SEQ ID NO:29 on TKC-rBLG complexes as compared to full length kappa casein. As shown in FIG. 13C, TEM microscopy revealed that 2.5% SEQ ID NO:29 -5% rBLG complex formed aggregates that were characterized as short fibril-like structures and there determined to be different from the amorphous soluble aggregates obtained for KC-rBLG (see FIG. 9B).Example 5. TKC-rBLG gel characterization and comparison to benchmarks (BLG only, BLG+CMP, BLG+full kappa casein)Gels formed with BLG, in the absence of any casein, tend to be elastic and very hard at high protein concentrations and this is primarily attributed to covalent network formed by thiol groups of BLG aggregates. It has been shown that when thiol groups are blocked with chemical compounds such as N-ethylmaleimide, hardness of BLG gels could be decreased. Additionally, it has been proven that kappa casein and BLG form covalent thiol bonds upon heat treatmentof milk.Gels formed with BLG, in the absence of any casein, seem to produce a grainy texture upon shear. Also, their texture in the absence of fat is watery. Such defects limit the application of BLG gels for applications such as stirred yogurt. Grainy texture could be due to large sized gel particles that are produced when the gel is sheared by stirring. Gels formed by TKC-BLG complex upon shearing may yield small particle size and produce a smooth texture due to decrease in thiol polymerization.Stable BLG aggregates can form a gel when the pH is adjusted towards the isoelectric point (pl =5.1). In this Example, the pH is adjusted slowly. By choosing a TKC sequence with desired pl, it is possible to modulate the pl of TKC-BLG complex and thereby the gelation pH. For example, SEQ ID NO: 21 has theoretical pl of 9.2 and this sequence when complexed with BLG would allow gelation at pH 6.0.A gel blend of 2.5% SEQ ID NO: 5 and 5% rBLG was produced by acidification. The TKC polypeptide SEQ ID NO: 5 was produced from Pichia pastoris as described above. The soluble aggregate SEQ ID NO: 5-rBLG was obtained according to the method described in example 4 (protein concentration 7.5 w / w%, 15 min, 80 °C, pH 6.7). Then, lOOmL of a solution was prepared with deionized water and adjusted at pH 7,0 with IM HC1, and 1% of Glucono Delta Lactone (GDL) was added. The sample was left at 27 °C until a pH 4.5 was reached. TKC- rBLG gels and rBLG gels were compared in a texturometer after 24 hours of storage time at 4°C to measure gel hardness and adhesiveness. The summary in Table 4 reveals a significant increase in gel hardness due to the blending of SEQ ID NO: 5 and rBLG as compared to rBLG alone. While not intending to be bound by theory, this may enable optimal gel texture at a lower inclusion rate. Therefore, blending TKC polypeptides with rBLG can avoid thermal gelation and improve heat stability of high protein formulations.Table 4: Characteristics of TKC-rBLG gels.Example 6A. TCP aggregate micelle-like formation without BLG blendingTCPs were expressed in a recombinant host cell and examined to determine whether self- aggregation could occur in the absence of rBLG. First, truncated bovine alpha casein polypeptides were produced by fermentation. Briefly, two truncated forms of cow alpha Sl-casein were expressed in Aspergillus oryzae. The alpha SI -casein polypeptides were truncated at both the N-terminus and the C-terminus and are represented by SEQ ID NOs: 62 and 63. The latter truncated alpha Sl-casein polypeptide had additional substitutions at A68T, P102L, and M75T. As determined by PAGE densitometry using cow alpha casein (mix of SI and S2 caseins) as the standard (Rehbein et al., « Integrated protocol for reliable and fast quantification and documentation of electrophoresis gels», 2015), both truncated polypeptides exhibited good titer (see FIG. 14 and Table 5).Table 5: Titer of truncated cow alpha SI casein polypeptides.SEQ ID NO: 62 gave a good titer (estimated at 4 g / L) and amino acid score. Unlike the full- length cow alpha casein (SI and S2 mix), neither truncated polypeptide was phosphorylated according to the mass spectrometry measurements of p-Serines. As shown in FIG. 15 A, purified SEQ ID NO: 62 showed solubility and pH similar to full-length cow alpha S1 / S2 casein purified from cow’s milk.Solubility profile of SEQ ID NO: 62 (A1C11) and SEQ ID NO: 63 (A1C13) was determined in the presence of different concentration of calcium by measuring the concentration of the supernatant after centrifugation. If the concentration in the supernatant was the same as initial protein concentration, solubility was 100%. If the concentration of supernatant after centrifugation was much lower this means that the protein has precipitated and low or no solubility was obtained.Further, as shown in FIG. 15B, the solubility of both truncated alpha polypeptides (SEQ ID NOs: 62 and 63) exhibited solubility in the presence of up to 10 mM calcium. On the other hand, full-length cow alpha S 1 / S2 casein was insoluble in concentrations of calcium higher than 5 mM.Despite the impact that calcium ions have on electrostatic interactions of proteins, and while not intending to be bound by theory, one reason associated to the solubility of full-length cow alpha S1 / S2 casein is the presence of phosphorylated groups in the sequence, which form complexes to calcium and precipitates. However, as shown in FIG. 16, the intact mass results of short alpha casein sequences (SEQ ID NOs: 62 and 63) revealed the absence ofphosphorylation, which enables the high solubility of the truncated alpha casein polypeptides in high concentrations of calcium.Next, 100 μl aqueous solution of 2.5% purified SEQ ID NO: 62 was acidified with 0.4% glucono-delta-lactone (“GDL”). After incubation at 27 °C until pH of the solution reached 4.5, a gel was obtained. Surprisingly, as shown in FIG. 17, this truncated cow alpha Sl-casein polypeptide formed a good gel after acidification with GDL, even without mixing with rBLG. In contrast, cow full alpha casein S1 / S2 polypeptides alone did not form any gel following acidification with GDL.The solution (formed without applying heat) was evaluated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Examination revealed that the SEQ ID NO: 62 self-aggregated without requiring the application of heat or the need to complex with rBLG. A main monodisperse population of aggregates was observed (83%) at about 38 nm and another population (17%) about 190 nm was observed (see FIG. 18A). As shown in FIG. 18B, the composition exhibited an amorphous shape when viewed using TEM. The imaging revealed pseudomicelles (loosely associated casein aggregate; these structures lack the strong internal organization seen in true micelles and they are less stable).In contrast, full-length cow alpha S1 / S2 casein did not undergo self-aggregation. As shown in FIG. 18C, DLS revealed that up to 99% of the population was present in monomeric form (about 2 nm). TEM imaging confirmed that no aggregates were formed (see FIG. 18D).These results demonstrated that SEQ ID NO: 62 self-aggregated without requiring functionalization or other triggering and presented texture potential in dairy-like products. Further, the SEQ ID NO: 62 acidic gel exhibited stability for more than 10 days. On the other hand, full length cow alpha S1 / S2 casein formed a gel only at 5% protein concentration (see Table 8). The results of the gelation analysis are summarized in Table 6.n / a: data not availableTable 6: Acidic gelation and texture potential (alpha TCP polypeptides).As noted above in Example 4, complexes containing SEQ ID NO: 5 (a TKC polypeptide produced from Pichia pastoris) and rBLG or SEQ ID NO: 29 (a TKC polypeptide produced from Bacillus subtilis) and rBLG produced aggregates. To determine whether TKCs could form aggregates in the absence of rBLG, SEQ ID NO :29 was used.Western blotting revealed an estimated titer of about 0.6 to 1 g / L after 3 days of fermentation. Mass spectrometry was used to measure the molecular weight of the purified SEQ ID NO: 29 TKC (FIG. 19B). An analysis of the measured molecular weight of SEQ ID NO: 29 as compared to the predicted or theoretical molecular weight based on amino acid sequence revealed a lack of glycosylation.On the other hand, when SEQ ID NO: 5 was produced in Pichia pastoris, the mass spectrometry data compared to the predicted / theoretical molecular weight showed increased glycosylation compared to SEQ ID NO: 29. As shown in FIG. 19A, mass spectrometry data for SEQ ID NO: 5 produced in Pichia pastoris showed a difference of mass corresponding to 162 Da related to glycosylation. Further, the ion spacing that was observed suggested that O-mannosylation occurred. In contrast, the mass spectrometry data for SEQ ID NO: 29 produced in Bacillus subtilis showed no glycosylation pattern (see FIG. 19B). FIG. 18C illustrates the presence of at least eight sites in SEQ ID NO: 5 that are prone to glycosylation. These glycosylation sites are also present in SEQ ID NO: 29. While not intending to be bound by theory, and given the sequence identity between the two TKCs tested, the difference in glycosylation was attributed to the host cell.Both TKC polypeptide preparations were submitted to a solubility versus pH assay from pH 4.0 to 7.2 and compared to full-length cow kappa casein polypeptide. As shown in FIG. 20, SEQ ID NO: 5 (produced in Pichia pastoris) exhibited increased solubility compared to full length kappa casein or SEQ ID NO: 29 (produced in Bacillus subtilis). While not intending to be bound by theory, the increased solubility of glycosylated SEQ ID NO: 5 can be attributed to the high glycosylation, which decreased the hydrophobicity of the protein as compared to the non-glycosylated SEQ ID NO: 29.Next, an unheated 2.5% solution of SEQ ID NO: 29 and a preheated 2.5% solution of SEQ ID NO:5 (85 °C for 15 minutes) were prepared and analyzed to determine if self-aggregation occurred. DLS and TEM analysis demonstrated that SEQ ID NO: 29 formed soluble aggregatesranging from 50 nm to 190 nm (data not shown). In contrast, a main population of SEQ ID NO: 5 (99%) showed aggregates with a size of only 27 nm (data not shown). As shown in FIGs. 21A (SEQ ID NO: 5) and 21B (SEQ ID NO: 29), 5% solutions of the two TKC polypeptides showed similar results. While not intending to be bound by theory, the differences in aggregate formation were attributed to the level of glycosylation, which increased electrostatic repulsion and prevented aggregate formation.FIG. 21C is a TEM image showing self-aggregates of SEQ ID NO: 29 at 2.5% solution. Self- aggregates of the SEQ ID NO: 29 solution were observed as two main populations formed by tube-like branch shapes of different sizes around 200 nm to 500 nm as well as smaller amorphous aggregates that were less than 100 nm. These aggregates formed without preheating the solution or forming a complex with BLG. Only micelle-like aggregates were observed in the full-length cow kappa casein solution (see FIG. 2 ID), and no aggregates were observed in the SEQ ID NO: 5 solution (see FIG. 2 IE).The comparison of aggregate formation of the two TKC polypeptides as compared to full length cow kappa casein, 5% cow milk protein, and rBLG stand-alone polypeptides is summarized in Table 7. As seen in Table 7, the SEQ ID NO:29 TKC polypeptide had aggregate sizes that were within the optimal size window for dairy-like product texture potential. Therefore, unglycosylated TKC polypeptides, such as SEQ ID NO: 29 show stand-alone gelation potential without the need for BLG blending.Table 7: Aggregate formation in TKC polypeptide solutions.Next, gels of the TKC polypeptides were formed after acidification with 0.42% or 0.84% of glucono-delta-lactone (GDL) and properties (elastic modulus (G’), viscosity modulus (G”), ratio viscosity / elasticity or loss factor (tanD)) were determined with a rheometer to measure the viscoelastic properties. Gels formed from both the SEQ ID NO: 5 and SEQ ID NO: 29 polypeptides were compared to 5% cow milk protein, 2.5% and 5% cold-gellable rBLG. The summary of the results is shown in Table 8A. The SEQ ID NO: 29 polypeptide exhibited high tanD and was therefore less elastic than milk casein protein and much less elastic thanglycosylated TKC. Table 8B is a summary of gel rheology data comparing SEQ ID NO: 62 and SEQ ID NO: 29 with full length bovine alpha SI -casein and kappa-casein. Both TCPs exhibited superior tanD as compared to their full-length counterparts and, therefore were less elastic. Given their tangent delta (lower than milk protein benchmark), SEQ ID NO: 5 and SEQ ID NO: 29 give elastic gels for yogurt application. But SEQ ID NO:62 gives a tangent delta close to the milk proteins benchmark (0,182 at 5% protein