Stable protein formulations

Abstract

To provide a stable protein solution having a pH of 7.0 or less and being stable against precipitation of the protein, a method of producing such stable protein solution, and a beverage and a beverage additive for human or animal consumption comprising such stable protein solution.SOLUTION: A stable protein solution is provided, the solution comprising (i) 0.1% to 30% w / v of a plant-derived protein based on the volume of the solution, (ii) 0.001% to 5% w / v of a stabilizer based on the volume of the solution, and (iii) 0.5 U to 50 U of protein deamidating enzyme activity, or 0.1% to 10% w / w of a protein deamidating enzyme based on the weight of the protein in the solution, having a pH of 3.5 to 5.0, and being stable against precipitation of the protein.SELECTED DRAWING: None

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 810,891, filed on February 26, 2019, the entire content of which is incorporated herein by reference.

Technical Field

[0002] Disclosed herein are stable protein solutions having a pH of 7 or less that are stable against protein precipitation, methods for producing such stable protein solutions, and their use in beverages or beverage additives.

Background Art

[0003] There is an increasing demand for protein - rich food products, as well as protein - rich vegetarian and vegan products. In particular, there is an increasing demand for protein - rich beverages and beverage additives that include plant - based proteins and other non - animal proteins. However, formulating such proteins into solutions, particularly solutions having a pH of 7 or less that are common in beverages, is difficult because the solubility of such proteins is limited at pH 7 or less. Accordingly, there is a need for protein solutions that are stable against protein precipitation at pH 7 or less.

Summary of the Invention

[0004] (i) A protein, (ii) a stabilizer, and (iii) a protein deamidase, wherein the stable protein solution has a pH of from about 3.5 to about 7.0 and is stable against protein precipitation. Protein solutions are provided herein. In some embodiments, the solution comprises (i) from about 0.1% to about 30% w / v of protein based on the volume of the solution, (ii) from about 0.001% to about 5% w / v of stabilizer based on the volume of the solution, and (iii) from about 0.5 U to about 50 U of protein deamidase activity or from about 0.1% to about 10% w / w of protein deamidase based on the weight of the protein in the solution. In some embodiments, the solution , (i) from about 0.1% to about 30% w / v of protein based on the volume of the solution, (ii) from about 0.001% to about 1% w / v of a stabilizer based on the volume of the solution, and (iii) from about 5 U to about 50 U of protein deamidase activity or from about 1% to about 10% w / w of protein deamidase based on the weight of the protein in the solution. In some embodiments, the solution comprises from about 5% to about 15% w / v of protein based on the volume of the solution. In some embodiments, the solution comprises from about 0.02% to about 0.5% w / v of a stabilizer based on the volume of the solution. In some embodiments, the solution comprises from about 1% w / w to about 5% w / w of protein deamidase based on the weight of the protein in the solution.

[0005] In some embodiments, the protein comprises one or more selected from plant proteins (such as soybeans, peas, lentils, chickpeas, legumes, hemp, rice, nuts, wheat, and gluten proteins (including peanut protein and almond protein)), milk proteins (such as whey protein), and insect proteins (such as one or more of cricket, keratin, silkworm, sago worm, locust, scorpion, centipede, cockroach, earthworm, mealworm, and spider protein).

[0006] In some embodiments, the stabilizer comprises one or more of one or more gums, polysaccharides, and collagen, such as xanthan gum, gellan gum, carrageenan gum, cassia gum, locust bean gum, tara gum, psyllium seed gum, gelatin, tamarind seed gum, gum arabic, alginate, propylene glycol alginate, pectin, galactomannan (guar gum), pullulan, methylcellulose (MC), carboxymethylcellulose (CMC), and one or more of any derivatives or combinations thereof.

[0007] In some embodiments, the protein deamidase is a protein glutaminase deamidase or a protein asparaginase deamidase that deamidates the amide groups of asparagine and / or glutamine residues of a protein. In some embodiments, the protein deamidase is produced by a bacterium selected from Chryseobacterium, Flavobacterium, Enpedobacter, Sphingobacterium, Aureobacterium, Myroides, Cytophagales, Actinomycetes, and Flavobacteriaceae. In some embodiments, the protein deamidase is produced by a Penicillium microorganism. In some embodiments, the protein deamidase is Protein Glutaminase Amano 500 (PGA500), which is a protein glutaminase deamidase. In some embodiments, the protein deamidase comprises the amino acid sequence of SEQ ID NO: 1 (which is a protein glutaminase deamidase), or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto and having protein deamidase activity. In some embodiments, the protein deamidase comprises a mutant amino acid sequence of SEQ ID NO: 1 having one or more substitutions or deletions at amino acid residues 35, 38-43, 45, 46, 49, 79-84, 103-106, 117, 142, 143, 146, 166, or 185 of SEQ ID NO: 1.In some embodiments, the protein deamidase has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and has one or more substitutions or deletions at amino acid residues 35, 38-43, 45, 46, 49, 79-84, 103-106, 117, 142, 143, 146, 166, or 185 of SEQ ID NO: 1, and includes a mutant amino acid sequence of SEQ ID NO: 1 having protein deamidase activity.

[0008] In some embodiments, the solution has a pH of from about 4.0 to about 7.0 or from about 4.0 to about 5.0.

[0009] In some embodiments, the solution is stable against visible precipitation of the protein after storage at 4 °C for a period selected from 7 days, 14 days, 21 days, 1 month, 2 months, and 6 months (including 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, and 12 months).

[0010] In some embodiments, the solution is formulated as a beverage or beverage additive for human or animal consumption.

[0011] Also provided is a beverage or beverage additive for human or animal consumption comprising the stable protein solution described herein. In some embodiments, the beverage or beverage additive is selected from nutritional beverages, sports beverages, functional protein beverages, milk beverages, milk smoothies, fruit beverages, fruit smoothies, coffee beverages, tea beverages, plant-based milks, milk creamers, and non-dairy creamers. In some embodiments, the beverage or beverage additive comprises one or more acidic or fruit juices, or acidic or fruit juice concentrates. In some embodiments, the beverage or beverage additive comprises one or more vegetable juices or vegetable concentrates. In some embodiments, the beverage or beverage additive comprises one or more acidic fruit or vegetable juices, or acidic fruit juice or vegetable juice concentrates.

[0012] A method for producing a stable protein solution described herein, or a beverage or beverage additive described herein, comprising: (a) adding a protein deamidase to a solution containing a protein and a stabilizer to obtain a mixture; (b) incubating the mixture; and (c) acidifying the mixture to obtain a solution having a pH of about 3.5 to about 7.0. In some embodiments, the solution is prepared by mixing (i) a solution containing a protein and (ii) a solution containing a stabilizer. In some embodiments, the incubation is carried out until the enzymatic reaction reaches a desired level of completion, which is optionally determined by the concentration of free ammonium ions in the solution. In some embodiments, the incubation is carried out at a temperature of about 30°C to about 70°C for about 0.5 hour to about 48 hours, optionally with stirring, and optionally at a pH of about 3.0 to about 8.0. In some embodiments, the incubation is carried out at a temperature of about 40°C to about 60°C for about 3 hours to about 24 hours, optionally with stirring, and optionally at a pH of about 5.0 to about 8.0. In some embodiments, the acidification comprises the addition of an acidic juice or juice concentrate. In some embodiments, the protein deamidase is Protein Glutaminase Amano 500 (PGA500) and / or has the amino acid sequence of SEQ ID NO: 1 described herein or a variant thereof, and the incubation is carried out at 50°C for 3 hours.

[0013] In some embodiments, the process further includes subjecting the solution to a heat treatment at about 85°C for about 10 minutes. In some embodiments, the process further includes subjecting the solution to one or more treatments selected from homogenization, pasteurization, and sterilization. In some embodiments, the homogenization is performed at a pressure of about 2,000 psi to about 20,000 psi, including from about 2,000 psi to about 2,500 psi. In some embodiments, the pasteurization is performed using high temperature short time (HTST) pasteurization at about 100°C for about 10 seconds to about 20 seconds, ultra-high temperature (UHT) pasteurization at about 120°C for about 1 second to about 3 seconds, or low temperature long time (LTLT) pasteurization at about 75°C to about 85°C for about 10 minutes to about 20 minutes. In some embodiments, the sterilization is performed using high pressure (high specific gravity) sterilization.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0015] Definition As used herein, technical and scientific terms have the meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains, unless otherwise defined. References are made herein to various methods known to those of ordinary skill in the art. Suitable materials and / or methods known to those of ordinary skill in the art can be utilized in practicing the present disclosure. However, specific materials and methods are described by way of example only. Materials, reagents, etc. referred to in the following description and examples can be obtained from commercial sources unless otherwise specified.

