Protein formulations

Abstract

The present invention relates to a protein formulation comprising a protein formulation comprising a protein and a protective agent comprising (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitant and / or chelating molecule.

Description

[0001] The present invention relates to a protein preparation, a food comprising the protein preparation, and a method for preparing the protein preparation.

[0002] Proteins, especially enzymes, are becoming increasingly widely used as active agents in the food industry, one reason being their positive effect on the gastrointestinal health of animals.

[0003] However, many proteins used in industrial processes lack sufficient stability under the conventional conditions for manufacturing protein preparations or foods comprising such protein preparations.

[0004] For example, many proteins are heat-sensitive and thus at least partially degrade upon heating, such as during extrusion of the protein with other food ingredients or pelletizing. This is a disadvantage because many types of foods, such as animal feed, are preferably formulated in the form of pellets for reasons of storage efficiency, stability, and ease of handling.

[0005] EP2497372 has addressed this problem by describing a granule comprising a core, an active agent (such as an enzyme), and at least one protective coating. The coating is intended to prevent moisture migration into the core containing the active agent. Generally, the coating comprises a moisture-barrier coating that slows down the rate of moisture migration into the granule and / or a moisture-retentive coating that absorbs moisture, thereby hindering or slowing down the extent or rate of external moisture entering the core.

[0006] However, such coated granules still suffer severe degradation after pelletizing at a high temperature of 90 °C, and thus an additional step of applying a coating on the core containing the enzyme is still required.

[0007] The inventors of the present invention have developed a protein preparation that overcomes one or more of the above disadvantages. Specifically, the inventors have developed a protein preparation having similar or improved stability compared to a reference protein preparation, specifically similar or improved heat stability of the protein and / or similar or improved storage stability. The protein preparation comprises a protective agent, which can be mixed with the protein and optionally other components to obtain the protein preparation according to the present invention without a coating step. The advantage is that the protein preparation can be prepared in a more time-saving and cost-effective manner.

[0008] Accordingly, the present invention relates to a protein preparation comprising a protein and a protective agent, the protective agent comprising (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule.

[0009] Drawings

[0010] Figure 1: Power input required to mix wheat flour with an increasing aqueous mixture of diammonium sulfate (A), sodium chloride (B), sodium sulfate (C), triammonium sulfate (D), and ammonium acetate (E). For various salts, as the amount of salt increases, the energy required for mixing decreases.

[0011] Figure 2 : Power input required to mix an aqueous mixture of wheat flour (A), starch (B), wheat semolina (C), and whole wheat flour (D) with an increasing amount of diammonium sulfate. The results show that in the presence of gluten and ammonium salts, the energy required for mixing decreases.

[0012] Figure 3 : Relative enzyme activity of phytase preparations after heat stability testing. The results show that in the presence of sodium chloride (A), ammonium acetate (B), ammonium chloride (C), or ammonium sulfate (D), the relative enzyme activity does not increase or even decreases, but in the presence of ammonium citrate (E), diammonium hydrogen citrate (F), diammonium hydrogen phosphate (G), and diammonium tartrate (H), the relative enzyme activity increases.

[0013] Figure 4 : Relative enzyme activity of peptidase preparations (A), cellulase preparations (B), and chitinase preparations (C) after heat stability testing in the presence of sodium chloride or ammonium citrate. The results show that in the presence of ammonium citrate, the enzyme activity after heat stability testing increases, but in the presence of sodium chloride, it does not increase.

[0014] Figure 5 : Relative phytase activity (A) of phytase preparations after exposure on a technical scale. Relative phytase activity loss (B) during extrusion at increasing pressures. The results show that at higher pressures, more phytase activity is lost.

[0015] Figure 6 : Relative phytase activity of spray-dried phytase preparations in the presence (striped columns) and absence (black columns) of ammonium citrate. The results show that the enzyme preparations according to the present invention can also be prepared by spray-drying means.

[0016] Figure 7 : Relative phytase activity with increasing glucose concentration. The results show that after heat stability testing, the phytase activity decreases with increasing glucose concentration.

[0017] Figure 8 : Relative phytase activity of enzyme preparations after heat stability testing at increasing concentrations of iron(III) chloride in the presence (squares) and absence (circles) of ammonium citrate. The results show that in the presence of ammonium citrate, the phytase activity can be significantly increased.

[0018] Figure 9 : Relative phytase activity after heat stability testing of the enzyme preparation in the presence of ammonium citrate (triangle), sodium chloride (circle), and ammonium chloride (square). The results show that in the presence of ammonium citrate, the storage stability of the enzyme preparation can be significantly improved. Detailed implementation manners

[0019] Unless otherwise specified, the term "or" as used herein is defined as "and / or".

[0020] Unless otherwise specified, the term "a" or "an" as used herein is defined as "at least one / species".

[0021] When a noun (such as a compound, an additive, etc.) is mentioned in the singular form, it is intended to include the plural.

[0022] The term "substantially" or "essentially" is generally used herein to indicate that it has the general characteristics or functions of the specified content. When referring to a quantifiable feature, this term is generally used to indicate that it is greater than 30% of the maximum value of the feature, specifically greater than 50%, specifically greater than 70%, more specifically at least 90%, more specifically at least 95%, and even more specifically at least 98%. The term "substantially free of" as used herein generally means that the substance is absent (below the detection limit achievable by analytical techniques available on the effective filing date) or present in a low content that will not significantly affect the characteristics of the product substantially free of the substance.

[0023] In the context of the present application, the term "about" generally means a deviation of 15% or less, specifically 10% or less, more specifically 5%, 4%, 3%, 2%, 1%, 0.5% or less from a given value.

[0024] As used herein, "protein" refers to a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding a polypeptide. Generally, the chain contains at least 10 or more amino acids, preferably at least 15 or more amino acids, such as 20 or more amino acids. There is no upper limit to the number of amino acids that may be present in a protein, but generally, a protein has at most 40,000 amino acid residues, such as 35,000 amino acid residues. On average, a protein generally contains about 100 to about 1500 amino acid residues.

[0025] As used herein, "enzyme" refers to a protein that has the ability to catalyze biochemical reactions under physiological conditions. The enzymes herein can be referred to by their corresponding Enzyme Commission (EC) numbers, which are determined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) (https: / / iubmb.qmul.ac.uk / enzyme / index.html; accessed June 17, 2022). Examples of enzymes include hydrolases such as phosphatases and peptidases.

[0026] In the art, "reducing sugar" is generally considered to refer to a sugar molecule that contains an aldehyde group with reducing agent activity in its open form. Generally, reducing sugars can exist either in a closed form, in which the reducing sugar forms a 5- or 6-membered ring with a hemiacetal or hemiketal functional group, or in an open form, in which the hemiacetal of the closed form is opened to form a linear structure with an aldehyde group at the end. Examples of reducing sugars include glucose, fructose, lactose, and maltose.

[0027] As used herein, "amine" is generally understood in the art to refer to a functional group having the structural formula NR 1 R 2 R 3 wherein R 1 、R 2 and R 3 can be the same or different and can be any group that can provide a chemically stable molecule under physiological conditions. One example of an amine is NH 3 , wherein each R 1 、R 2 and R 3 represents a hydrogen group ("H").