concentration); its G' is higher, indicating that a firm gel was obtained.Table 8A: Gel rheology of TKC polypeptide gels.Table 8B: Gel rheology of TCP polypeptide gels.Table 9 is a summary of the gelation and texture potential of the TKC polypeptides described above. Non-glycosylated SEQ ID NO: 29 produced in Bacillus Subtilis exhibited stand-alone texture potential, even without the need for heat application of blending / complexing with BLG.n / a: data not availableTable 9: Acidic gelation & texture potential (TKC polypeptides).Example 6B. TCPs and TKCs for use in nutritional products, dairy products, or dairy- like products without BLG blending.TCP compositions comprising truncated alpha Sl-casein polypeptides SEQ ID NO: 62 and SEQ ID NO: 63 and TKC polypeptides SEQ ID NO: 27 and SEQ ID NO: 29 were assessed for selected traits, including acidic gelling capacity, curd capacity, heat stability, viscoelasticity, amino acid score, and phenylalanine content. For instance, TCPs and TKCs with optimal values for TanD, elasticity and viscosity moduli, and aggregate sizes exhibited good gelling and / or curd capacity in acidic environments, which demonstrate these polypeptides may be selected for use in yogurt and / or cheese products. TCPs and TKCs with high amino acid scores (at or above 94%) may be selected for high nutritional products. TCPs and TKCs with no phenylalanine residues may be selected to produce medical nutritional products for individuals with abnormal phenylalanine processing caused by metabolic disorders, such as phenylketonuria (PKU) or hyperphenylalaninemia (HPA). TCPs and TKCs with high heat stability may be selected for use in producing sports nutritional products, such as high protein ultra heat treatment beverages (UHT). As shown below, while each of the TCPs and TKCs of the disclosure that were tested had at least one trait suitable for producing particular nutritional products, dairy products, or dairy-like products, one having ordinary skill in the art would readily appreciate that the TCPs and TKCs disclosed below having at least one of the desired traits may be selected for producing a particular nutritional product, dairy product, or dairy - like product, e.g., acid-gelling capacity and texture optimal for spoonable yogurt products.A kappa-casein polypeptide was truncated at the N-terminus and represented by SEQ ID NO: 27. This truncated kappa casein polypeptide additionally had an amino acid substitution at S176R as compared to full length mature cow alpha Sl-casein (see SEQ ID NO: 53). TKC SEQ ID NO: 27 was expressed in A. oryzae and produced by fermentation according to the above-described methods.The truncated alpha Sl-casein polypeptides SEQ ID NO: 62 and SEQ ID NO: 63 were compared to full length alpha S 1 -casein polypeptide (produced in A. oryzae; “Full Alpha AO”) and animal-derived full-length alpha casein. Similarly, TKC polypeptides SEQ ID NO: 27 and SEQ ID NO: 29 were compared to full length kappa casein polypeptide (produced in A. oryzae; “Full Kappa OA”) and animal -derived full length kappa casein polypeptide. The polypeptideswere purified using art-standard techniques, for instance, SEQ ID NO: 63 was purified using ion exchange chromatography, SEQ ID NOs: 62, 27, and 29 were purified using immobilized metal affinity chromatography, Full Alpha AO was purified using microfiltration and ultrafiltration, and Full Kappa AO was purified using anion exchange chromatography. The purity levels and the physico-chemical properties of each are summarized in Table 10 below.Table 10: Physical-Chemical Properties of TCP and TKC polypeptides.For the acidic gelling capacity analysis, samples containing 2.5% or 5% of the polypeptide were acidified with 0.42% or 0.84% of GDL.The samples were left at 27 °C until a pH of 4.5 was reached. The presence of a gel was determined visually in an Eppendorf tube (see FIG. 22). While both Full Alpha AO and SEQ ID NO: 62 exhibited gelling potential at 5%, only SEQ ID NO: 62 exhibited gelling potential at the lower 2.5% concentration (see Table 8 and FIGS. 22A and 22B). Thus, SEQ ID NO: 62 exhibited excellent gelling potential even at lower concentration and without heating, functionalization, or complexing with BLG (data not shown). On the other hand, while SEQ ID NO: 63 exhibited increased viscosity, it did not form a gel at either concentration.Similarly, the acidic gelling capacities for TKC polypeptides SEQ ID NO: 27 and SEQ ID NO: 29 were determined and compared to Full Kappa AO and animal-derived kappa-casein. Full Kappa AO formed only a weak gel at 5% protein due to low water holding capacity (FIG. 22D). However, SEQ ID NO: 29 formed a gel at both 2.5% and 5% (FIG. 22C), while SEQ ID NO: 27 did not form a gel at either concentration. The results are summarized in Table 10 above. Thus, compositions comprising the SEQ ID NO: 62 and SEQ ID NO: 29 polypeptides exhibited good acidic gelling capacity.Rheological measurements were taken on the acidic gels described above. Rheological measurements were performed simultaneously with slow GDL-induced acidification of the protein solution using an Anton Paar MCR92 rheometer equipped with coaxial cylinder geometry (CC27) in oscillation mode. The visco-elastic properties (elastic modulus G’, viscous modulus G”, ratio viscosity / elasticity (tanD)) were monitored at 27 °C over time. The deformation was set at 1 % and the frequency was set to 1 Hz to operate in linear visco-elasticity range until the visco-elastic measurements plateaued and these final values were recorded in Table 10 above.Rheological analysis was conducted on gel compositions containing 3.25% or 5% SEQ ID NO: 62 and SEQ ID NO: 29 and compared to Full Alpha AO, skimmed milk and cold-gellable rBLG. As shown in FIG. 23 A, SEQ ID NO: 62 exhibited excellent viscoelasticity (elastic modulus G’) at both concentrations, and SEQ ID NO: 29 exhibited the desired viscoelasticity at 5% concentration. Both were comparable to skimmed milk and superior to cold-gellable rBLG and Full Alpha AO. Notably, Full Alpha AO exhibited very poor viscoelasticity. SEQ ID NO: 62 also showed good ratio of viscosity to elasticity as measured by tanD (see FIG. 23B).Briefly, SEQ ID NOs: 27, 29, 62, and 63 at protein concentrations of 2.5% and 5% were examined for the potential to form curds in the presence of either rennet enzyme, which is