[0016] As used herein, the singular forms "a", "an", and "the" specify both the singular and the plural unless explicitly stated otherwise to specify only the singular.

[0017] As used herein, the term "about" does not limit a number or range to the exact number or range recited, but rather includes values around the recited number or range as would be understood by those of ordinary skill in the art depending on the context in which the number or range is used. Unless otherwise apparent from the context or convention of the art, "about" means plus or minus 10% of the particular term.

[0018] Described herein are stable protein solutions comprising a protein, a stabilizer, and a protein deamidase, having a pH of from about 3.5 to about 7.0 and being stable against precipitation of the protein. Also described herein are beverages and beverage additives comprising such solutions. Also described herein are methods of making such stable protein solutions and methods of making beverages or beverage additives comprising them.

[0019] As used herein, "stable against protein precipitation" means the absence of visible protein precipitation. In some embodiments, the absence of visible precipitation is confirmed by evaluating the absorbance at about 280 nm, where an increase in absorbance correlates with protein solubilization and the absence of precipitation. In a solution (without precipitation) having the protein concentration described herein, the typical absorbance at about 280 nm is in the range of about 8 to 50 mg protein / mL.

[0020] The stable protein solutions described herein address the challenges of protein formulation in solutions at pH 7 or less, which is the typical pH of beverages and beverage additives. For example, many beverages, including functional and sports beverages, contain fruit and / or vegetable juices or juice flavors and have a pH of 7 or less, such as a pH of about 7 to about 3.5. When protein is formulated into such beverages, it tends to precipitate out of solution and sediment. Without being bound by theory, this precipitation is thought to be due to the beverage's pH being close to the protein's isoelectric point, causing protein destabilization and its precipitation and sedimentation. Protein precipitation not only is unacceptable to consumers but also limits flavor masking options and other formulation choices. The stability of the solutions described herein at acidic pH enables the formulation of protein solutions into acidic juices such as fruit juices. As shown in the following examples, the solutions described herein are stable against precipitation even at acidic pH. Thus, the solutions described herein can be used to formulate a protein solution in or with an acidic juice such as a fruit juice to provide, for example, a fruit juice-based or fruit-flavored or fruit juice-flavored beverage or beverage additive containing protein.

[0021] The stable protein solution described herein uses a unique combination of a protein deamidase and a stabilizer to address this problem. Protease enzymes have been used previously, but their use is limited by the formation of compounds with undesirable flavors resulting from the enzymatic degradation of the substrate protein. Certain gum stabilizers and emulsifiers have been used previously, but they are only effective at high concentrations (e.g., 2 - 5% w / v) that exhibit other undesirable effects such as coagulation, stratification, and even precipitation. Further, using stabilizers at these high concentrations results in a final product with an undesirably high viscosity for the consumer. In contrast, the solutions described herein have acceptable viscosity characteristics, e.g., a viscosity in the range of about 10 to about 250 mPa·s, for use in beverages and beverage additives or as beverages and beverage additives. (For reference, milk has a viscosity of about 2 - 3 mPa·s, most vegetable oils have a viscosity of about 40 - 50 mPa·s, and chocolate sauce can have a viscosity of 280 mPa·s.)

[0022] Without being bound by theory, the protein deamidase described herein is thought to deamidate amino acid residues such as glutamine and / or asparagine residues in the protein, thereby increasing the negative charge of the protein, decreasing the isoelectric point of the protein, and increasing its solubility at acidic pH values. As a result, protein solubility at acidic pH is improved. Also without being bound by theory, the specific protein deamidase described herein increases protein solubility without generating undesirable flavor compounds by deamidating the amide groups of amino acid residues in the protein, including, for example, converting glutamine residues in the protein to glutamic acid and / or converting asparagine residues in the protein to aspartic acid, without cleaving peptide bonds.

[0023] The solubility of the protein can be increased to some extent by enzymatic treatment alone, but below pH 7, for example, over a long period exceeding the typical storage conditions for consumer beverages and beverage additive products, additional formulation approaches are needed to provide a solution that is stable against protein precipitation. Thus, the solutions described herein further promote the stability of the protein solution and, under refrigerated conditions for a long period, for example, under storage at 4°C for 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, and 12 months, for example, 4 months or 8 months, contain stabilizers that enable the preparation of a solution having a pH of about 3.5 to about 7 that is stable against protein precipitation. Unlike the formulations described above, the solutions described herein require only a relatively small amount of stabilizer, and as a result, the use of the stabilizers described herein does not impair the physicochemical properties of the solution, and no unacceptable effects for consumers such as coagulation, layer formation, precipitation, or high viscosity occur.

[0024] As shown in the following examples, the solutions described herein may be subjected to homogenization and may exhibit stability against protein precipitation after homogenization. Thus, even if the homogenization process may cause changes in protein-protein interactions, the proteins formulated in the solutions described herein may remain in solution even after homogenization.

[0025] As described above, according to certain embodiments, there is provided a stable protein solution comprising (i) a protein, (ii) a stabilizer, and (iii) a protein deamidase, having a pH of about 3.5 to about 7.0 and being stable against protein precipitation. Specific aspects and specific embodiments are described in more detail below.

[0026] Protein In this specification, the proteins that can be formulated are not limited, but the target embodiments include proteins suitable for human or animal intake, such as proteins from animals, plants, milk, and insects suitable for human or animal intake. In some embodiments, the solution described herein contains one or more proteins selected from vegetable proteins, milk proteins, and insect proteins.

[0027] Examples of suitable vegetable proteins include, but are not limited to, soybeans, peas, lentils, chickpeas, legumes, hemp, rice, nuts, wheat, and gluten proteins. In some embodiments, the vegetable protein is selected from one or more of soybeans, peas, lentils, chickpeas, legumes, hemp, rice, nuts, wheat, and gluten proteins. In some embodiments, the nuts are peanuts, almonds, or hazelnuts. In some embodiments, the protein includes pea protein. In some embodiments, the protein includes soy protein. In some embodiments, the protein includes peanut protein. In some embodiments, the protein includes hemp protein.

[0028] An example of a suitable milk protein includes, but is not limited to, whey protein. In some embodiments, the protein includes whey protein.

[0029] Examples of suitable insect proteins include, but are not limited to, crickets, mealworms, silkworms, waxworms, locusts, scorpions, fireflies, cockroaches, termites, mealworms, and spider proteins. In some embodiments, the protein includes insect proteins selected from one or more of crickets, mealworms, silkworms, waxworms, locusts, scorpions, fireflies, cockroaches, termites, mealworms, and spider proteins. In some embodiments, the protein includes cricket protein.

[0030] Stabilizer As described above, the solution described in this specification contains a stabilizer. Examples of suitable stabilizers include, but are not limited to, hydrophilic colloids (gums), polysaccharides, and collagen. In some embodiments, the stabilizer comprises one or more of gums, polysaccharides, and collagen. In some embodiments, the stabilizer comprises one or more of xanthan gum, gellan gum, carrageenan gum, cassia gum, locust bean gum, tara gum, psyllium seed gum, gelatin, tamarind seed gum, gum arabic, alginate, propylene glycol alginate, pectin, galactomannan (guar gum), pullulan, carboxymethyl cellulose (CMC), methyl cellulose (MC), and derivatives or combinations of any of these. In certain embodiments, the stabilizer is selected from xanthan gum, gellan gum, carrageenan gum, tara gum, pectin, alginate, and CMC. In some embodiments, the stabilizer comprises gellan gum. In some embodiments, the stabilizer comprises carrageenan gum. In some embodiments, the stabilizer comprises pectin gum. In some embodiments, the stabilizer comprises xanthan gum. As shown in the following examples, different stabilizers may be more effective at different pH ranges or different pH values. Thus, the choice of stabilizer can be guided in part by the pH of the final product.

[0031] Protein deamidase As described above, the solution described in this specification contains a protein deamidase. As used herein, "protein deamidase" is an enzyme that deamidates the amide group of an amino acid residue of a protein. In some embodiments, the protein deamidase deamidates the amide group of asparagine and / or glutamine residues of the protein. In some embodiments, the protein deamidase deamidates the amide group of glutamine residues of the protein. In some embodiments, the protein deamidase deamidates the amide group of asparagine residues of the protein. Examples of suitable protein deamidases include those described in U.S. Patent No. 6,756,221, U.S. Patent No. 6,251,651, U.S. Patent No. 7,462,477, and U.S. Patent No. 8,735,131, which are hereby incorporated by reference in their entirety, particularly with respect to the protein deamidases disclosed therein.