[0028] As used herein, "ammonium" is generally understood in the art to refer to a functional group having the structural formula N + R 1 R 2 R 3 H, wherein R 1 、R 2 and R 3 can be the same or different and can be any group that can provide a chemically stable molecule under physiological conditions. One example of ammonium is the NH 4 + ion, wherein each R 1 、R2 and R 3 represents a hydrogen group ("H").

[0029] As used herein, "metal" generally refers to elements in Groups 1 to 13 of the periodic table, excluding hydrogen and boron, or elements selected from tin (Sn), lead (Pb), bismuth (Bi), and polonium (Po). Examples of metals include transition metals, alkali metals, alkaline earth metals, lanthanides, and actinides.

[0030] As used herein, the term "transition metal" generally refers to elements selected from the following groups: Group 3, Periods 4 and 5; Group 4, Periods 4 to 7; Group 5, Periods 4 to 7; Group 6, Periods 4 to 7; Group 7, Periods 4 to 7; Group 8, Periods 4 to 7; Group 9, Periods 4 to 6; Group 10, Periods 4 to 6; Group 11, Periods 4 to 6; and Group 12, Periods 4 to 7. The transition metal can be in any oxidation state, but is preferably a divalent or trivalent metal ion.

[0031] A salt is defined as a compound formed by the chemical combination of an acid and a base or by neutralization. When ions are bonded together by ionic bonds, a salt can be formed. When dissolved in a solvent (such as water), a salt can dissociate into ions (except H + or OH - ). In the art, "inorganic salt" is generally understood to mean a salt whose backbone does not contain C-H bonds. Examples of inorganic salts include sodium chloride, ammonium phosphate, etc. In the art, "organic salt" is generally understood to mean a salt that is not inorganic, i.e., a salt whose backbone does contain C-H bonds. Examples include citrate or tartrate.

[0032] The term "nucleophile" or "nucleophilic group" is generally understood in the art to refer to a molecule or functional group that can supply electrons by reacting with an electrophilic group. Generally, a nucleophile has a high electron density, for example, due to the presence of a free electron pair (also called a lone electron pair) and / or an adjacent electron-donating group. Herein, an "electrophile" or "electrophilic group" is understood in the art to refer to a molecule or group that can accept electrons. Generally, an electrophile has a low electron density, for example, due to the presence of an adjacent electron-withdrawing group. An "electron-withdrawing" group is generally understood to refer to a group that can reduce the electron density of an adjacent group, for example, by polarization (induction) or by stabilization through electron delocalization. In the art, an electron-donating group is generally understood to refer to a group that can increase the electron density of an adjacent group, for example, by polarization (induction) or delocalization of electrons.

[0033] In the context of the present application, the terms "chelating agent", "chelating molecule", or "chelator" are used interchangeably herein and generally refer to a molecule capable of binding to a metal ion (preferably a transition metal ion) to form a complex. Generally, a chelating molecule is a multidentate or bidentate molecule that contains at least two functional groups capable of coordinating to a metal ion (preferably a transition metal ion, more preferably a divalent or trivalent transition metal ion). Examples of divalent transition metal ions (M 2+ ) that can be coordinated by a chelating molecule include Cu 2+ , Co 2+ , Ni 2+ , Mn 2+ , Zn 2+ and Fe 2+ . One example of a trivalent metal ion (M 3+ ) is Fe 3 + . Examples of functional groups capable of coordinating to a transition metal ion include carboxylate groups and amines.

[0034] In the context of the present application, a "metal precipitant" generally refers to a reagent capable of forming a complex or salt with a metal ion (preferably a transition metal ion), which complex or salt is insoluble or has limited solubility in an aqueous medium at about 25 °C, atmospheric pressure, and physiological pH. Solubility in an aqueous medium can be determined by the naked eye, for example, by observing precipitates, crystals, haze, turbidity, etc., or can be detected using analytical means, such as using high performance liquid chromatography.

[0035] Those skilled in the art will understand that in the context of the present application, the metal precipitant can form a salt or complex with a metal ion (preferably a transition metal ion) that has limited solubility in any suitable aqueous medium. Examples of suitable aqueous media include limited solubility in an aqueous solution (such as an aqueous enzyme solution), and limited solubility in the aqueous phase of an aqueous emulsion or aqueous suspension. One example of an aqueous suspension is a dough containing protein, water, and flour. Herein, limited solubility can refer to limited solubility in the aqueous phase of the dough.

[0036] The term "dough" as used herein generally refers to a mixture containing at least flour, water, protein, and a protecting agent, which has a viscoelastic consistency. Dough can generally be kneaded using a suitable tool (such as a hand or a mechanical mixer). Dough is generally capable of substantially maintaining its shape without being fixed by a tool (such as a container).

[0037] In the context of the present application, "food" refers to any product that can be consumed by animals (including humans), which means that the ordinary health authorities (such as the FDA and / or EFSA) do not recommend dietary intake.

[0038] Protein preparation

[0039] It is believed that the instability of proteins, specifically their instability to heat (usually a problem at 40 °C or higher) or over time at ambient temperature (usually at least 24 hours), may be at least partially due to degradation caused by reducing sugars and / or metal ions present in the protein preparation, for example, as residues in the protein production process.

[0040] Without being bound by any theory, it is contemplated that the amines present in the protein amino acid residues can react with the aldehyde groups of reducing sugars. This is known in the art as the "Maillard reaction". (Kaufmann, 2018, doctoral thesis; 'Dynamik der Zuckertautomerie und ihr Einfluss auf die Kinetik der Maillard-reaktion').

[0041] The Maillard reaction can be accelerated by metal ions, specifically transition metal ions (Kato et al., 1981. J. Agric. Food Chem. 29, 540 - 543).

[0042] Thus, by introducing a protecting agent into the protein preparation and at least partially removing reducing sugars and metal ions as active components from the protein preparation, the proteins present in the preparation can be prevented from participating in the Maillard reaction. Compared with other protein preparations known in the art, this significantly improves the tolerance of the protein preparation to the presence of reducing sugars and metal ions, specifically transition metal ions. Therefore, the stability of the protein over time and / or at high temperatures can be significantly improved.

[0043] Accordingly, the present invention relates to a protein preparation comprising a protein and a protecting agent, the protecting agent comprising (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule.

[0044] Protein

[0045] The protein preparation according to the present invention can comprise any protein of interest, i.e., any protein having relevant commercial, nutritional, pharmaceutical or scientific uses.

[0046] For example, the protein can be an antibody, a peptide, preferably a therapeutic peptide, such as an antimicrobial peptide or an enzyme.

[0047] Preferably, the protein is a natural protein, more preferably an enzyme. Preferably, the protein is an enzyme that has value as a food ingredient in food, more preferably an enzyme in animal feed, in the household industry (such as detergents), in leather processing, or in waste fermentation (such as textile waste, waste paper, waste water, etc.).

[0048] Preferably, the protein in the protein preparation according to the present invention is an enzyme belonging to the following classes: oxidoreductases (EC1), transferases (EC2), hydrolases (EC3), lyases (EC4), isomerases (EC5), ligases (EC6), or translocases (EC7). Most preferably, the enzyme in the enzyme preparation according to the present invention belongs to the class of hydrolases (EC3).

[0049] The protein in the protein preparation according to the present invention may be an enzyme from the class of oxidoreductases, such as an enzyme from subclasses EC1.1, EC1.2, EC1.3, EC1.4, EC1.5, EC1.6, EC1.7, EC1.8, EC1.9, EC1.10, EC1.11, EC1.12, EC1.13, EC1.14, EC1.15, EC1.16, EC.1.17, EC1.18, EC1.19, EC1.20, EC1.21, EC1.22, EC1.23, or EC1.97.