an enzyme used to coagulate milk into cheese in the presence of heat and is referred to herein as “rennet conditions,” or lactic acid and compared to recombinant full-length casein and animal- derived casein polypeptides. Curd capacity was distinguished from gelling capacity on the basis of water holding capacity. As one having ordinary skill in the art would understand, curds have much less water holding capacity as compared to gels. The water holding capacity was quantitated by weighing the gels before and after centrifugations. Briefly, approximately 5 grams of each gel was aliquoted and pre-weighed in 30 ml centrifuge tubes. Samples were then centrifuged at 5,000 rpm for 10 minutes and the water holding capacity was calculated according to Equation 3.Each of these TCPs and TKCs were placed under rennet conditions (Food Science & Nutrition, 2024, 12, 1399-1412) or in solution with lactic acid and visually observed to determine whether curd formation occurred. None of SEQ ID NO: 62, SEQ ID NO: 63, Full Alpha AO, or animal- animal derived alpha casein formed a curd under rennet conditions. However, as shown in Table 8, SEQ ID NO: 62 formed a curd under lactic acid conditions comparable to Full Alpha AO and animal-derived alpha casein. Microscopic imagery revealed that the curd structure of SEQ ID NO: 62 was similar to that of Full Alpha AO (data not shown), thus demonstrating that SEQ ID NO: 62 exhibits good cheese making traits. SEQ ID NO: 63 did not form a curd under rennet or lactic acid conditions.TKC polypeptides SEQ ID NO: 27 and SEQ ID NO: 29 at protein concentrations of 2.5% and 5% were also assessed for curd capacity under both rennet and lactic acid conditions. Neither TKC formed a curd under any of the conditions evaluated. However, SEQ ID NO: 29 exhibited coagulation under lactic acid conditions, but formed a gel rather than a curd as qualitatively determined by water holding capacity (see Table 8). As such, in some embodiments, compositions containing SEQ ID NO: 62 or SEQ ID NO: 29 are used to make cheese dairy or dairy-like products.For nutritional products of the disclosure, the amino acid scores for each of the tested TCPs and TKCs was determined, it being understood that an amino acid score of greater than or equal to 0.94 (94%) may be desired for high nutritional products. As shown in Tables 8 and 9, SEQ ID NO: 62 had an amino acid score of 125.3%, which was comparable to Full Alpha AO. The amino acid score was calculated based on the limiting amino acid quantified by HPLC (in mg / g of protein) divided by the scoring pattern of the same amino acid required by the Food andAgricultural Organization of the United Nations (FAO reference protein). This is a score to evaluate if a protein is completed and presents all the essential amino acids required for a nutrition stand point. In fact, except for lysine and leucine, SEQ ID NO: 62 had a superior essential amino acid score compared to Full Alpha AO (see Table 11).The digestibility of the SEQ ID NO: 62 was determined as a measure of the degree of hydrolysis in the gastric and intestinal phase (proteolysis) and compared to Full Alpha AO. The INFOGEST in vitro gastrointestinal food digestion simulation model was used as described in Brodkorb et al., Nature Protocols 14:991-1014 (2019), the entire contents of which are incorporated herein by reference. Briefly, the parameters for the oral phase were set at pH 7, 37 °C, in the presence of salivary amylase; the parameters for the gastric phase were set at pH 3, 37 °C and mixing for 2 hours in the presence of pepsin; and the parameters for the intestinal phase were set at pH 7 at 37 °C and incubating for an additional 2 hours in the presence of pancreatin and bile sales. As summarized in Table 10 and Table 12, both SEQ ID NO: 62 and Full Alpha AO exhibit low proteolysis in the gastric phase similar to micellar casein. Further, SEQ ID NO:62 exhibited comparable digestibility to Full Alpha AO and higher digestibility than micellar casein (compare 85.6% to 75%) by the end of the intestinal phase. In addition, the in vitro protein digestibility corrected amino acid score (“PDCAAS”) for SEQ ID NO:62 revealed a high nutrition content of 1 (see Tables 10 and 12). The PDCAAS values shown in Table 12 refer to the degree of hydrolysis (digestibility) at the end of the intestinal phase multiplied by the limiting amino acids.Further, the solubility profile for SEQ ID NO: 62 was determined at acidic pH (below 3.0). As shown in FIG. 24, SEQ ID NO: 62 exhibited high solubility in acid conditions and in a manner similar to sodium caseinate and micellar casein, suggesting that products comprising SEQ ID NO: 62 would have a faster amino acid release in the gastric environment. As such, in some embodiments, compositions containing SEQ ID NO: 62 are used to make nutritional products.Table 11: Essential Amino Acid.S.D.: standard deviationTable 12: INFOGEST digestibility of SEQ ID NO:62.Other nutritional products in the medical nutrition area include products with reduced phenylalanine content for individuals with phenylalanine processing defects caused by metabolic disorders, such as PKU and PHA. As described herein, some of the TCPs and TKCs of the disclosure have no phenylalanine amino acid residues, which content can be determined by art standard techniques, such as, but not limited to mass spectrometry. The phenylalanine content of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 62, and SEQ ID NO: 63 was determined and summarized in Table 10. The polypeptide sequence of SEQ ID NO: 63 does not have any phenylalanine residues, which makes this TCP suitable for medical products for individuals with PKU, PHA, or other similar metabolic disorders. Other TCPs and TKCs of the disclosure that do not contain phenylalanine residues include SEQ ID NOs: 116, 117, and 118. As such, in some embodiments, compositions containing SEQ ID NOs: 62, 116, 117, or 118 are used to make medicinal products for individuals with PKU or PHA.The TCPs and TKCs were further assessed for heat stability. SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 62, SEQ ID NO: 63, Full Alpha AO, animal-derived alpha casein, Full Kappa AO, and animal-derived kappa casein at 5% protein concentration were assessed for heat stability for 5 minutes at 90 °C (2000 uS / cm). SEQ ID NO: 62 and SEQ ID NO: 27 exhibited good