[0032] In some embodiments, the protein deamidase is produced by bacteria selected from Chryseobacterium, Flavobacterium, Enpedobacter, Sphingobacterium, Aureobacterium, Myroides, Cytophagales, Actinomycetes, and Flavobacteriaceae, or by Penicillium microorganisms. In some embodiments, the protein deamidase is produced by bacteria derived from Chryseobacterium. In some embodiments, the protein deamidase is produced by bacteria derived from Flavobacterium. In some embodiments, the protein deamidase is produced by bacteria derived from Enpedobacter. In some embodiments, the protein deamidase is produced by bacteria derived from Sphingobacterium. In some embodiments, the protein deamidase is produced by bacteria derived from Aureobacterium. In some embodiments, the protein deamidase is produced by bacteria derived from Myroides. In some embodiments, the protein deamidase is produced by bacteria derived from Cytophagales. In some embodiments, the protein deamidase is produced by bacteria derived from Actinomycetes. In some embodiments, the protein deamidase is produced by bacteria derived from Flavobacteriaceae.

[0033] In some embodiments, the protein deamidase is the protein glutaminase deamidase "Protein Glutaminase Amano 500 (PGA500)" commercially available from Amano Enzyme.

[0034] In some embodiments, the protein deamidase has an amino acid sequence of SEQ ID NO: 1 (which is a protein glutaminase deamidase), or has or comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto and having protein deamidase activity. The degree of protein deamidase activity is not particularly limited as long as the function of the protein deamidase can be exerted, but is preferably equal to or greater than that of the enzyme having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the protein deamidase comprises a mutant amino acid sequence of SEQ ID NO: 1 having one or more substitutions or deletions at amino acid residues 35, 38-43, 45, 46, 49, 79-84, 103-106, 117, 142, 143, 146, 166, or 185 of SEQ ID NO: 1. In some embodiments, the protein deamidase has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1, has one or more substitutions or deletions at amino acid residues 35, 38-43, 45, 46, 49, 79-84, 103-106, 117, 142, 143, 146, 166, or 185 of SEQ ID NO: 1, and comprises a mutant amino acid sequence of SEQ ID NO: 1 having protein deamidase activity. In some embodiments, the protein deamidase has or comprises a mutant amino acid sequence of SEQ ID NO: 1 having one or more substitutions or deletions at amino acid residues 39, 40, 41, 43, 79-82, 142, 143, 146, 166 or 185 of SEQ ID NO: 1, such as one or more substitutions or deletions at amino acid residues 35, 38, 40-43, 45, 46, 49, 80-84, 103-106 or 117 of SEQ ID NO: 1, as described in U.S. Patent No. 8,735,131. In some embodiments, the protein deamidase has or comprises a mutant amino acid sequence of SEQ ID NO: 1 having one or more substitutions or deletions at amino acid residue 82 or 84 of SEQ ID NO: 1, as described in U.S. Patent No. 8,735,131.In some embodiments, the protein deamidase has, or comprises, a variant amino acid sequence of SEQ ID NO:1, such as a substitution at amino acid residue 82 of SEQ ID NO:1, such as a serine substitution at amino acid residue 82, and / or a substitution at amino acid residue 84 of SEQ ID NO:1, such as an aspartic acid substitution at amino acid residue 84, as described in U.S. Patent No. 8,735,131.

[0035] Stable protein solution As noted above, in some embodiments, the stable protein solution described herein (i) contains from about 0.1% to about 30% w / v of protein, based on the volume of the solution, (ii) from about 0.001% to about 5%, such as from about 0.001% to about 1% w / v of a stabilizer, based on the volume of the solution, and (iii) from about 0.5 U to about 50 U of protein deamidase activity, or from about 0.1% to about 10%, such as from about 1% to about 10% w / w of protein deamidase, based on the weight of the protein in the solution, where the protein deamidase activity can be determined according to the assay of Example 16 below.

[0036] Thus, in some embodiments, the solution contains from about 0.1% to about 30% w / v, or from about 0.5 to about 30% w / w, or from about 5 to about 25% w / w, or from about 10 to about 20% w / w of protein, based on the volume of the solution (e.g., the final volume of the solution). In some embodiments, the solution contains from about 1% to about 15% w / v of protein, based on the volume of the solution. In some embodiments, the solution contains from about 5% to about 15% w / v of protein, based on the volume of the solution. In some embodiments, the solution contains, based on the volume of the solution, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 25%, or about 30% w / v of protein, such as, based on the volume of the solution, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, or 30% w / v of protein.

[0037] In some embodiments, the solution comprises a stabilizer in an amount of from about 0.001% to about 5% w / v, based on the volume of the solution, such as from about 0.001% to about 1.5%, from about 0.001% to about 2%, from about 0.001% to about 3%, and from about 0.001% to about 4% w / v of the stabilizer. In some embodiments, the solution comprises a stabilizer in an amount of from about 0.001% to about 1% w / v, based on the volume of the solution, such as from about 0.01% to about 1%, from about 0.01% to about 0.5%, and from about 0.02% to about 0.5% w / v of the stabilizer. In some embodiments, the solution comprises a stabilizer in an amount of about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5% w / v of the stabilizer, such as 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% w / v of the stabilizer. In some embodiments, the solution comprises a stabilizer in an amount of about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2%, about 3%, about 4% or about 5% w / v of the stabilizer, such as 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2%, 3%, 4%, and 5% w / v of the stabilizer. In some embodiments, the relatively small amount of stabilizer used results in a solution having a viscosity that is acceptable to consumers for beverages and beverage additives, such as a viscosity of from about 10 to about 250 mPa·s.

[0038] In some embodiments, the solution comprises a protein deamidase of from about 0.1% w / w to about 10% w / w, by weight of the protein in the solution, such as from about 0.1% w / w to about 1.0% w / w, from about 0.5% w / w to about 1.0% w / w, about 0.1% w / w, about 0.2% w / w, about 0.3% w / w, about 0.4% w / w, about 0.5% w / w, or about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, or about 0.9% w / w, by weight of the protein in the solution. In some embodiments, the solution comprises a protein deamidase of from about 1% w / w to about 10% w / w, by weight of the protein in the solution. In some embodiments, the solution comprises a protein deamidase of from about 1% w / w to about 5% w / w, by weight of the protein in the solution, such as about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, or about 5% w / w (including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%), by weight of the protein in the solution.

[0039] In some embodiments, the solution comprises a protein deamidase activity of from about 0.5 U to about 50 U, such as from about 0.5 U to about 5.0 U, from about 2.5 U to about 5.0 U, about 0.5 U, about 1.0 U, about 2.0 U, about 2.5 U, about 3.0 U, about 4.0 U or about 5.0 U. In some embodiments, the solution comprises a protein deamidase activity of from about 5 U to about 50 U. In some embodiments, the solution comprises a protein deamidase activity of from about 5 U to about 25 U, such as about 5 U, about 10 U, about 15 U, about 20 U, or about 25 U (including 5 U, 10 U, 15 U, 20 U, 25 U, 30 U, 35 U, 40 U, 45 U, or 50 U). The protein deamidase activity can be determined as described in Example 16 below.

[0040] In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) has a pH of from about 3.5 to about 7 (including 3.5 to 7), for example from about 3.5 to about 5.5 (including 3.5 to 5.5). In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) has a pH of from about 4.0 to about 5.0 (including 4.0 to 5.0). In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) has a pH of from about 4.0 to about 7.0 (including 4.0 to 7.0), for example, a pH of about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0 (including 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0).

[0041] In some embodiments, the viscosity of the solution (or a beverage or beverage additive containing the solution described herein) is from about 10 to about 250 mPa·s (including 10 to 250 mPa·s). In some embodiments, the viscosity of the solution (or a beverage or beverage additive containing the solution described herein) is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, or about 250 mPa·s (including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 mPa·s). The viscosity can be measured using an AMETEK BROOKFIELD viscometer with spindle S61 at room temperature (~20 °C).