[0050] Alternatively, the protein in the protein preparation according to the present invention is an enzyme from the class of transferases, such as an enzyme from subclasses EC2.1, EC2.2, EC2.3, EC2.4, EC2.5, EC2.6, EC2.7, EC2.8, EC2.9, and EC2.10.

[0051] Preferably, the protein in the protein preparation according to the present invention is an enzyme from the class of hydrolases, such as an enzyme from the following: hydrolase subclasses acting on ester bonds (EC3.1), glycosidases (EC3.2), hydrolases acting on ether bonds (EC3.3), hydrolases acting on peptide bonds (EC3.4), hydrolases acting on carbon-nitrogen bonds other than peptide bonds (EC3.5), hydrolases acting on acid anhydrides (EC3.6), hydrolases acting on carbon-carbon bonds (EC3.7), hydrolases acting on halide bonds (EC3.8), hydrolases acting on phosphorus-nitrogen bonds (EC3.9), hydrolases acting on sulfur-nitrogen bonds (EC3.10), hydrolases acting on carbon-phosphorus bonds (EC3.11), hydrolases acting on sulfur-sulfur bonds (EC3.12), and hydrolases acting on carbon-sulfur bonds (EC3.13).

[0052] Most preferably, the protein is an enzyme from subclasses EC3.1, EC 3.2, or EC 3.4.

[0053] Examples of enzymes from subclass EC 3.1 include carboxyl ester hydrolases (EC 3.1.1), thioester hydrolases (EC 3.1.2), phosphoric monoester hydrolases (EC 3.1.3), phosphoric diester hydrolases (EC 3.1.4), triphosphoric monoester hydrolases (EC 3.1.5), sulfuric ester hydrolases (EC 3.1.6), diphosphoric monoester hydrolases (EC 3.1.7), triphosphoric ester hydrolases (EC 3.1.8), exodeoxyribonucleases producing 5'-phosphomononucleotides (EC 3.1.11), exodeoxyribonucleases producing 5'-phosphomononucleotides (EC 3.1.12), exoribonucleases producing 5'-phosphomononucleotides (EC 3.1.13), exoribonucleases producing 3'-phosphomononucleotides (EC 3.1.14), exonucleases acting on ribo- or deoxyribonucleic acid and producing 5'-phosphomononucleotides (EC 3.1.15), exonucleases acting on ribo- or deoxyribonucleic acid and producing 3'-phosphomononucleotides (EC 3.1.16), endodeoxyribonucleases producing 5'-phosphomononucleotides (EC 3.1.21), endodeoxyribonucleases producing 3'-phosphomononucleotides (EC 3.1.22), site-specific endodeoxyribonucleases for modified bases (EC 3.1.25), endoribonucleases producing 5'-phosphomononucleotides (EC 3.1.26), endoribonucleases producing 3'-phosphomononucleotides (EC 3.1.27), endoribonucleases that can act on ribo- or deoxyribonucleic acid and produce 5'-phosphomononucleotides (EC 3.1.30), and endoribonucleases that can act on ribo- or deoxyribonucleic acid and produce 3'-phosphomononucleotides (EC 3.1.31).

[0054] Examples of enzymes from subclass EC 3.2 include glycosidases (EC 3.2.1) or enzymes hydrolyzing N-glycosyl compounds (EC 3.2.2).

[0055] Examples of enzymes from subclass EC 3.4 include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl and tripeptidyl peptidases (EC 3.4.14), peptidyl dipeptidases (EC 3.4.15), serine-type carboxypeptidases (EC 3.1.16), metallo-carboxypeptidases (3.4.17), cysteine-type carboxypeptidases (3.4.18), omega peptidases (3.4.19), serine endopeptidases (3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metallo endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), and endopeptidases with unknown catalytic mechanism (EC 3.4.99).

[0056] Most preferably, the protein in the protein preparation according to the present invention is 3 - phytase (EC 3.1.3.8), 4 - phytase (also known in the art as 6 - phytase (EC 3.1.3.26)), 5 - phytase (EC 3.1.3.72), chitinase (EC 3.2.1.14), cellulase (EC 3.2.1.4), peptidase (EC 3.4), xylanase (EXC 3.2.1), amylase (EC 3.2.1), lipase (EC 3.1.1), mannanase (EC 3.2.1), pectinase (EC 3.2.1.15, β - glucanase (EC 3.2.1.6), α - galactosidase (EC 3.1.2.22).

[0057] In one aspect, the protein in the protein preparation according to the present invention is an enzyme from the lyase class, such as enzymes from subclasses EC4.1, EC4.2, EC4.3, EC4.4, EC4.5, EC4.6, EC4.7 and EC4.99.

[0058] Alternatively, the protein in the protein preparation according to the present invention is an enzyme from the isomerase class, such as enzymes from subclasses EC5.1, EC5.2, EC5.3, EC5.4, EC5.5 and EC5.99.

[0059] Alternatively, the protein in the protein preparation according to the present invention is an enzyme from the ligase class, such as enzymes from subclasses EC6.1, EC6.2, EC6.3, EC6.4, EC6.5 and EC6.6.

[0060] The protein in the protein preparation according to the present invention can be an enzyme from the translocase class, such as enzymes from subclasses EC7.1, EC7.2, EC7.3, EC7.4, EC7.5 and EC7.6.

[0061] The protein in the protein preparation according to the present invention can be produced in any manner known in the art. Preferably, the protein (preferably an enzyme) is expressed by a suitable microorganism in a culture medium or in cells, and then isolated from the culture medium or cells. Microorganisms suitable for expressing the protein (preferably an enzyme) include filamentous fungi, yeasts, bacteria and algae. The microorganism can be a wild - type microorganism or can be genetically modified, for example to increase the yield of the protein present in the protein preparation according to the present invention.

[0062] During protein expression, the protein (preferably an enzyme) is typically expressed in a culture medium or in cells and can subsequently be processed to obtain the protein from the culture medium or cells. Thus, the obtained protein (preferably an enzyme) can be obtained as a mixture that contains one or more other components derived from the microbial culture medium or cells, such as other proteins, peptides, amino acids, nucleic acids (such as DNA or RNA), residues of the microbial cells that produce the protein, carbohydrates, and lipids expressed by the microorganism.

[0063] The obtained protein (preferably an enzyme) can be at least partially purified to remove one or more of these components, or the crude protein mixture can be used directly in the protein preparation according to the present invention without extensive purification. Examples of purification include filtration, centrifugation, and column chromatography, including combinations thereof.

[0064] Examples of commercially available 6-phytases suitable for the present invention include Enzy Phostar from Kessler Nutrition GmbH.

[0065] Preferably, the protein preparation according to the present invention contains residues from the microorganism that produces the protein, and more preferably, the residues include cellular material (such as carbohydrates), including reducing sugars, lipids, including phospholipids and glycolipids, or proteins.

[0066] Preferably, the protein preparation according to the present invention contains one or more reducing sugars, such as galactose, glucose, mannose, fructose, rhamnose, galactosamine, xylose, or sialic acid. Preferably, the one or more reducing sugars include one or more of glucose, galactose, maltose, rhamnose, and fructose.