heat stability comparable to their full-length counterparts (see Table 10).Example 7. TCP (alpha)-rBLG characterizationBlends were prepared with truncated alpha SI -casein polypeptides (SEQ ID NO: 62) produced by Aspergillus oryzae. Briefly, 2.5% SEQ ID NO: 62 polypeptide or cow full-length alpha S1 / S2 casein was mixed with 5% rBLG and heated at 85 °C for 15 minutes. The solubility, DLS, and acidic gelation measurements as described in Example 6 were performed to assess the gelation and texture potential of the SEQ ID NO: 62-rBLG blend. As summarized in Table 13, the SEQ ID NO: 62-rBLG blend exhibited aggregation and produced stable gels after acidification. Depicted in FIG 25 are the TEM images for the SEQ ID NO: 62-rBLG as compared to the cow full length alpha casein- rBLG blend and the rBLG alone. The SEQ IDNo: 62-rBLG blend gels produced short fibril structures, and this structure was not impacted by the addition of calcium (see Table 13). On the other hand, while full-length cow alpha casein-rBLG blends produced similar short fibril structures, the addition of calcium reduced the size of the aggregates (see Table 13). Thus, the SEQ ID NO: 62 polypeptide was not impacted by calcium and thus demonstrated good texture potential.Table 13: Acidic gelation & texture potential (TCP-rBLG blends).Example 8. Emulsification capacityThe emulsification capacity of different casein proteins as full alpha recombinant (Full alpha AO), A1C11 (SEQ ID NO: 62, recombinant truncated alpha Sl-casein), produced from a precision fermentation process, was studied and compared to the animal protein bovine a- casein as benchmark using the miniaturized microfluidic system.Microfluidic systems have been introduced for measurement of emulsion properties. These systems have specifically designed geometries to form dispersed droplets in the continuous phase, to measure the dynamic and equilibrium IFT, and to ascertain the stability of the droplets against coalescence. Miniaturized microfluidic system flow focusing device was used in this study. A unique approach was developed to create a two-dimensional (2D) array of droplets enabling visualization of the influence of droplet size on partial coalescence and destabilization. The V-FFD had a flow-focusing junction with a membrane valve on both sides of the orifice. When air pressure was applied to the membrane valves, the width of the flow- focusing orifice decreased, thereby reducing the size of generated droplets. Using this setup and the DOE described information about IFT and interfacial load and coalescence can be obtained (Journal of Colloid and Interface Science, 2019, 533, pages 59-70; Applied Physics Letters, 2009, 94, 023503).Interfacial tension, coalescence and interfacial load were measured and are reported in table 14 and figure 26.Interfacial tension (IFT}Interfacial tension is the force per unit length that exists at the interface between two immiscible phases. Low interfacial tension facilitates mixing and dispersion of one phase into another and it is critical for creating stable emulsions in food products. Higher interfacial tension encourages faster coalescence by making it easier for the thin film to break.Table 14: IFT values of a-casein, recombinant full a-AO and SEQ ID NO: 62 at a concentration of 0.05 and 0.1 wt%, pH of 3, 5, or 7 and a concentration of salt (phosphate buffer saline PBS) of 0, 0.1 or 0.4M.The interfacial tension values for full Alpha AO are significantly higher than a-casein at both concentrations 0.05 wt% and 0.1 wt% at pH 3, 5 and 7. The SEQ ID NO: 62 shows high similarity to a-casein where IFT values looks very close at 0.05% and 0.1% at pH 3, 5 and 7. The interfacial tension of SEQ ID NO: 62 is similar to the interfacial tension of a-casein. However, the value is lower than the one of full Alpha AO.In the presence of salt, the interfacial tension increases whatever the pH of the solution or the protein concentration. The low IFT of SEQ ID NO: 62 facilitates the dispersion of one phase into another leading to more stable emulsions with no phase separation.Interfacial loadThe interfacial load arises due to the distribution of particles at the interface between two immiscible liquids. Lower interfacial tension often facilitates better distribution of stabilizers, increasing interfacial load.Results of the experiments are presented in figure 26.The interfacial load of SEQ ID NO: 62 is higher than the interfacial load of the full Alpha AO, at a concentration of 0.1 wt% or at a lower concentration of 0.05 wt%, and under acidic pH of 5.5 or under neutral pH 7. However, under the same conditions, the interfacial load value of alpha-casein remains higher.Low interfacial tension facilitates mixing and dispersion of one phase into another and leads to better emulsion properties, and higher interfacial load means more emulsifier molecules are present at the interface, stabilizing the emulsion. As interfacial load increases, interfacialtension (y) decreases and emulisification capacity is more stable.A well-formed emulsion enhances dairy product quality by ensuring stability, smooth texture, whereas poor emulsification can lead to phase separation, off-flavors, graininess, and textural defects. A well-emulsified dairy product resists phase separation (e.g., creaming in milk or syneresis in yogurt). In addition, milk has a smooth texture due to finely dispersed fat droplets. Poor emulsification leads to a grainy or greasy mouthfeel.Example 9. Acid-gelling propertiesAcid gels were prepared comprising a concentration of 3.25 wt% or 5 wt% of SEQ ID NO: 62 protein or SEQ ID NO: 29 protein. ImL solution of purified SEQ ID NO: 62 and SEQ ID NO: 29 protein were prepared with deionized water and adjusted at pH 7,0 with IM HCL. Conductivity was adjusted with IM NaCl. The heat treatment was done at 90°C during 5 minutes using a thermocycler, then GDL was added to slowly acidify the solution. Gelation step and gel characterization are performed in rheometer: visco-elastic properties (Elastic modulus (G’), Viscous modulus (G”), ratio elasticity / viscosity or loss factor (tan D)) of the gels were monitored at 27°C (GDL's active temperature) as a function of time during acidification, using an Anton Paar MCR92 rheometer equipped with coaxial cylinder geometry (CC27) in oscillation mode. Deformation was set at 1% and frequency at 1 Hz