[0042] In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for a period selected from 7 days, 14 days, 21 days, 1 month, 2 months, and 6 months. In some embodiments, the solution is stable against visible precipitation of the protein after storage at 4°C for a period selected from 7 days, 14 days, 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, and 12 months, for example, 4 months or 8 months. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 7 days. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 14 days. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 21 days. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 1 month. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 2 months. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 4 months. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 6 months. In some embodiments, the solution (or a beverage or beverage additive containing the solution described herein) is stable against visible precipitation of the protein after storage at 4°C for 8 months.As shown above and in the Examples below, stability against precipitation can also be assessed by measuring absorbance at 280 nm, with an increase in absorbance correlating with protein solubilization, i.e., reduced or absent precipitation.

[0043] Beverage or beverage additive The protein solutions described herein may be formulated as or used to prepare beverages or beverage additives for human or animal consumption. Examples of beverages or beverage additives include, but are not limited to, nutritional drinks, sports drinks, functional protein drinks, dairy drinks, dairy smoothies, fruit drinks, fruit smoothies, coffee drinks, tea drinks, plant-based milks, dairy creamers, and non-dairy creamers. In some embodiments, the beverage or beverage additive is a nutritional drink, sports drink, functional protein drink, dairy drink, dairy smoothie, fruit drink, fruit smoothie, coffee drink, tea drink, plant-based milk, dairy creamer, and non-dairy creamer. The beverage or beverage additive may further comprise one or more of a fruit juice, a vitamin, and a flavoring agent.

[0044] As described above, in some embodiments, the beverage or beverage additive includes one or more acidic juices, such as a mixture with fruit and vegetable juices including acidic fruit juice and / or acidic vegetable juice, and includes one or more fruit or vegetable juices. Examples of such juices include apple juice, cherry juice, cranberry juice, grape juice, pineapple juice, pomegranate juice, grapefruit juice, guava juice, honeydew juice, lime juice, lemon juice, blackberry juice, orange juice, pineapple juice, raspberry juice, banana puree, apricot juice, peach juice, acai puree, acai juice, kiwi fruit juice, sugarcane juice, strawberry juice, watermelon juice, passion fruit juice, celery juice, carrot juice, potato juice, beet juice, parsley juice, tomato juice, cress juice, and turnip juice. As described above, in the present disclosure regarding a protein solution that is stable against precipitation at acidic pH, the protein solution can be formulated in or with fruit and / or vegetable juices in order to provide a fruit and / or vegetable juice-based, or fruit and / or vegetable-, or fruit and / or vegetable juice-flavored beverage or beverage additive containing the protein.

[0045] Preparation method Also described herein are methods for producing a stable protein solution as described herein, as well as methods for producing beverages and beverage additives. The method can include (a) adding a protein deamidase to a solution containing a protein and a stabilizer to obtain a mixture, (b) incubating the mixture, and (c) acidifying the mixture to obtain a solution having a pH of from about 3.5 to about 7.0. In some embodiments, the solution is prepared by mixing (i) a solution containing a protein and (ii) a solution containing a stabilizer. The method can include preparing a mixture containing a protein and a stabilizer, adding a protein deamidase to the mixture, and incubating the mixture. In some embodiments, the incubation is carried out until the enzyme reaction reaches a desired level of completion, which is optionally determined by the concentration of free ammonium ions in the solution. The method generally can include mixing a solution containing a protein with a solution containing a stabilizer to obtain a mixture containing the protein and the stabilizer, adding a protein deamidase to the mixture, and incubating the mixture. Mixing and adding can be carried out in any order. In some embodiments, mixing is completed before the enzyme is added.

[0046] The method also can include adjusting the pH of the solution to a pH of from about 3.5 to about 7.0, for example, by acidifying the solution to a pH of from about 3.5 to about 7.0. In some embodiments, acidifying the solution includes adding an acidic juice or juice concentrate, such as an acidic fruit juice or acidic fruit juice concentrate, and / or an acidic vegetable juice or acidic vegetable juice concentrate. In some embodiments, the solution is acidified by two or more acidifying agents, such as an acidic additive and an acidic juice or juice concentrate. In some embodiments, the acidifying agent is added for other purposes, such as flavoring the solution or enhancing the nutritional or functional food content, and the acidic pH results from the amount of acidifying agent added for that purpose.

[0047] The incubation conditions can be any incubation conditions suitable for the particular protein deamidation enzyme used, such as any temperature and pH at which the enzyme is active, and any time necessary to achieve the desired deamidation level. In some embodiments, the progress of the deamidation reaction is monitored, for example, by measuring the concentration of free ammonium ions in the solution. For example, when the concentration of free ammonium ions in the solution reaches a specific level, the reaction may be considered complete. For a solution having the protein amount described herein, when the concentration of free ammonium ions in the solution reaches from about 0.002% to about 0.07% w / v based on the volume of the solution, for example, from 0.002% to 0.07% w / v based on the volume of the solution (about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, or about 0.07% w / v, or including 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06% or 0.07% w / v), the reaction can be considered complete. The incubation conditions can include agitation. The agitation can be at a low speed (e.g., about 150 to about 250 rpm) or a high speed (e.g., about 3,000 to about 5,000 rpm). In some embodiments, the agitation is performed using a shaker at an agitation in the range of about 150 to about 250 rpm. In some embodiments, the agitation is performed using a shaker at an agitation in the range of about 3,000 to about 5,000 rpm.

[0048] The incubation step may be carried out at a temperature of about 30°C to about 70°C for about 0.5 hour to about 48 hours at a pH of about 3.0 to about 8.0. Generally, incubation is carried out at a temperature of about 40°C to about 60°C for about 3 hours to about 24 hours at a pH of about 5.0 to about 8.0. In some embodiments, incubation is carried out at a temperature of about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C. In some embodiments, incubation is carried out for about 0.5 hour, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or about 48 hours. In some embodiments, incubation is carried out at a pH of about 3, about 3.5, about 4, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0.

[0049] In some embodiments, the protein deamidase is protein glutaminase Amano 500 (PGA500), and incubation is carried out at 50°C for 3 hours at a pH of about 5.0 to about 8.0. In some embodiments, the protein deamidase has or comprises the amino acid sequence of SEQ ID NO: 1, and incubation is carried out at 50°C for 3 hours at a pH of about 5.0 to about 8.0. In some embodiments, the protein deamidase is a variant of SEQ ID NO: 1 described herein, and incubation is carried out at 50°C for 3 hours at a pH of about 5.0 to about 8.0.

[0050] The solution may be subjected to one or more further processing steps, such as addition of one or more flavor or nutritional components, heat treatment, homogenization, filtration, sterilization, and pasteurization.

[0051] In some embodiments, the method further comprises subjecting the solution to a heat treatment, for example, a heat treatment at about 75°C to about 95°C for about 5 minutes to about 20 minutes. In some embodiments, the heat treatment is performed at about 75°C, about 80°C, about 85°C, about 90°C, or about 95°C for about 5 minutes, 10 minutes, 15 minutes, or about 20 minutes. In some embodiments, the heat treatment is performed at about 85°C for about 10 minutes.

[0052] In some embodiments, the process further comprises subjecting the solution to homogenization. In some embodiments, the homogenization is performed at a pressure of about 2,000 psi to about 20,000 psi, for example, about 2,000 psi to about 2,500 psi. In some embodiments, the homogenization is performed at a pressure of about 2,000 psi, about 5,000 psi, about 10,000 psi, about 15,000 psi, or about 20,000 psi. In some embodiments, the homogenization is performed at a pressure of about 2,000 psi, about 2,500 psi, about 3,000 psi, about 3,500 psi, about 4,000 psi, about 4,500 psi, or about 5,000 psi.

[0053] In some embodiments, the process further includes subjecting the solution to sterilization. In some embodiments, the sterilization is performed using high temperature short time (HTST) sterilization, ultra-high temperature (UHT) sterilization, or low temperature long time (LTLT) sterilization. In some embodiments, the sterilization is performed using high temperature short time (HTST) sterilization. In some embodiments, the sterilization is performed using high temperature short time (HTST) sterilization at about 90°C to about 110°C for about 5 seconds to about 30 seconds. In some embodiments, the sterilization is performed using high temperature short time (HTST) sterilization at about 100°C for about 10 seconds to about 20 seconds. In some embodiments, the sterilization is performed using ultra-high temperature (UHT) sterilization. In some embodiments, the sterilization is performed using ultra-high temperature (UHT) sterilization at about 110°C to about 130°C for about 1 second to about 10 seconds. In some embodiments, the sterilization is performed using ultra-high temperature (UHT) sterilization at about 120°C for about 1 second to about 3 seconds. In some embodiments, the sterilization is performed using low temperature long time (LTLT) sterilization. In some embodiments, the sterilization is performed using low temperature long time (LTLT) sterilization at about 65°C to about 95°C for about 5 minutes to about 30 minutes. In some embodiments, the sterilization is performed using low temperature long time (LTLT) sterilization at about 75°C to about 85°C for about 10 minutes to about 20 minutes.