[0067] In a preferred embodiment, the protein preparation according to the present invention contains one or more reducing sugars produced by a microorganism, specifically the microorganism that expresses the protein present in the protein preparation, or where the one or more reducing sugars are present as residues from the culture medium.

[0068] The protein preparation preferably contains the cellular material of the microorganism that produces the protein, such as residues of the microorganism and / or compounds excreted by the microorganism. As used herein, the term "residue" refers to a compound that is generated or present during the protein production process, including derivatives of such compounds. The term "protein production process" includes any step related to at least partially separating the protein from the cells or culture medium, such as extracting the protein from the culture medium or cells.

[0069] Preferably, the protein preparation according to the invention comprises compounds present in microbial cell walls (such as cell walls of bacteria, yeast, fungi or algae), such as glycoproteins, chitin or glucans, or derivatives of such compounds, including their degradation products.

[0070] In another preferred aspect, the protein preparation according to the present invention comprises compounds present in microbial cell membranes (such as cell membranes of bacteria, fungi, yeast or algae), including phospholipids and proteins, including fragments thereof.

[0071] The protein preparation according to the present invention further preferably comprises compounds present in the cytoplasm of microorganisms, including nucleic acid molecules (such as plasmids, DNA and RNA ribosomes), intracellular membranes, proteins (including enzymes), lipids and carbohydrates.

[0072] In another preferred aspect, the protein preparation according to the present invention comprises microbially secreted compounds such as proteins, for example host cell proteins and derivatives thereof such as peptides and amino acids.

[0073] The protein preparation according to the invention further preferably comprises culture medium residues.

[0074] Examples of such compounds include nitrogen sources (e.g., peptone, glutamine, or nitrates), carbon sources (such as fermentable carbon sources, e.g., glucose, lactose, molasses, corn syrup), growth factors (such as vitamins), minerals (such as calcium, potassium, magnesium), metal ions (such as Zn 2+ , Cu 2+ , Fe 2+ or Fe 3+ ) and other decomposition products of these compounds.

[0075] Removing protein residues from fermentation or substratum requires one or more purification steps and challenging, especially removing such residues of traces. In addition, purification steps usually result in lower protein yields, because usually at least part of the protein will be lost during purification. Therefore, the advantage of protein formulations according to the present invention is that a certain amount of fermentation residues can be tolerated, specifically metal ions (such as transition metal ions and reducing sugars). This allows the use of crude protein or partially purified protein in protein formulations according to the present invention. Therefore, before protein is added to protein formulations according to the present invention, it is not necessary to fully purify it, thereby saving time and cost and avoiding unnecessary losses.

[0076] Those skilled in the art will recognize that the protein preparation complies with the legal requirements in the food context established by relevant authorities such as the Food and Drug Administration (FDA) or the European Food Safety Authority (EFSA). Thus, if there are cell substances, fermentation residues or other compounds, their contents are relatively low to ensure that the protein preparation complies with the legal requirements stipulated by the relevant agencies.

[0077] Alternatively or additionally, the reducing sugar and / or metal ions can be added, for example, in the form of a carrier or excipient, to the protein in the protein preparation. Examples of carriers include flour, specifically wheat flour and lactose.

[0078] The protein can be added to the protein preparation according to the present invention in any form, for example, in a dry form (such as powder or granule) or in a solution form.

[0079] Preferably, the protein is added to a solution, specifically an aqueous solution. The aqueous solution may contain one or more other components, for example, to stabilize the protein in the solution. Preferably, the aqueous solution contains a stabilizer, a solubilizer, a cofactor or a cosolvent. Preferably, the aqueous solution further contains glycerol. Glycerol can advantageously stabilize the protein by preventing the protein from aggregating during refolding.

[0080] Preferably, the amount of the protein present in the protein preparation according to the present invention is about 0.001% to about 50% by weight, preferably about 0.01% to about 40% by weight, about 0.1% to about 25% by weight, and most preferably about 0.2% to about 10% by weight based on the total dry weight of the protein preparation.

[0081] Protectant

[0082] The protein preparation according to the present invention further contains a protectant, which contains (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitant and / or chelating molecule.

[0083] Without being bound by any theory, it is believed that the amine or ammonium group of the protectant can undergo a Maillard or Maillard-type reaction with the reducing sugar (Sengar and Sharma, J Food Sci Technol. 2014. 51(9):1686 - 1696). Thus, any reducing sugar that may be present in the protein preparation will be inactivated, and the protein will no longer or at least to a lesser extent participate in the Maillard reaction, thereby avoiding the degradation of the protein.

[0084] Those skilled in the art will understand that the protectant can be present in the protein preparation according to the present invention in a molecular form containing an amine group. In this form, the protectant can include a chemical formula of NR 1 R2 R 3 The molecule of. In this form, the amine group contains a lone pair of electrons capable of reacting as a nucleophile with the aldehyde of a reducing sugar.

[0085] Alternatively or additionally, the protecting agent may be present in the protein preparation according to the present invention in the form of an ammonium group. In this form, the protecting agent may include a molecule with the chemical formula NR 1 R 2 R 3 H + X - The molecule of. In this form, the ammonium group can undergo a reversible reaction with the amine group. Generally, this reversible reaction can be represented by the following formula: Where HA represents an acidic group capable of providing H+ ions, and A- represents a basic group capable of accepting H + Ions. Those skilled in the art will understand that the substance A- does not necessarily carry a net negative charge and can be represented by any substance capable of accepting H+ ions (such as the carbonyl group or alcohol group present in a reducing sugar). Similarly, the substance HA is not necessarily neutral and can be represented by any substance capable of providing H + Ions, such as ammonium ions or ammonium salts.

[0086] Therefore, without being bound by any theory, it is believed that when the protecting agent contains an ammonium group, it can in-situ generate the corresponding amine, which can then participate in the Maillard-type reaction as described above. However, as those skilled in the art will understand, the electrons in the N-H bond can also directly react with the carbonyl group without generating an amine as an intermediate.

[0087] In the general formulas NR 1 R 2 R 3 And NR 1 R 2 R 3 H + X - In, the substituents R 1 、R 2 And R 3 In principle, can be any substituent capable of making the molecule chemically stable.

[0088] For example, the substituents R 1 、R 2 And R 3 Can each independently be selected from the following groups: hydrogen (H), alkyl groups, alkenyl groups, alkynyl groups, or aryl groups.

[0089] The alkyl can be a straight-chain alkyl group or a branched alkyl group, usually represented by the general formula C n H 2n+1It is represented that, where n can be any integer, preferably an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Preferably, the alkyl group is a methyl, ethyl, propyl, isopropyl or butyl group.

[0090] The alkyl group can also be a cyclic group, usually represented by the general formula C n H 2n It is represented that, where n can be any integer, preferably an integer selected from 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Examples of the cyclic alkyl group include pentyl, hexyl and heptyl groups.

[0091] The alkenyl group refers to any unsaturated aliphatic group that contains at least one double bond in its backbone. Thus, the alkenyl group can contain one double bond, or multiple double bonds, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 double bonds. The alkenyl can be straight-chain or branched. Examples of the alkenyl group include vinyl, propenyl, butenyl, etc.

[0092] The alkynyl group refers to any unsaturated aliphatic hydrocarbon that contains at least one triple bond in its backbone. Thus, the alkynyl can contain one triple bond, or multiple triple bonds, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 triple bonds. Examples of the alkynyl group include ethynyl, propynyl, butynyl, etc.