to operate in Linear Visco-Elasticity (LVE) range. Results were compared to values of acid gels prepared from skimmed milk and from full alpha AO and are reported in table 15.Acid gels comprising 3.25 wt% of protein (SEQ ID NO: 62 or SEQ ID NO: 29) have viscoelasticity close to the one of skimmed milk. However, at a concentration of 5 wt%, the viscoelasticity is high, leading to a strong gel. On the contrary, the use of full alpha AO leads to gel having low elastic modulus value making them not suitable for the production of yogurts.Treatment at 90°C for 5 min, make the gel stronger, thus increasing the visco-eslasticity modulus. This phenomenon is also observed when the gel is treated with a conductivity. In conclusion, viscoelasticity properties of SEQ ID NO: 62 makes it suitable for yogurt applications. Concerning SEQ ID NO:29, at 3,25 wt% and 5 wt% the acid gels obtained are too elastic for yogurts application (tangent delta of 0,08-0,09), which gives a jelly texture.A 3.25 wt% SEQ ID NO :62 gel seems to have the same firmness has an acid gel done at 5% with skimmed milk after heat treatment, and tangent delta is close. At this concentration, SEQ ID NO: 62 seems thus to be interesting for yogurt applications.Table 15: Mechanical properties of acid gels prepared from SEQ ID NO:62, SEQ ID NO:29, skimmed milk and full alpha AO.Example 10. Comparison of yogurts properties: yogurts prepared from SEQ ID NO:62 versus plant-based yogurtsYogurts were prepared from SEQ ID NO: 62. 1 mL solution is made with 3.25% of SEQ ID NO: 62 with coconut cream in order to reach a fat concentration of 2%. pH is adjusted at pH 7,0 with IM HCL. After mixing, the solution is heated at 90°C during 5 minutes using a thermocycler to mimic protein functionalization that is done in yogurt industry. 0.,55% of GDL is added to the sample, and gelation step and gel characterization are performed in rheometer: visco-elastic properties (Elastic modulus G’, Viscous modulus G”, ratio viscosity / elasticity or loss factor (tan D)) of the gels were monitored at 27°C (GDL's active temperature) as a function of time during acidification, using an Anton Paar MCR92 rheometer equipped with coaxial cylinder geometry (CC27) in oscillation mode. Deformation was set at 1% and frequency at 1 Hz to operate in Linear Visco-Elasticity (LVE) range.Gel structure of the obtained yogurt was compared to gel structure of plant-based yogurts. In this aim, elastic modulus (G’) and the ratio viscosity / elasticity (tan D) were measured at 25°C using a rheometer. Results are reported in table 16.Table 16: Elastic modulus and viscosity / elasticity ratio (tanD) of yogurts prepared from SEQ ID NO: 62 and plant-based protein.Yogurts prepared from SEQ ID NO:62 comprising 3.25 wt% of protein makes a stronger gel than plant-based yogurts (FIG. 27).Example 11. Gel properties of gels prepared from a complex comparing SEQ ID NO: 62 and rBLGAcid gels were prepared with 3.25 wt% SEQ ID NO: 62 protein and rBLG at different concentrations (0%, 3.25 wt% and 6.25 wt%) according to the process described in example 9. Microparticulated rBGL (mrBLG) were obtained according to the process described in patent application PCT / EP2024 / 077629. Gelation step and gel characterization are performed inrheometer: visco-elastic properties (Elastic modulus G’, Viscous modulus G”, ratio viscosity / elasticity or loss factor (Tan D)) of the gels were monitored at 27°C as a function of time during acidification, using an Anton Paar MCR92 rheometer equipped with coaxial cylinder geometry (CC27) in oscillation mode. Deformation was set at 1% and frequency at 1 Hz to operate in Linear Visco-Elasticity (LVE) range.Results are reported in table 17.Table 17: Elastic modulus, and viscosity / elasticity ratio (tan D), of gel prepared from SEQID NO:62 and rBLGThe addition of microparticulated rBLG (without heat treatment) makes the gel weaker but does not affect the viscosity / elasticity modulus. Over time, the elastic modulus of a gel prepared from SEQ ID NO: 62 and microparticulated rBLG (3.25 wt% or 6.25 wt%) becomes weaker. On the contrary, in the absence of microparticulated rBLG, the gel prepared from SEQ ID NO: 62 is stronger over time. This property makes SEQ ID NO: 62 particular suitable for the preparation of yogurts having a skyr-like texture.Yogurts were prepared from SEQ ID NO: 62 and microparticulated rBLG (mrBLG) and compared to yogurts prepared from full alpha AO and microparticulated rBLG (mrBLG), cold- gellable rBLG and microparticulated rBLG (mrBLG), Aria®, or to Skyr yogurt (Siggi’s®). Elastic modulus (G’) and the ratio viscosity / elasticity (tan D) were measured for yogurts and are reported in table 18. Yogurt texture is also defined in table 18 and pictures are found in figures 28A, 28B and 28C.Preparation of yogurts: For each protein, a ImL solution is made with 4 wt% protein concentration of the protein and 5 wt% of mrBLG (to reach a high protein content). pH is adjusted at pH 7.0 with IM HC1 or IM NaOH. After mixing, the solution is heated at 90°Cduring 5 minutes using a thermocycler to mimic protein functionalization that is done in yogurt industry. 1.7% of GDL is added to the sample, and gelation step and gel characterization are performed in rheometer: visco-elastic properties (Elastic modulus G’, Viscous modulus G”, the ratio viscosity / elasticity (Tan D)) of the gels were monitored at 27°C (GDL's active temperature) as a function of time during acidification, using an Anton Paar MCR92 rheometer equipped with coaxial cylinder geometry (CC27) in oscillation mode. Deformation was set at 1% and frequency at 1 Hz to operate in Linear Visco-Elasticity (LVE) range.Aria and Siggi’s® are 2 skyr yogurts commercially available. Skyr is made by fermenting skim milk with live cultures, similar to yogurt. The milk is first heated, then cooled before adding bacterial cultures. After fermentation, the mixture is strained to remove whey, resulting in a thick, creamy texture. This straining process gives skyr its high protein content.Table 18: Elastic modulus and tan D of yogurts prepared from SEQ ID NO:62SEQ ID NO: 62 combined with mrBLG allows the production of high protein yogurts with skyr texture, although Tan D is slightly lower. Cold-gellable rBLG+ mrBLG gives a firmer gel, whereas full alpha AO + mrBLG gives a gel too weak for yogurt applications.