[0054] In some embodiments, the process further includes subjecting the solution to aseptic treatment. In some embodiments, the aseptic treatment is performed using high pressure (high specific gravity) aseptic treatment.

[0055] The method for manufacturing the beverage or beverage additive described in this specification may include adding the stable protein solution described in this specification to a beverage or beverage additive composition, or preparing the solution described in this specification as a beverage or beverage additive. For example, the stable protein solution described in this specification can be added to a pre-prepared nutritional beverage, sports beverage, functional protein beverage, milk beverage, milk smoothie, fruit beverage, fruit smoothie, coffee beverage, tea beverage, plant-based milk, milk creamer or non-dairy creamer in an amount that imparts the desired amount of protein in the beverage or beverage additive. Alternatively, the stable protein solution described in this specification can be prepared as a nutritional beverage, sports beverage, functional protein beverage, milk beverage, milk smoothie, fruit beverage, fruit smoothie, coffee beverage, tea beverage, plant-based milk, milk creamer or non-dairy creamer, for example, containing other ingredients of such beverages and beverage additives, as well as the desired amount of protein.

[0056] In some embodiments, the final solution, beverage or beverage additive has a protein content of up to about 30% w / w (including about 30% w / w) on a weight basis of the protein in the solution. In some embodiments, the final solution, beverage or beverage additive has a protein content of about 0.5 to about 30% w / w (including 0.5 to 30% w / w, or about 5 to about 25% w / w, or about 10 to about 20% w / w) on a weight basis of the protein in the solution. In some embodiments, the final solution, beverage, or beverage additive has a protein content of about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% w / w on a weight basis of the protein in the solution.

[0057] In any embodiment, the beverage or beverage additive may further contain one or more components commonly present in beverages or beverage additives that include one or more of fruit or vegetable juice, vitamins, dietary supplements, flavoring agents, coloring agents, and preservatives. In some embodiments, the beverage or beverage additive includes one or more acidic fruit juices, including one or more selected from apple juice, cherry juice, cranberry juice, grape juice, pineapple juice, pomegranate juice, grapefruit juice, guava juice, honeydew juice, lime juice, lemon juice, blackberry juice, orange juice, pineapple juice, raspberry juice, banana puree, apricot juice, peach juice, acai puree, acai juice, kiwifruit juice, sugarcane juice, strawberry juice, watermelon juice, passion fruit juice, celery juice, carrot juice, potato juice, beet juice, parsley juice, tomato juice, cress juice, and turnip juice, and includes one or more acidic juices or one or more fruit and vegetable juices.

[0058] The following specific examples are included as illustrations of the compositions and methods described herein. These examples are in no way intended to limit the scope of the disclosure. Other aspects of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Example

[0059] Example 1: Mung Bean Protein Solution (Formulations 1 - 4) As shown in the following table, the mung bean protein solution was prepared using mung bean protein isolate in powder form (NOW Foods) as the protein, with and without gellan gum (Ticagel® Gellan HS NGMO, a high acyl gellan gum from TIC Gums) as the stabilizer, and with and without PGA500 (Amano Enzyme Inc.) as the protein deamidase.

[0060] For example, a 10% (w / v) aqueous solution of pea protein was prepared and mixed with a 0.2% (w / v) aqueous solution of gellan gum to obtain an aqueous solution containing 3% (w / v) pea protein and 0.03 - 0.05% (w / v) gellan gum. To the PGA500-containing solution, an enzyme was added in an amount of 2% (w / w) of the protein and incubated at 50°C for 3 hours. For the PGA500-free formulation, the solution was heated to 50°C without incubation. After acidifying to pH 4.0 - 4.5 with citric acid, it was heat-treated at 85°C for 10 minutes. Other formulations were prepared by a similar process.

[0061] The resulting solution was stored at 4°C in a VWR laboratory refrigerator, and after 24 hours and 72 hours, evaluations were performed by (i) visual inspection, (2) measurement of the absorbance at 280 nm of the soluble protein content in the supernatant, (3) viscosity measurement using a viscometer (AMETEK BROOKFIELD), and (4) pH measurement. The results are reported in the following table.

Table 1

[0062] This result indicates that the formulations according to the present disclosure (containing PGA500 and gum) (Formulation 2) were stable against protein precipitation at a pH of about 4.5, as shown by the appearance of dispersion versus separation and the higher absorbance (reflecting an increase in protein solubility).

[0063] Example 2: Soybean protein solutions (Formulations 5 - 12) As shown in the following table, soybean protein solutions were prepared using soy protein isolate in powder form (NOW Foods) as the protein, with and without gellan gum (Ticagel® Gellan HS NGMO from TIC Gums) as a stabilizer, and with and without PGA500 (Amano Enzyme Inc.) as a protein deamidating enzyme.

[0064] For example, a 10% (w / v) aqueous solution of soy protein was prepared and mixed with a 0.2% (w / v) aqueous solution of gellan gum to obtain an aqueous solution containing 3% (w / v) soy protein and 0.10% (w / v) gellan gum. To the PGA500-containing formulation, an enzyme was added in an amount of 2% (w / w) of the protein and incubated at 50 °C for 3 hours. For the PGA500-free formulation, the solution was heated to 50 °C without incubation. After acidifying to pH 4.0 - 4.6 with citric acid, it was heat-treated at 85 °C for 10 minutes. Other formulations were prepared by a similar process.

[0065] The resulting solution was stored at 4 °C in a VWR laboratory refrigerator and evaluated as described in Example 1 above. The results are reported in the following table.

Table 2

[0066] This result indicates that the formulations according to the present disclosure (containing PGA500 and gum) (Formulations 6 and 10) were stable against protein precipitation at acidic pH (about 4.0 - 4.5), as shown by the appearance of dispersion versus separation and higher absorbance.

[0067] Example 3: Peanut protein solutions (Formulations 13 - 16) As shown in the following table, peanut protein solutions were prepared using peanut protein powder (Tru-Nut Company) as the protein, with and without gellan gum (Ticagel® Gellan HS NGMO from TIC Gums) as a stabilizer, and with and without PGA500 (Amano Enzyme Inc.) as a protein deamidase.

[0068] For example, a 10% (w / v) aqueous solution of peanut protein was prepared and mixed with a 0.2% (w / v) aqueous solution of gellan gum to obtain an aqueous solution containing 3% (w / v) peanut protein and 0.02% (w / v) gellan gum. For the PGA500-containing formulation, an enzyme was added in an amount of 2% (w / w) of the protein and incubated at 50 °C for 3 hours. For the PGA500-free formulation, the solution was heated to 50 °C without incubation. After acidifying to pH 4.0 - 4.5 with citric acid, it was heat-treated at 85 °C for 10 minutes. Other formulations were prepared by a similar process.

[0069] The resulting solution was stored at 4 °C in a VWR laboratory refrigerator and evaluated as described in Example 1 above. The results are reported in the following table.

Table 3

[0070] This result shows that the formulation according to the present disclosure (containing PGA500 and gum) (Formulation 14) was more stable against protein precipitation at acidic pH (about 4.0), as indicated by the higher absorbance (reflecting an increase in protein solubility) compared to other formulations. The slight sedimentation observed may be due to the nature of the peanut protein and the fact that the processing conditions were not optimized for the peanut protein.

[0071] Example 4: Owl protein solution (Formulations 17 - 24) As shown in the following table, the owl protein solutions were prepared using owl powder (LITHIC) containing 68% (w / w) owl protein, with and without gellan gum (Ticagel® Gellan HS NGMO from TIC Gums) as a stabilizer, and with and without PGA500 (Amano Enzyme Inc.) as a protein deamidase.

[0072] For example, a 10% (w / v) aqueous solution of cricket powder was prepared and mixed with a 0.2% (w / v) aqueous solution of gellan gum to obtain an aqueous solution containing 3% (w / v) cricket powder and 0.03% (w / v) gellan gum. To the PGA500-containing solution, an enzyme was added in an amount of 2% (w / w) of the protein and incubated at 50 °C for 3 hours. For the PGA500-free formulation, the solution was heated to 50 °C without incubation. After acidifying to pH 4.0 - 4.5 with citric acid, it was heat-treated at 85 °C for 10 minutes. Other formulations were prepared by a similar process.