[0093] The aryl group refers to any group that contains an aromatic functional group in its backbone. An aromatic group known in the art refers to a planar cyclic group containing unsaturated bonds, where the unsaturated bonds are fully conjugated, which means that electrons can freely pass through the p-orbitals present in the aromatic ring. If a group or molecule has 4n + 2 π electrons (also known as the "Hückel rule" in the art), it is usually aromatic. The aryl group can include one aromatic group or multiple, such as two, three, four or five aromatic groups. Examples of the aromatic group include benzyl, phenyl, naphthyl or imidazole.

[0094] Wherein, the X - can be represented by any suitable negatively charged ion or molecule that is capable of interacting with the positively charged NR 1 R 2 R 3 H +The substance forms an ionic bond. Generally, the X− represents a small molecule. Preferably, the small molecule has a molecular weight of less than 1000 g / mol, more preferably less than 900 g / mol, less than 700 g / mol, less than 500 g / mol, less than 350 g / mol, and specifically less than 250 g / mol. Preferably, the small molecule has a molecular weight in the range of about 100 g / mol to about 900 g / mol, more preferably in the range of about 110 g / mol to about 500 g / mol, even more preferably in the range of about 120 g / mol to 350 g / mol, and most preferably in the range of about 140 g / mol to about 200 g / mol.

[0095] Preferably, X - represents an organic acid. More preferably, the organic acid is selected from citric acid, malic acid, malonic acid, oxalic acid, tartaric acid, lignosulfonate, humic acid, fulvic acid, pectin, amidated pectin, lactic acid, and uric acid. Most preferably, X - represents citric acid or tartaric acid.

[0096] Alternatively, X - may represent an inorganic molecule, preferably a metal ion precipitation group, and most preferably a phosphate group.

[0097] Preferably, the substituents R 1 、R 2 and R 3 are independently selected from hydrogen or hydrocarbon. Alternatively, the substituents may include one or more functional groups such as carbonyl, ester, carboxylic acid, halogen, amine, amide, nitrile, nitro group, or ether group.

[0098] Those skilled in the art will understand that the substituents R 1 、R 2 and R 3 at least partially determine the nucleophilicity and basicity of the amino group.

[0099] For example, when R 1 、R 2 and R 3 represent electron-donating groups, the nucleophilic properties of the amino group are enhanced because the electron density of the lone pair of electrons present on the amino group increases. Examples of electron-donating groups include alkyl groups such as linear alkyl groups including methyl, ethyl, propyl, or isobutyl groups and cyclic alkyls (such as pentyl or hexyl) as well as aromatic groups (such as benzyl or phenyl groups).

[0100] In addition, when R 1 、R 2 or R 3 represents an electron-withdrawing group, the nucleophilicity of the amino group is reduced because the density of the lone pair of electrons present on the amino group is reduced. One example of an electron-withdrawing group is the carbonyl group.

[0101] In addition, those skilled in the art know that due to steric hindrance caused by the presence of one or more substituents R 1 、R 2 and R 3 , compared with amines without said substituents, the presence of one or more substituents R 1 、R 2 and R 3 increases the basicity of the amine and reduces the nucleophilicity of the amine group compared with amines without said substituents.

[0102] Therefore, in the general formula NR 1 R 2 R 3 , it is preferred that at least one of R 1 、R 2 and R 3 is a hydrogen atom, more preferably at least two of R 1 、R 2 and R 3 are hydrogen atoms, and most preferably R 1 、R 2 and R 3 are all hydrogen atoms.

[0103] When one of R 1 R 2 R 3 in the general formula NR 1 、R 2 and R 3 is a hydrogen atom, the general formula is NR 1 R 2 H, where R 1 and R 2 are as defined above, provided that R 1 and R 2 are not hydrogen. This is referred to as a secondary amine in the art.

[0104] When two of R 1 R 2 R 3 in the general formula NR 1 、R 2 and R 3 are hydrogen atoms, the general formula is NR 1 H 2 , where R 1 is as defined above, provided that it is not a hydrogen atom. This is referred to as a primary amine in the art.

[0105] When R 1 R 2 R 3 in the general formula NR 1 、R 2 and R3 When all three are hydrogen atoms, the general formula is NH 3 , which is also called ammonia in the art. Preferably, the preservative herein contains ammonia.

[0106] In one aspect, the preservative contains amino acids, specifically amino acids selected from methionine, phenylalanine, tryptophan, asparagine, glutamine, serine, threonine, cysteine, lysine, arginine and histidine or their conjugate acids.

[0107] Preferably, the preservative in the protein preparation according to the present invention contains ammonia (NH 3 ), ammonium ions (NH 4 + ) or both. If the preservative contains ammonium ions, the ammonium ions are usually present in the form of salts, having negatively charged counterions or negatively charged molecules (X - ) as described above.

[0108] Preferably, the preservative in the protein preparation according to the present invention includes ammonium salts of organic acids, preferably ammonium salts of citric acid, malic acid, malonic acid, oxalic acid, tartaric acid, lactic acid, uric acid, lignosulfonic acid, humic acid, fulvic acid, pectin, amidated pectin, or ammonium salts of inorganic salts, preferably phosphates, carbonates, sulfides.

[0109] The preservative further contains at least one metal precipitant and / or chelating molecule.

[0110] Preferably, the metal precipitant and / or chelating molecule can bind transition metal ions, more preferably transition metals in the 4th or 5th period of the periodic table, even more preferably transition metal ions in the 4th period of the periodic table.

[0111] The transition metal is preferably divalent or trivalent transition metal ions.

[0112] The transition metal is preferably selected from scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc. Preferably, the transition metal ions are selected from Cu 2+ , Fe 2+ , Fe 3+ , Zn 2+ , Mn 2+ or Mb 2+ substances. These are transition metal ions that are often present in protein preparations, specifically as residues of the culture medium for culturing microorganisms to produce proteins.

[0113] Preferably, the preservative contains chelating molecules, more preferably metal ion chelating molecules, specifically transition metal ion chelating molecules, specifically Fe 3+ , Fe 2+, Cu 2+ , Mn 2+ , Mb 2+ and / or Co 2+ chelating molecules.

[0114] Without being bound by any theory, it is believed that chelating molecules facilitate the formation of stable, soluble complexes with metal ions (preferably transition metal ions). Thus, the metal ions can be prevented from accelerating the Maillard reaction that causes protein degradation in protein formulations.

[0115] Preferably, the protecting agent comprises citrate or tartrate, more preferably an ammonium citrate salt, including diammonium hydrogen citrate, diammonium tartrate, ammonium hydrogen tartrate, dihydrogen ammonium citrate, and ammonium citrate. Most preferably, the protecting agent comprises ammonium citrate or diammonium hydrogen citrate

[0116] Alternatively or additionally, the protecting agent comprises a metal precipitant, preferably a metal ion precipitant, most preferably a transition metal ion precipitant, specifically Fe 3+ , Fe 2+ , Cu 2+ , Mn 2+ , Mb 2+ , Co 2+ precipitants.

[0117] The metal precipitant preferably has the ability to form a complex or salt with a transition metal ion that has limited solubility in an aqueous medium (such as dough). Without being bound by any theory, it is believed that the metal precipitant has the ability to prevent the metal ions from diffusing in an aqueous medium (such as dough) and form a stable complex or salt with the metal ions that have limited solubility in the aqueous medium.