Claims

CLAIMS1. A composition comprising: a truncated casein polypeptide truncated (TCP) relative to a full length asl-casein (alpha Sl-casein), as2-casein (alpha S2-casein), β-casein (beta-casein), or K- casein (kappa-casein) polypeptide or a mixture thereof.2 The composition according to claim 1, wherein the casein polypeptide is truncated relative to SEQ ID NOs: l and 51 to 61.

3. The composition according to any one of claims 1 or 2, wherein the casein polypeptide is truncated at the C-terminus, the N-terminus, or a combination thereof.

4. The composition according to any one of claims 1 to 3, wherein the casein polypeptide is truncated by 10 to 150 amino acids relative to a full length asl-casein, as2-casein, β-casein, or K-casein polypeptide.

5. The composition according to any one of claims 1 to 4, wherein the truncated casein polypeptide (TCP) comprises at least one cysteine, the cysteine preferably being a free or a modified cysteine.

6. The composition according to any one of claims 1 to 5, wherein the truncated casein polypeptide (TCP) comprises an amino acid sequence between 28 and 199 amino acids.

7. The composition according to claim 6, wherein the truncated casein polypeptide (TCP) is a kappa-casein polypeptide (TKC) comprising between 56 and 161 amino acids or a truncated alpha Sl-casein polypeptide comprising between 49 and 180 amino acids or a truncated alpha S2-casein polypeptide comprising between 39 and 199 amino acids or a truncated beta casein polypeptide comprising between 28 and 188 amino acids.

8. The composition according to any one of claims 1 to 7, wherein the truncated casein polypeptide (TCP) comprises at least one additional modification, said modification being selected from substitution, deletion, truncation, insertion, phosphorylation, glycosylation, or a combination thereof.

9. The composition according to any one of claims 1 to 8, wherein the truncated casein polypeptide (TCP) is non-glycosylated or is less than about 20% glycosylated, preferably less than about 10% glycosylated, and more preferably less than 5% glycosylated.

10. The composition according to any one of claims 1 to 9, wherein the truncated casein polypeptide (TCP) is truncated alpha SI -casein polypeptide and comprises SEQ ID NO: 62 or SEQ ID NO: 63 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 62 or to SEQ ID NO: 63.

11. The composition according to any one of claim 1 to 9, wherein the composition comprises a truncated casein polypeptide (TCP) that does not comprise a phenylalanine amino acid residue.

12. The composition according to any one of claim 1 to 9, wherein the truncated casein polypeptide (TCP) is a truncated kappa-casein polypeptide (TKC) and comprises between 56 and 161 amino acids and the TKC does not comprise SEQ ID NO: 17.

13. The composition according to any one of claims 1 to 9, wherein the truncated casein polypeptide (TCP) is a truncated kappa-casein polypeptide (TKC) and comprises SEQ ID NO: 5, SEQ ID NO: 27, or SEQ ID NO: 29 or an amino acid sequence with at least 90% identity to SEQ ID NO: 5, SEQ ID NO: 27, or SEQ ID NO: 29.

14. The composition according to any one of claims 1 to 13, wherein the truncated casein polypeptide (TCP) or the truncated kappa-casein polypeptide (TKC) comprises an amino acid sequence according to any one of SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118 or comprises an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 5, 10, 12, 16, 21, 26, 27, 29, 33, 40, 62, 63, 116, 117, or 118.

15. The composition according to any one of the claims 1 to 14, wherein the composition comprises at least one additional polypeptide, wherein the at least one additional polypeptide is selected from a beta-casein, an alpha lactalbumin, a kappa casein, an alpha SI -casein, an alpha S2-casein, a lactoferrin, a transferrin, a beta-lactoglobulin, and a serum albumin.

16. The composition according to claim 15, wherein the additional polypeptide is a truncated polypeptide, preferably truncated at the C-terminus, the N-terminus, or both, relative to a full- length beta-casein, alpha lactalbumin, kappa casein, alpha SI -casein, alpha S2-casein, lactoferrin, transferrin, beta-lactoglobulin, or serum albumin.

17. The composition of any of claims 1 to 16, wherein the alpha SI -casein (aSl-casein), alpha S2-casein (aS2-casein), beta-casein (β-casein), or kappa-casein (K-casein) polypeptide relative to which the casein polypeptide is truncated comprises a cow, human, sheep, goat, buffalo,bison, horse, yak, reindeer, donkey, moose, lemur, panda, guinea pig, squirrel, bear, macaque, gorilla, chimpanzee, mountain goat, monkey, ape, cat, dog, wallaby, rat, mouse, elephant, rabbit, whale, baboons, gibbons, orangutan, mandrill, pig, wolf, fox, lion, tiger, echidna, or camel casein polypeptide, preferably a cow, buffalo, goat, sheep, human, camel, donkey, or horse casein polypeptide .

18. The composition according to any one of claims 1 to 17, further comprising at least one recombinant beta-lactoglobulin polypeptide.

19. The composition according to claim 18, wherein the truncated casein polypeptide (TCP) comprises at least one cysteine involved in a disulfide bond with the recombinant beta- lactoglobulin polypeptide.

20. A dairy product or dairy-like product comprising the composition of any one of claims 1 to 19.

21. The dairy product or dairy-like product according to claim 20, wherein the dairy product of dairy-like product is milk, yogurt, curd, cheese, cream, cream cheese, butter, or ice cream.

22. The dairy product or dairy-like product according to any one of claims 20 or 21, further comprising one or more additional components selected from proteins, texture agents, lipids, flavor compounds, sweetening agents, color balancing agents, ashes, vitamins, and any combination thereof.

23. The dairy product or dairy-like product according to any one of claims 20 to 22, wherein the one or more additional components is animal-derived, non-animal derived, or a combination thereof.

24. The dairy product or dairy-like product according to any one of claims 20 to 23, wherein the composition comprises from 1 to 10%, preferably from about 2% to about 5% by weight of a truncated casein polypeptide (TCP) and a plurality of aggregates.

25. The dairy product or dairy-like product according to claim 24, wherein the aggregates comprise a diameter ranging from about 20 to 200 nm, preferably from about 40 to 100 nm as measured by dynamic light scattering.

26. The dairy product or dairy-like product according to any one of claims 20 to 25, wherein the composition comprises from about 2% to about 5% by weight of a truncated casein polypeptide (TCP), wherein the TCP comprises an elastic modulus (G’) of about 0,1 mPa to about 2,000 Pa, preferably of about 100 Pa to about 1,500 Pa when the TCP is measured using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

27. The dairy product or dairy-like product of any one of claims 20 to 26, wherein the composition comprises about 2% to about 5% by weight of a truncated casein polypeptide (TCP), wherein the TCP comprises a viscosity modulus (G”) of about 1 mPa to about 500 Pa, preferably of about 10 Pa to about 300 Pa when the TCP is measured using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

28. The dairy product or dairy-like product according to claims 20 to 27, wherein the composition comprises about 2% to about 5% by weight of a truncated casein polypeptide (TCP) , wherein the composition has a ratio of elasticity to viscosity for the TCP (tanD) of about 0.05 to about 100, preferably of about 0.05 to about 0.5, and more preferably about 0.1 to about 0.4 when the TCP is measured using a rheometer at 25 °C to 30 °C using coaxial cylinder geometry oscillating at 1 Hz.