[0073] The resulting solution was stored at 4 °C in a VWR laboratory refrigerator and evaluated as described in Example 1 above. The results are reported in the following table.

Table 4

[0074] This result shows that the formulations according to the present disclosure (containing PGA500 and gum) (Formulations 18 and 22) were more stable against protein precipitation at acidic pH (about 4.0 - about 4.5), as indicated by having higher absorbance values (reflecting an increase in protein solubility) than other formulations. Since Formulations 21 - 24 are near pH 4.0 while Formulations 17 - 20 were prepared near pH 4.5 (with some measurement error), the lower pH of Formulation 22 (pH 4.0) compared to Formulation 18 (pH 4.5) indicates that the decrease in pH reduced the protein in the suspension, resulting in lower absorbance and viscosity being observed for Formulation 22.

[0075] Example 5: Hemp protein solution (Formulations 25 - 28) As shown in the following table, hemp protein solutions were prepared using hemp protein powder (Nutiva) as the protein, with and without carrageenan gum (Ticaloid® 750 from TIC Gums) as a stabilizer, and with and without PGA500 (Amano Enzyme Inc.) as a protein deamidase.

[0076] For example, a 10% (w / v) aqueous solution of hemp protein was prepared and mixed with a 1.0% (w / v) aqueous solution of carrageenan gum to obtain an aqueous solution containing 3% (w / v) hemp protein and 0.5% (w / v) carrageenan gum. To the PGA500-containing solution, an enzyme was added in an amount of 2% (w / w) of the protein and incubated at 50°C for 3 hours. For the PGA500-free formulation, the solution was heated to 50°C without incubation. After acidifying to pH 4.0 - 4.5 with citric acid, it was heat-treated at 85°C for 10 minutes. Other formulations were prepared by a similar process.

[0077] The resulting solution was stored at 4°C in a VWR laboratory refrigerator and evaluated as described in Example 1 above. The results are reported in the following table.

Table 5

[0078] This result shows that the formulation according to the present disclosure (containing PGA500 and gum) (Formulation 26) was stable against protein precipitation at acidic pH (about 4.5), as indicated by the appearance of dispersion versus separation and higher absorbance (reflecting an increase in protein solubility).

[0079] Example 6: Pea protein solutions at pH 3.5 - 7.0 (Formulations 29 - 37) As shown in the following table, pea protein solutions were prepared using pea protein isolate in powder form (NOW Foods) as the protein, with and without gums as stabilizers, and with and without PGA500 (Amano Enzyme Inc.) as the protein deamidase. The following gums from TIC Gums were used: gellan (Ticagel® Gellan HS NGMO), pectin (Pre-Hydrated® Pectin 1694 Powder), carboxymethyl cellulose (CMC, Pre-Hydrated® Ticalose® CMC 2500 Powder), alginate (TICA-algin® HG-400 Powder), and tara gum (TIC Pretested® Tara Gum 100).

[0080] For example, a 10% (w / v) aqueous solution of pea protein was prepared and mixed with an aqueous gum solution to obtain an aqueous solution containing 3% (w / v) pea protein and the gums at the concentrations (% w / v) shown in the following table. For the PGA500-containing formulations (formulation "A" in the following table), the enzyme was added in an amount of 2% (w / w) of the protein and incubated at 50 °C for 3 hours. For the formulations without PGA500 (formulation "B" in the following table), the solution was heated to 50 °C without incubation. The solution was acidified to pH 3.5 - 7 with citric acid (as shown in the table) and then heat-treated at 85 °C for 10 minutes. Other formulations were prepared by a similar process.

[0081] The resulting solutions were stored at 4 °C in a VWR laboratory refrigerator and evaluated first after 24 hours and then periodically for up to 2 months by (i) visual inspection, (ii) measurement of the absorbance at 280 nm of the soluble protein content of the supernatant, and (iii) pH measurement. The results are reported in the following table and shown in Figure 1.

Table 6

Table 7

[0082] This result indicates that the formulations according to the present disclosure (including PGA500 and gum) (Formulations 29A - 37A) were stable against protein precipitation at a pH of about 3.5 to about 7, as indicated by the appearance of dispersion versus separation and higher absorbance (reflecting an increase in protein solubility). On the other hand, formulations without protein deamidase were less stable at a pH of less than about 4.0.

[0083] Example 7: Soy Protein Solutions at pH 3.5 - 7.0 (Formulations 38 - 45) Soy protein isolate in powder form (NOW Foods) was used as the protein, and soy protein solutions were prepared and evaluated as described in Example 6. The results are reported in the following table and shown in Figure 2.

Table 8

Table 9

[0084] This result indicates that the formulations according to the present disclosure (including PGA500 and gum) (Formulations 38A - 45A) were stable against protein precipitation in the pH range of about 3.5 to about 7, as indicated by the appearance of dispersion versus separation and higher absorbance (reflecting an increase in protein solubility). In contrast, other formulations (including gum but not PGA500) were not stable against protein precipitation at a pH of less than 5.2 (see the results reported for pH 4.4 - 3.5).

[0085] Example 8: Hemp Protein Solutions at pH 3.5 - 6.5 (Formulations 46 - 52) Hemp protein powder (Nutiva) was used as the protein, and hemp protein solutions were prepared and evaluated as described in Example 6. The results are reported in the following table and shown in Figure 3.

Table 10

Table 11

[0086] As shown by this result, as indicated by the dispersed appearance and higher absorbance (reflecting an increase in protein solubility), the formulations according to the present disclosure (including PGA500 and gum) (Formulations 46A - 52A) were more stable against protein precipitation in the pH range of about 3.5 to about 7.

[0087] Example 9: Peanut protein solutions at pH 3.5 - 7.0 (Formulations 53 - 59) Peanut protein powder (Tru - Nut Company) and xanthan gum (Pre - Hydrated® Ticaxan® Xanthan EC NGMO from TIC Gums) were used as the protein, and peanut protein solutions were prepared and evaluated as described in Example 6. The results are reported in the following table and shown in Figure 4.

Table 12

Table 13

[0088] As shown by this result, as indicated by the dispersed appearance and higher absorbance (reflecting an increase in protein solubility), the formulations according to the present disclosure (including PGA500 and gum) (Formulations 53A - 59A) are stable against protein precipitation in the pH range of about 3.5 to about 7, while other formulations were less stable, especially at more acidic pH values.

[0089] Example 10: Kangaroo protein solutions at pH 3.5 - 7.0 (Formulations 60 - 67) Using cricket powder (LITHIC) containing 68% (w / w) cricket protein, a cricket protein solution was prepared and evaluated in the same manner as described in Example 6. The results are reported in the following table and shown in Figure 5.

Table 14

Table 15

[0090] This result indicates that the formulations according to the present disclosure (including PGA500 and gums) (Formulations 60A - 67A) were stable against protein precipitation in the pH range of about 3.5 to about 7, as indicated by the dispersed appearance and higher absorbance (reflecting increased protein solubility). Sedimentation was observed in Formulation 62A, which may be due to the fact that the specific amount of gellan gum used at pH 5.1 was not sufficient to completely prevent protein sedimentation. Most of the other formulations were less stable, especially at more acidic pH values (e.g., less than about 4.5). Without wishing to be bound by theory, it is possible that the cricket protein used contained impurities such as non - protein impurities and was not pure cricket protein.

[0091] Example 11: Homogenized pea protein solution (Formulations 68 - 71) As shown in the following table, pea protein solutions were prepared using pea protein isolate in powder form (NOW Foods) as the protein, with and without gums (gellan: Ticagel® Gellan HS NGMO; pectin: Pre - Hydrated® Pectin 1694 Powder, both from TIC Gums) as stabilizers, and with and without PGA500 (Amano Enzyme Inc.) as a protein deamidase.

[0092] For example, a solution was prepared by hydrating 0.075% (w / w) of gellan gum and 0.45% (w / w) of pectin in water. Soybean protein was added to obtain soybean protein solutions having different amounts of soybean protein as shown in the following table. To the PGA500-containing formulations, the enzyme was added in an amount of 0.67% - 1.8% (w / w) of the protein and incubated at 50 °C for 3 hours. For the PGA500-free formulations, the solution was heated to 50 °C without incubation. For acidification, 1 M (molar) citric acid was used to adjust to a specific pH. The acidified solution was homogenized at 2,000 - 2,500 psi and then heat-treated at 85 °C for 10 minutes. Other formulations were prepared by a similar process.