[0118] Thus, since the transition metal ions are removed from the reaction, the adverse effects of the transition metal ions on enhancing the Maillard reaction can be prevented.

[0119] Preferably, the solubility of the metal precipitant-metal ion complex or salt in water at 25 °C, atmospheric pressure, and physiological pH is at most 10,000 μg / l, preferably 5000 μg / l, more preferably at most 1000 μg / l, more preferably 500 μg / l, more preferably at most 100 μg / l, at most 80 μg / l, at most 60 μg / l, at most 40 μg / l, most preferably at most 25 μg / l.

[0120] Preferably, the solubility of the salt or complex of the metal precipitant and the transition metal ion in water at 25 °C, atmospheric pressure, and physiological pH is from about 5 to about 10,000 μg / l, such as from about 10 to about 1000 μg / l, preferably from about 15 to about 100 μg / l, from about 20 to about 50 μg / l, most preferably from about 21 to about 40 μg / l.

[0121] Generally, physiological pH herein refers to a pH of about 7.4, preferably from about 4 to about 10, more preferably from about 5 to about 9, from about 6 to about 8, and most preferably about 7, specifically 7 to 8.

[0122] Those skilled in the art will understand that solubility in water is a property of metal precipitant-metal ion complexes and is generally available in suitable substance databases such as "Gestis-Stoffendatenbank", https: / / gestis.dguv.de / list (accessed on July 20, 2022).

[0123] Experimentally, the solubility in water can be determined by conventional methods known in the art. For example, at 25 °C, atmospheric pressure, and physiological pH, the component whose solubility is to be measured can be gradually mixed into water until precipitation, haze, turbidity, etc. appear, which indicates that the maximum solubility in water has been reached.

[0124] Solubility can also be determined using analytical means such as high performance liquid chromatography.

[0125] Preferably, the metal precipitant is diammonium hydrogen phosphate, ammonium phosphate, ammonium dihydrogen phosphate, ammonium carbonate, ammonium bicarbonate, ammonium hydrogen sulfide, or ammonium sulfide.

[0126] Preferably, the metal precipitant is not a sulfate or a chloride.

[0127] The protective agent preferably comprises molecules capable of increasing the surface tension of water. Such molecules (usually salts) are also referred to in the art as "kosmotropic molecules". Different from chaotropic molecules, kosmotropic molecules generally contribute to the stability and structure of water-water interactions. Therefore, kosmotropic molecules can stabilize the intramolecular interactions in molecules such as proteins, thereby making the protein have a more stable and tighter fold. When the concentration of such kosmotropic molecules is high enough, the protein usually salts out from the aqueous phase in a non-denatured, tightly folded form. The protein salted out in a tight form usually contains a hydration layer around the protein, in which (kosmotropic) salt molecules are embedded.

[0128] As used herein, the term "salting out" refers to the precipitation of a protein from an aqueous medium induced by salt. Natural salting out of proteins can be distinguished, in which the protein salts out in a non-denatured form, and salting out of proteins in a denatured form. The latter usually occurs in the presence of salts that promote protein unfolding. Unfolded proteins are generally less soluble in water because the hydrophobic parts of the protein are exposed to water, thereby reducing solubility.

[0129] Without being bound by any theory, the inventors believe that by using a kosmotropic salt to increase the surface tension of water, the hydrophobic interaction between the protein and water will be enhanced. It is expected that the protein will respond to this by adopting a more compact protein fold to reduce its surface area, in order to minimize contact with the protectant. The inventors believe that this compact fold increases the stability of the protein, making the protein more resistant to external pressures such as mechanical and thermal stress.

[0130] Accordingly, the protectant preferably comprises a kosmotropic molecule, specifically a kosmotropic salt. This is particularly advantageous in the case where the protein preparation is solid, since solid protein preparations are often exposed to mechanical stress, for example during preparation or during downstream processing, for example in order to prepare a food or feed product comprising the protein preparation.

[0131] The ability of a molecule (usually a salt) to increase surface tension is determined by the Hofmeister series (Wingfield, Curr Protoc Protein Sci May 2001). Accordingly, a person skilled in the art can select a protectant that can increase the surface tension of water based on common general knowledge and the information provided herein.

[0132] A person skilled in the art will understand that the at least one amine and / or ammonium group may be comprised within the same molecule as the at least one metal precipitant and / or chelating molecule.

[0133] Accordingly, the present invention preferably relates to a protein preparation comprising a protein (preferably an enzyme) and a protectant, wherein the protectant is a molecule, preferably a salt, comprising (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating group.

[0134] Preferably, the protectant is a small molecule. In the art, a molecule is generally referred to as a small molecule if it is an organic molecule having a molecular weight below 1000 g / mol, preferably below 900 g / mol, more preferably below 700 g / mol, below 500 g / mol, below 350 g / mol, specifically below 200 g / mol. Preferably, the small molecule has a molecular weight of from about 80 g / mol to about 700 g / mol, more preferably from about 90 g / mol to about 500 g / mol, such as from about 100 g / mol to about 350 g / mol, specifically from about 110 g / mol to about 200 g / mol.

[0135] Preferably, the molecule is a salt comprising a cation and an anion, wherein the cation comprises ammonium and wherein the anion is a chelating agent or a metal precipitant.

[0136] Preferred examples of such molecules include ammonium citrate tribasic, diammonium hydrogen citrate, ammonium dihydrogen citrate, diammonium tartrate, ammonium hydrogen tartrate, ammonium phosphate, ammonium dihydrogen phosphate, and diammonium hydrogen phosphate.

[0137] Alternatively, the protective agent may comprise at least two molecules, wherein the first molecule comprises at least one amine and / or ammonium group, and the second molecule comprises at least one metal precipitant and / or chelating molecule. For example, the protective agent may comprise a first molecule containing an amine or ammonium group and a second chelating molecule, such as citric acid or its salts. Here, the citrate salt may be an ammonium salt or a different salt, such as a sodium salt.

[0138] Those skilled in the art will understand that in-situ formed protective agents are also covered by the present invention.

[0139] In the protein preparation according to the present invention, the weight ratio between the protein, preferably an enzyme, and the protective agent is preferably about 1:5, preferably about 1:10, preferably about 1:20, preferably about 1:30, preferably about 1:50, more preferably 1:100.

[0140] The present invention also preferably relates to a protein preparation that is at least partially tolerant to reducing sugars and / or metal ions, heat-stable and / or storage-stable. Preferably, after the protein preparation according to the present invention is subjected to a temperature of at least 50 °C, at least 20% of the protein present in the protein preparation still has activity. Most preferably, after the protein preparation according to the present invention is subjected to a temperature of at least 50 °C, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and most preferably 100% of the protein present in the protein preparation still has activity.

[0141] The activity of the protein can be determined using an appropriate activity assay, such as those described in Examples 3 and 4. To test the activity of 6-phytase, for example, the protocol according to ISO method 30024:2009 can be employed.

[0142] Preferably, the protein preparation according to the present invention is stable at a temperature of at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, at least 95 °C, at least 100 °C, at least 105 °C, at least 110 °C, at least 110 °C, and most preferably at least 120 °C. These are the conditions typically employed in extrusion or steam granulation processes.