29. The dairy- product or dairy-like product according to any one of claims 20 to 28, wherein the composition comprises SEQ ID NO:62 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO:62, SEQ ID NO:29 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO:29, SEQ ID NO:63 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO:63 or SEQ ID NO:27 or an amino acid sequence with at least 90% sequence identity to SEQ ID NO:27.

30. The dairy product or dairy-like product according to any one of claims 20 to 29, wherein the dairy product or dairy-like product is yogurt or cheese, and wherein the composition comprises: (i) a truncated alpha SI -casein (aSl-casein) polypeptide comprising SEQ ID NO:62 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:62; or (ii) a truncated kappa casein (K-casein) polypeptide (TKC) comprising SEQ ID NO:29 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:29.

31. The dairy product or dairy-like product according to any one of claims 20 to 29, wherein the dairy product or dairy-like product is an ultra-heat treatment beverage, and wherein thecomposition comprises: (i) a truncated alpha Sl-casein (aSl-casein) polypeptide comprising SEQ ID NO:62 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:62; or (ii) a truncated kappa casein (K-casein) polypeptide (TKC)-comprising SEQ ID NO:27 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:27.

32. A nutritional product comprising the composition according to any one of claims 1 to 19.

33. The nutritional product according to claim 32, wherein it comprises a truncated casein polypeptide (TCP) with an amino acid score of at least 80%, preferably with an amino acid score of at least 94%.

34. The nutritional product according to claim 32, wherein the composition comprises a truncated casein polypeptide (TCP) with a protein digestibility corrected amino acid score (PDCAAS) of at least 0.94.

35. The nutritional product according to any one of claims 32 to 34, wherein the truncated casein polypeptide (TCP) is an alpha Sl-casein (aS 1 -casein) polypeptide truncated at the N- terminus and C-terminus relative to a full length alpha Sl-casein (aS 1 -casein) polypeptide and comprising SEQ ID NO:62 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:62, preferably comprising SEQ ID NO:62.

36. The nutritional product according to any one of claims 32 to 34 wherein the composition comprises a truncated casein polypeptide (TCP), wherein the TCP is an alpha Sl-casein (aSl- casein) polypeptide truncated at the N-terminus and C-terminus relative to a full length alpha Sl-casein (aSl-casein) polypeptide and comprising SEQ ID NO:63 or an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID 63, preferably comprising SEQ ID NO:63.

37. The nutritional product according to any one of claims 32 to 34, wherein the composition comprises: (i) a truncated casein polypeptide (TCP), truncated at the N-terminus, the C- terminus, or a combination of both relative to a full-length casein polypeptide, wherein the TCP comprises SEQ ID NOs: 116 or 117, or comprises an amino acid sequence with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 116 or 117; or (ii) a truncated kappa casein (K-casein) polypeptide (TKC) truncated at the N-terminus relative to a full-length kappa casein polypeptide, wherein the TKC comprises SEQ ID NO: 118, orcomprises an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 118.

38. The nutritional product according to any one of claims 32 to 37 for treating a metabolic disorder in an individual.

39. The nutritional product according to claim 38, wherein the metabolic disorder is phenylketonuria or hyperphenylalaninemia.

40. A method for manufacturing a truncated casein polypeptide (TCP) relative to a full length asl-casein, as2-casein, β-casein, or K-casein polypeptide, wherein the method comprises:(i) expressing the truncated casein polypeptide in a recombinant host;(ii) culturing the recombinant host under conditions suitable for the production of the truncated casein polypeptide; and(iii) harvesting the truncated casein polypeptide.

41. The method according to claims 40, wherein the truncated casein polypeptide (TCP) is truncated at the N-terminus, at the C-terminus, or a combination of both relative to a full-length alpha SI -casein polypeptide or a full-length kappa-casein polypeptide.

42. The method according to claim 40, wherein the truncated casein polypeptide (TCP) is a kappa-casein polypeptide truncated (TKP) at the N-terminus, the C-terminus, or a combination of both, relative to a full length K-casein polypeptide, and wherein the TKC comprises an amino acid sequence between 56 and 161 amino acids; and wherein the TKC does not comprise SEQ ID NO: 17.

43. The method according to claim 40, wherein the truncated casein polypeptide (TCP) is an alpha Sl-casein polypeptide truncated at the N-terminus, the C-terminus, or a combination of both, relative to a full-length alpha Sl-casein polypeptide, wherein the truncated alpha Sl- casein polypeptide comprises an amino acid sequence between 49 and 180 amino acids.

44. The method according to any one of claims 40 to 43, wherein the truncated casein polypeptide (TCP) comprises at least one cysteine.

45. The method according to any one of claims 40 to 44, further comprising one or more of the steps of combining the truncated casein polypeptide (TCP) with a recombinant beta-lactoglobulin (“rBLG”), heating and / or agitating the combination, followed by optionally cooling, concentrating and / or drying the combination.

46. The method according to any one of claims 40 to 45, wherein the truncated casein polypeptide (TCP) has an increased titer of casein compared to a recombinant full-length casein.

47. The method according to any one of claims 40 to 46, wherein the recombinant host is a microbial host, a plant host, or a mammalian host.

48. The method according to claim 47, wherein the recombinant host is a microbial host, preferably a bacteria, and more preferably a bacteria selected from Lactococci sp., Lactococcus lactis, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Brevibacillus choshinensis, Mycobacterium smegmatis, Rhodococcus erythropolis, Corynebacterium glutamicum, Lactobacilli sp., Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Synechocystis sp. PCC6803, Escherichia coli, or a combination thereof, and preferably the bacteria is Bacillus subtilis.

49. The method according to claim 47, wherein the microbial host is a fungus, and preferably a fungus selected from Aspergillus niger, Aspergillus niger var. awamori, Aspergillus oryzae, Candida guilliermondii, Candida lipolytica, Candida pseudotropicalis, Candida utilis, Endothia parasitica, Eremothecium ashbyii, Fusarium moniliforme, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis, Morteirella vinaceae var. raffinoseutilizer, Mucor miehei, Mucor miehei var. Cooney et Emerson, Mucor pusillus Lindt, Penicillium roquefortii, Pichia pastoris (also named Komagataella phaffii), Rhizopus niveus, Saccharomyces cervisea, Saccharomyces fragilis, Trichoderma reesei, or a combination thereof, more preferably the fungus is Pichia pastoris or Aspergillus oryzae.