[0093] The activity of the protein deamidase enzyme was measured according to Example 16.

[0094] The resulting solutions were stored at 4 °C in a laboratory VWR refrigerator and evaluated as described in Example 6 above. The results are reported in the following table and shown in Figure 6.

Table 16

[0095] These results indicate that the formulations according to the present disclosure (including PGA500 and gums) (Formulations 68B - 71B) were stable against protein precipitation at acidic pH (about 4.0 - about 4.5), as indicated by the dispersed appearance and higher absorbance (reflecting increased protein solubility). These results show that the improved stability achieved with the formulations disclosed herein is maintained even after homogenization.

[0096] Example 12: Homogenized Soybean Protein Solutions Containing Juice Concentrates (Formulations 72 - 74) As shown in the following table, soy protein solutions were prepared using soy protein isolate in powder form (NOW Foods) as the protein, with and without gums (gellan: Ticagel® Gellan HS NGMO; pectin: Pre-Hydrated® Pectin 1694 Powder, both from TIC Gums) as stabilizers, and with and without PGA500 (Amano Enzyme Inc.) as the protein deamidase enzyme.

[0097] For example, solutions were prepared by hydrating 0.06% or 0.075% (w / w) gellan gum and 0.45% (w / w) pectin in water. Soy protein isolate was added to obtain soy protein solutions with different amounts of soy protein as shown in the following table. For the PGA500-containing formulations, the enzyme was added in an amount of 0.67% - 2.4% (w / w) of the protein and incubated at 50 °C for 3 hours. For the PGA500-free formulations, the solution was heated to 50 °C without incubation. For acidification, a berry juice concentrate (100% Juice Berry Blend concentrate with a Brix value of 65, from Old Orchard) was added at a 1:2 w / w ratio. The acidified solution was homogenized at 2,000 - 2,500 psi and then heat-treated at 85 °C for 10 minutes. Other formulations were made by a similar process. The activity of the protein deamidase enzyme was measured according to Example 16.

[0098] The resulting solutions were stored at 4 °C in a VWR laboratory refrigerator and evaluated as described in Example 6 above. The results are reported in the following table and shown in Figure 7.

Table 17

[0099] These results indicate that the formulations according to the present disclosure (including PGA500 and gums) (Formulations 72B - 74B) were stable against protein precipitation at acidic pH (from about 4.0 to about 4.5), as indicated by the dispersed appearance and higher absorbance (reflecting an increase in protein solubility). These results show that the improved stability achieved with the formulations disclosed herein is maintained even after homogenization.

[0100] Example 13: Homogenized Peanut Protein Solutions Containing Juice Concentrates (Formulations 75 - 77) As shown in the following table, peanut protein solutions were prepared using peanut protein powder (Tru-Nut Company) as the protein, with and without gums (gellan: Ticagel® Gellan HS NGMO; pectin: Pre-Hydrated® Pectin 1694 Powder, both from TIC Gums) as stabilizers, and with and without PGA500 (Amano Enzyme Inc.) as the protein deamidase.

[0101] For example, solutions were prepared by hydrating the amounts of gellan gum and pectin shown in the following table in water. Peanut protein was added to obtain peanut protein solutions with different amounts of peanut protein as shown in the following table. For the PGA500-containing formulations, the enzyme was added in an amount of 0.67% - 3.6% (w / w) of the protein and incubated at 50 °C for 3 hours. For the PGA500-free formulations, the solution was heated to 50 °C without incubation. For acidification, a berry juice concentrate (100% Juice Berry Blend concentrate with a Brix value of 65, from Old Orchard) was added at a 1:2 w / w ratio. The acidified solutions were homogenized at 2,000 - 2,500 psi and then heat-treated at 85 °C for 10 minutes. Other formulations were made by a similar process. The activity of the protein deamidase was measured according to Example 16.

[0102] The resulting solution was stored at 4 °C in a VWR laboratory refrigerator and evaluated as described in Example 6 above. The results are reported in the table below and shown in Figure 8.

Table 18

[0103] These results indicate that the formulations according to the present disclosure (including PGA500 and gums) (Formulations 75B - 77B) were stable against protein precipitation at acidic pH (about 4.0 - about 4.5), as indicated by the dispersed appearance and higher absorbance (reflecting increased protein solubility). These results show that the improved stability achieved with the formulations disclosed herein is maintained even after homogenization.

[0104] Example 14: Homogenized almond protein solutions (Formulations 78 - 79) As shown in the table below, almond protein solutions were prepared using almond protein powder (Noosh Brands) as the protein, with and without gums (gellan: Ticagel® Gellan HS NGMO; pectin: Pre - Hydrated® Pectin 1694 Powder, both from TIC Gums) as stabilizers, and with and without PGA500 (Amano Enzyme Inc.) as a protein deamidase.

[0105] For example, a solution was prepared by hydrating 0.06% (w / w) of gellan gum or 0.45% (w / w) of pectin in water. Almond protein was added to obtain an almond solution containing 3% (w / w) of almond protein. To the PGA500-containing formulation, an enzyme was added in an amount of 3.3% (w / w) of the protein and incubated at 50 °C for 3 hours. For the PGA500-free formulation, the solution was heated to 50 °C without incubation. For acidification, a berry juice concentrate (100% Juice Berry Blend concentrate with a Brix value of 65, from Old Orchard) was added at a 1:2 w / w ratio. The acidified solution was homogenized at 2,000 - 2,500 psi and then heat-treated at 85 °C for 10 minutes. The activity of the protein deamidase enzyme was measured according to Example 16.

[0106] The resulting solution was stored at 4 °C in a VWR laboratory refrigerator and evaluated as described in Example 6 above. The results are reported in the following table.

Table 19

[0107] These results indicate that the formulations according to the present disclosure (containing PGA500 and gum) (Formulations 78B - 79B) were stable against protein precipitation at acidic pH (about 4.0), as shown by the dispersed appearance and higher absorbance (reflecting increased solubility of the protein). These results indicate that the improved stability achieved with the formulations disclosed herein is maintained even after homogenization.

[0108] These results show that at a pH of about 4.0 to about 4.2, the comparative formulations (Formulations 78A and 79A) that do not contain PGA500 but contain gum exhibit separation and lower absorbance values (decrease in protein solubility), and in comparison, both of the PGA500 formulations containing gum (Formulations 78B and 79B) are shown to be stable, as indicated by higher absorbance values (increase in protein solubility) and no precipitation or sedimentation. These results indicate that the stability observed with the combination of PGA500 and gum is maintained even upon homogenization.

[0109] Example 15: Long-Term Stability Test The long-term stability test was conducted as follows. The formulations described below were mixed in a certified commercial pilot plant, aseptically packaged to produce a commercially sterilized stable product, and stored under refrigerated conditions (4 °C) for up to 6 months. These products would be stable against protein precipitation. [Table 20]

[0110] For example, the following process can be used to prepare a commercially sterilized formulation. 1. Weigh pectin gum, gellan gum, water, and pea protein. 2. Mix the gum and water under high shear conditions. 3. Heat to 85 °C and hold to activate the gum in the solution. 4. Transfer the gum aqueous solution to a 100 L vat and add the protein. 5. Add the enzyme to the vat and mix well. Transfer the solution to a container and incubate at 50 °C for 3 hours. 6. Weigh the juice concentrate and food coloring. 7. Return the solution to the 100 L vat and add the juice concentrate and food coloring. 8. Once well mixed, send the mixture to an ultra-high temperature (UHT; e.g., about 120 °C for about 1 second to about 3 seconds) / high temperature short time (HTST, e.g., about 100 °C for about 10 seconds to about 20 seconds) system for sterilization. 9. Send the sterilized product to a homogenizer to homogenize at 2000 psi. 10. Pack the product in bottles in a sterile environment, cap it, and transfer it to a storage box.

[0111] To date, stability tests have shown that the above protein deamidase formulation is stable after storage at 4 °C for 25 weeks.

[0112] Example 16: Protein Deamidase Activity Assay The activity of the protein deamidase can be measured by the following method, exemplified by citing the protein glutaminase deamidation activity. A similar assay can be performed for protein asparaginase deamidation activity using a substrate suitable for protein asparaginase deamidation (e.g., Z-Asn-Gly).