[0143] Preferably, the protein preparation according to the present invention is stable at a temperature of 55°C to 150°C, more preferably at a temperature of 60°C to 140°C, 65°C to 130°C, 70°C to 125°C, 75°C to 120°C, 80°C to 115°C, 85°C to 110°C, 90°C to 105°C, 95°C to 100°C.

[0144] Preferably, at such a temperature, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and most preferably 100% of the enzyme still has activity. In a specific embodiment, after the protein preparation according to the present invention is subjected to a temperature of at least 90°C for at least 2 minutes, preferably at least 15 minutes, at least 50%, preferably at least 60% of the enzyme still has activity.

[0145] Generally, the protein preparation according to the present invention is subjected to heating, for example, heated to at least 50°C, preferably heated to at least 90°C, and the heating time is about 10 seconds to about 5 hours, preferably about 20 seconds to about 3 hours, preferably about 30 seconds to about 2 hours, about 45 seconds to about 1 hour, about 1 minute to about 45 minutes, about 1.5 minutes to about 30 minutes, and most preferably heated about 2 minutes to about 15 minutes.

[0146] The protective agent can be present in the protein preparation according to the present invention in any amount sufficient to enhance the thermal stability of the protein.

[0147] Preferably, the protective agent is present in an amount of about 5% to about 50% by weight, preferably about 10% to about 40% by weight, and most preferably about 15% to about 30% by weight based on the dry weight of the protein preparation.

[0148] Preferably, the protective agent is present in an amount of at least 5% by weight based on the dry weight of the protein preparation, more preferably at least 10% by weight, at least 15% by weight, at least 20% by weight, at least 25% by weight, at least 30% by weight, at least 35% by weight, at least 40% by weight, at least 45% by weight, and most preferably at least 50% by weight based on the dry weight of the protein preparation.

[0149] Preferably, the protective agent is present in an amount of at most 50% by weight based on the dry weight of the protein preparation, more preferably at most 45% by weight, at most 40% by weight, at most 35% by weight, at most 30% by weight, at most 25% by weight based on the dry weight of the protein preparation.

[0150] In a protein preparation containing a liquid medium, the protectant is preferably present in an amount of at least 1.3 mol / L, more preferably at least 1.4 mol / L, at least 1.6 mol / L, and even more preferably at least 1.8 mol / L.

[0151] Preferably, in a protein preparation containing a liquid medium, the protectant is present in an amount of at most 5 mol / L, more preferably at most 4 mol / L, at most 3.5 mol / L, such as at most 3 mol / L, specifically at most 2.5 mol / L.

[0152] Typical amounts of the protectant in such protein preparations are from about 1.3 mol / L to about 5 mol / L, specifically from about 1.5 mol / L to about 3 mol / L. Here, the concentration is determined by calculating the total number of moles of the protectant present in the protein preparation / total volume of the liquid (in liters).

[0153] When this protectant concentration is reached in the protein preparation, the folding of the protein is usually tighter, and the protein usually precipitates out of the aqueous medium in such a tightly folded and non-denatured state.

[0154] The protein preparation according to the present invention may further comprise one or more carriers. In principle, any carrier that complies with the relevant regulations for protein preparations is suitable for the protein preparation according to the present invention. Preferably, the protein preparation according to the present invention contains flour, most preferably flour containing gluten, such as wheat flour, barley flour, rye flour, spelt wheat flour, triticale flour, and combinations thereof. Most preferably, the protein preparation according to the present invention contains wheat flour.

[0155] Such protein preparations are particularly suitable for extrusion because the protein preparation forms a dough at least in part due to the presence of gluten in the flour. Such dough can advantageously undergo one or more steps of an extrusion, (steam) granulation, or tableting process, as described below.

[0156] Preferably, the protein preparation containing flour contains at least 10% by weight of flour based on the total dry weight of the protein preparation, more preferably at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight of flour.

[0157] Preferably, the protein preparation containing flour contains from about 10% by weight to about 90% by weight of flour based on the total dry weight of the protein preparation, more preferably from about 20% by weight to about 80% by weight of flour, from about 30% by weight to about 60% by weight of flour, from about 40% by weight to about 50% by weight of flour.

[0158] Preferably, the protein preparation according to the present invention further comprises an excipient. In principle, any excipient that complies with the relevant regulations for protein preparations is applicable to the protein preparation according to the present invention. Preferably, the protein preparation according to the present invention further comprises microcrystalline cellulose.

[0159] Preferably, the protein preparation containing flour comprises at least 1% by weight of microcrystalline cellulose based on the total dry weight of the protein preparation, more preferably at least 2% by weight, at least 3% by weight, at least 4% by weight, at least 5% by weight of microcrystalline cellulose.

[0160] Preferably, the protein preparation containing flour comprises at most 30% by weight of microcrystalline cellulose based on the total dry weight of the protein preparation, more preferably at most 25% by weight, at most 20% by weight, at most 18% by weight, at most 15% by weight of microcrystalline cellulose.

[0161] Preferably, the protein preparation containing flour comprises about 1 to about 30% by weight of microcrystalline cellulose based on the total weight of the protein preparation, more preferably about 2% by weight to about 25% by weight of microcrystalline cellulose, about 5% by weight to about 20% by weight of flour.

[0162] Preferably, the protein preparation according to the present invention comprises about 3% by weight to about 25% by weight of a protective agent based on the total dry weight, preferably about 5% by weight to about 20% by weight of a protective agent, most preferably about 10% by weight to about 15% by weight of a protective agent, and about 30% by weight to about 70% by weight of flour, preferably wheat flour, preferably about 40% by weight to about 60% by weight of flour, preferably wheat flour, and about 5% by weight to about 25% by weight of microcrystalline cellulose, preferably about 10% by weight to about 20% by weight of microcrystalline cellulose, more preferably about 15% by weight to about 18% by weight of microcrystalline cellulose, and about 0.001% by weight to about 50% by weight of protein, more preferably 0.1% by weight to about 40% by weight of protein, about 1% by weight to about 30% by weight of protein, about 5% by weight to about 20% by weight of protein. Preferably, the protein is an enzyme, more preferably phytase, chitinase, cellulase or protease.

[0163] The protein preparation according to the present invention can be in any shape. For example, the protein preparation can be formulated as a solid, liquid, emulsion or suspension. Preferably, the protein preparation according to the present invention is formulated as a solid, as this is the densest form, can be stored efficiently and is easy to handle. In addition, solid protein preparations generally have longer storage stability compared to similar liquid protein preparations of the same protein.

[0164] Preferably, the protein preparation according to the present invention is formulated as (compressed) tablets, granules, powders, capsules or particulates.

[0165] Preferably, the protein preparation according to the present invention is formulated as a mixture of a protective agent and a protein as defined herein. More preferably, the protein preparation is formulated as a solid mixture to form a powder or granules.

[0166] Preferably, the particle size of such powders or granules is from about 10 μm to about 3 mm, preferably from about 50 μm to about 2 mm, more preferably from about 100 μm to about 1 mm, from about 200 μm to about 900 μm, from about 350 μm to about 800 μm, and most preferably from about 500 μm to about 75000 μm.

[0167] In the protein preparation according to the present invention, the protein, preferably the enzyme and the protective agent as defined herein, is preferably at least substantially uniformly distributed in the protein preparation according to the present invention. As used herein, the term "uniformly" generally means that the components are substantially uniformly distributed in the protein preparation according to the present invention. This can be tested by taking at least two samples from the protein preparation according to the present invention, and the samples should be substantially the same, for example having substantially the same composition and / or properties.