[0113] Prepare a test solution by adding 0.1 mL of an aqueous solution containing protein deamidase to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly (substrate for protein glutaminase deamidation activity assay), and incubate at 37 °C for 10 minutes. Terminate the reaction by adding 1 mL of 0.4 M trichloroacetic acid (TCA) solution. Prepare a blank solution by adding 0.1 mL of an aqueous solution containing protein deamidase to a solution containing 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly (for protein glutaminase deamidation activity assay) and 1 mL of 0.4 M trichloroacetic acid (TCA) solution, and incubate at 37 °C for 10 minutes. The amount of ammonia generated in the test solution by the reaction is measured using an ammonia test wako (manufactured by Wako Pure Chemical Industries, Ltd.) by determining the ammonia concentration using a calibration curve of ammonia concentration against absorbance (at 630 nm) prepared using an ammonia standard solution (ammonium chloride). The activity of the protein deamidase can be calculated as follows (1 unit = the amount of enzyme required to generate 1 μmol of ammonia per minute). Enzyme activity (U / mL) = (Ammonia concentration (mg / L) in the reaction solution) × (1 / 17.03) × (2.1 / 0.1) × (1 / 10) × Df In the formula, 17.03 is the molecular weight of ammonia, and 2.1 is the volume (mL) of the enzyme reaction system in the above protocol, and 0.1 is the volume (mL) of the enzyme solution in the above protocol, and 10 is the reaction time (minutes) in the above protocol, and Df is the dilution rate of the enzyme solution.

[0114] In summary, these examples demonstrate that stable protein solutions can be prepared for various proteins from various sources, as described herein, and that such protein solutions are stable against precipitation of proteins at acidic pH, including formulations acidified with fruit juice concentrate. The examples also show that the protein solutions formulated as described herein are stable after homogenization. [Sequence Listing] SEQ ID NO: 1 LASVIPDVATLNSLFNQIKNQSCGTSTASSPCITFRYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

Claims

1. 1. A stable protein solution comprising: (i) 0.1% to 30% w / v vegetable protein based on the volume of the solution; (ii) 0.001% to 5% w / v of a stabilizer based on the volume of the solution; and (iii) 0.5 U to 50 U of protein deamidase activity, or 0.1% to 10% w / w of protein deamidase based on the weight of the protein in the solution; A solution having a pH of 3.5 to 5.0 and which is stable against protein precipitation.

2. The solution according to claim 1 , wherein the protein deamidase is a protein glutaminase deamidase that deamidates an amide group of a glutamine residue of a protein.

3. The solution according to claim 1 , wherein the protein deamidase is a protein asparaginase deamidase that deamidates an amide group of an asparagine residue of a protein.

4. 4. The solution of any one of claims 1 to 3, wherein the vegetable protein comprises a vegetable protein selected from one or more of soy, pea, lentil, chickpea, legume, hemp, rice, nut, wheat and gluten protein.

5. 5. The solution of claim 4, wherein the nuts are peanuts, almonds, or hazelnuts.

6. The solution of any one of claims 1 to 5, wherein the stabilizing agent comprises one or more of a gum, a polysaccharide, and a collagen.

7. 7. The solution of claim 6, wherein the stabilizer comprises one or more of xanthan gum, gellan gum, carrageenan gum, cassia gum, locust bean gum, tara gum, psyllium seed gum, gelatin, tamarind seed gum, gum arabic, propylene glycol alginate, pectin, galactomannan (guar gum), pullulan, carboxymethylcellulose (CMC), methylcellulose (MC), and any derivatives or combinations thereof.

8. 8. The solution according to any one of claims 1 to 7, wherein the protein deamidase is produced by a bacterium selected from Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, Myroides, Cytophagales, Actinomycetes and Flavobacteriaceae.

9. The solution according to any one of claims 1 to 8, wherein the protein deamidase comprises an amino acid sequence of SEQ ID NO: 1 or a sequence having at least 85% identity thereto and having protein deamidase activity.

10. 9. The solution according to any one of claims 1 to 8, wherein the protein deamidase has protein deamidase activity and comprises a mutant amino acid sequence of SEQ ID NO: 1 having one or more substitutions or deletions at amino acid residues 35, 38 to 43, 45, 46, 49, 79 to 84, 103 to 106, 117, 142, 143, 146, 166, or 185 of SEQ ID NO: 1, optionally wherein the mutant sequence has at least 85% identity to SEQ ID NO:

1.

11. The solution according to any one of claims 1 to 10, (i) 0.001% to 1% w / v of a stabilizer based on the volume of the solution; and (ii) A solution comprising 5 U to 50 U of protein deamidase activity, or 1% to 10% w / w of protein deamidase based on the weight of protein in the solution.

12. The solution according to any one of claims 1 to 11, having a viscosity of 10 to 250 mPa·s.

13. 13. The solution of claim 12 comprising 5% to 15% w / v vegetable protein based on the volume of the solution.

14. 13. The solution of claim 12, comprising 0.01% to 1% w / v or 0.02% to 0.5% w / v of a stabilizer based on the volume of the solution.

15. 13. The solution according to claim 12, comprising 5 U to 25 U of protein deamidation activity, or 1% w / w to 5% w / w of protein deamidating enzyme based on the weight of protein in the solution.

16. 16. The solution according to any one of claims 1 to 15, having a pH of 4.0 to 5.

0.

17. 17. The solution of any one of claims 1 to 16, which is stable to visible precipitation of protein after storage at 4°C for a period selected from 7 days, 14 days, 21 days, 1 month, 2 months, 4 months, 6 months, and 8 months.

18. 18. The solution of any one of claims 1 to 17 formulated as a beverage or beverage additive for human or animal consumption.

19. A beverage or beverage additive for human or animal consumption comprising a solution according to any one of claims 1 to 18.

20. 20. The beverage or beverage additive of claim 19 selected from nutritional drinks, sports drinks, functional protein drinks, dairy drinks, dairy smoothies, fruit drinks, fruit smoothies, coffee drinks, tea drinks, plant-based milks, dairy creamers, and non-dairy creamers.

21. 21. The beverage or beverage additive of claim 19 or 20, further comprising one or more of fruit juice, fruit juice concentrate, vegetable juice, and vegetable juice concentrate.

22. 22. The beverage or beverage additive of claim 21, wherein the composition comprises an acidic juice or juice concentrate.

23. A method for producing a solution according to any one of claims 1 to 18 or a beverage or beverage additive according to any one of claims 19 to 22, comprising the steps of: (a) adding the protein deamidase to a solution containing the vegetable protein and the stabilizer to obtain a mixture; (b) incubating the mixture; and (c) acidifying the mixture to obtain a solution having a pH of 3.5 to 5.

0.

24. 24. The method of claim 23, further comprising the step of mixing (i) the solution comprising the vegetable protein and (ii) the solution comprising the stabilizer prior to step (a).

25. 24. The method of claim 23, wherein the incubation is carried out at a temperature between 30° C. and 70° C. for a period between 0.5 h and 48 h, at a pH between 3.0 and 8.0, and with stirring.

26. 26. The method of claim 25, wherein the incubation is carried out at a temperature between 40° C. and 60° C. for a period between 3 hours and 24 hours, at a pH between 5.0 and 8.0, and with slow stirring.

27. 27. The method of any one of claims 23 to 26, wherein the incubation is carried out until the enzymatic reaction reaches a desired level of completion, which is determined by the concentration of free ammonium ions in the solution.

28. 28. The method of any one of claims 23 to 27, wherein the acidification comprises adding an acidic juice or juice concentrate.

29. The method according to any one of claims 23 to 28, wherein the protein deamidase has the amino acid sequence of SEQ ID NO: 1, and the incubation is performed at 50°C for 3 hours.

30. 30. The method of any one of claims 23 to 29, further comprising sterilizing the solution and subjecting it to heat treatment at 85°C for 10 minutes.

31. The method of any one of claims 23 to 30, further comprising subjecting the solution to one or more treatments selected from homogenization, pasteurization, and sterilization.

32. 32. The method of claim 31, wherein the solution is subjected to homogenization at a pressure of from 2,000 psi to 20,000 psi.

33. 32. The method of claim 31 , wherein the solution is subjected to high temperature short time (HTST) sterilization at 100° C. for 10 seconds to about 20 seconds, ultra high temperature (UHT) sterilization at 120° C. for 1 second to 3 seconds, or low temperature long time (LTLT) sterilization at 75° C. to 85° C. for 10 minutes to 20 minutes.

34. 32. The method of claim 31 , wherein the solution is subjected to high pressure (high specific gravity) sterilization.

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