[0168] Preferably, in the protein preparation according to the present invention, the protective agent as defined herein does not exist as a coating, or the protein does not exist as a core containing a coating. Most preferably, the protein preparation according to the present invention is not formulated as a core containing a protein that is at least partially coated with the protective agent as defined herein.

[0169] The solid protein preparation according to the present invention may further optionally comprise a moisture-proof coating and / or a gas-proof coating, preferably comprising sugar.

[0170] Preferably, the protein preparation is an additive for food or animal feed.

[0171] (Food) product

[0172] The present invention further relates to a food, preferably animal feed, which comprises the protein preparation according to the present invention and one or more other food ingredients, such as carbohydrates, fats and protein substances. In principle, the food can be a food that benefits from the protein preparation according to the present invention.

[0173] Any fat suitable for consumption by animals (including humans) is suitable for the food according to the present invention. Such fats include animal fats (such as lard or butter) or vegetable oils. The fats may comprise saturated fatty acids, unsaturated fatty acids and polyunsaturated fatty acids and combinations thereof.

[0174] Examples of vegetable oils include rapeseed oil, sunflower oil, corn oil, soybean oil, coconut oil, palm oil, palm kernel oil, linseed oil, safflower oil, peanut oil and olive oil.

[0175] The fat may further comprise one or more omega-3 fatty acids, such as eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid.

[0176] Any carbohydrate suitable for consumption by animals, including humans, is applicable to the food according to the present invention, including digestible and indigestible carbohydrates.

[0177] Any protein suitable for consumption by animals, including humans, is applicable to the food according to the present invention. For example, a protein or any part of a protein, such as a non-hydrolyzed protein, a native protein, a hydrolyzed protein, a peptide (such as an oligopeptide, i.e., a peptide containing 2 to 50 amino acids), and free amino acids from any source can be used in the food according to the present invention. Preferably, the protein is a coagulated protein. The total content of the proteinaceous substance in the food according to the present invention can be determined by the Kjeldahl method known in the art. As understood by those skilled in the art, the Kjeldahl method measures the total nitrogen content, so to determine the protein content, the total content of the protecting agent needs to be subtracted.

[0178] The food may further comprise other food ingredients beneficial to humans (including animals), such as vitamins, minerals, and / or probiotics.

[0179] Examples of minerals suitable for addition to the food according to the present invention are sodium, chloride, potassium, calcium, phosphorus, magnesium, sulfur, iron, zinc, iodine, selenium, copper, manganese, fluoride, chromium, and / or molybdenum.

[0180] Examples of essential vitamins suitable for addition to the food preparation according to the present invention are vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6, vitamin B8 (biotin), vitamin B9, vitamin B10 (factor R), vitamin B11 (folic acid), vitamin B12, vitamin C, vitamin D, vitamin E, and / or vitamin K.

[0181] Preferably, the food is animal feed. Most preferably, the food relates to pet food (such as dog food, cat food, hamster food, guinea pig food, rodent food, mouse food, and rabbit food), or livestock food (such as horse food, cattle food, pig food, goat food, sheep food), and poultry food (such as chicken, duck, goose, and turkey food).

[0182] The food can be formulated into any form or shape, such as solid, such as in particulate form, tablet form, capsule form, powder form, or fluid form (such as gel, suspension, emulsion, or drink). Preferably, the food is solid, and most preferably particulate.

[0183] The present invention further relates to household products, preferably detergents comprising a protein preparation according to the invention and one or more surfactants, sequestering agents or bleaching agents.

[0184] The surfactant is preferably an anionic surfactant, such as a branched or linear anionic surfactant, or a non-ionic surfactant (such as a polyalkylene oxide condensate of an alkylphenol).

[0185] The sequestering agent can be any suitable sequestering agent, such as an aluminosilicate material, a silicate, a polycarboxylate or a fatty acid.

[0186] The bleaching agent can be any suitable bleaching agent, such as an oxygen bleaching agent or a halogen bleaching agent. Specifically, a bleaching agent capable of releasing hydrogen peroxide, such as a perborate or a hypohalite bleaching agent (such as trichloroisocyanuric acid).

[0187] The household product, preferably the detergent, may optionally comprise one or more other components, such as a foaming agent, a stabilizer, a solubilizer, a corrosion inhibitor, a dye transfer inhibitor, an aromatic, a brightening agent or a softening agent.

[0188] Methods and uses

[0189] The present invention further relates to a method for preparing a protein preparation according to the invention, which comprises mixing a protein with a protecting agent, the protecting agent comprising (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule. Preferably, the method further comprises mixing one or more reducing sugars and / or metal ions, specifically one or more transition metal ions.

[0190] The protein and the protecting agent comprise (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule, as described above.

[0191] The mixing of the protein with the protecting agent as defined herein can be carried out using any method in the art (such as by stirring, beating or agitating). Preferably, a mixture of the protein and the protecting agent that is at least substantially homogeneous is obtained, the protecting agent comprising (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule.

[0192] Preferably, the method further comprises the step of mixing water to form an aqueous mixture, which contains protein, a protecting agent and preferably one or more reducing sugars and / or metal ions. Preferably, the aqueous mixture contains at least 25% by weight of water, more preferably at least 40% by weight of water, at least 55% by weight of water, and most preferably at least 70% by weight of water, based on the total weight of the aqueous mixture. Preferably, the aqueous mixture contains about 25% by weight to about 70% by weight of water, specifically about 30% by weight to about 50% by weight of water, based on the total weight of the aqueous mixture.

[0193] Preferably, the concentration of the protecting agent in the aqueous mixture is about 1.3 mol / L to about 5 mol / L, specifically about 1.5 mol / L to about 3 mol / L.

[0194] Preferably, the method further comprises mixing a carrier to obtain a protein preparation containing flour, and the carrier is preferably flour selected from the group consisting of wheat flour, barley flour, rye flour, spelt wheat flour, triticale flour, and combinations or derivatives thereof (such as starch).

[0195] Preferably, the method further comprises the step of mixing one or more excipients, specifically microcrystalline cellulose.

[0196] Generally, the protein preparation containing flour and optionally microcrystalline cellulose is in the form of dough, which is suitable for further processing (such as extrusion, granulation, tablet pressing, etc.).

[0197] Preferably, the dough contains protein (preferably an enzyme), a protecting agent, flour, water and microcrystalline cellulose.

[0198] In the preferred method according to the present invention, the protein, the protecting agent, the carrier (preferably the wheat flour), the water and optionally the excipient (preferably microcrystalline cellulose) are subjected to a wet granulation process. In the wet granulation process, a mixture containing protein, a protecting agent, water, a carrier (preferably wheat flour) and optionally an excipient (preferably microcrystalline cellulose) is mixed to form dough, and then extruded in a suitable extruder to form extruded dough particles (also called particulate matter).

[0199] Conventional wet granulation processes generally require a large amount of energy to prepare dough containing protein and flour, which is not desirable from the perspectives of durability and economy. The inventors unexpectedly found that in the presence of salt, the energy required to prepare dough containing protein and flour can be significantly reduced. This is shown in Examples 1 and 2 of this application and Figure 1 and 2In addition, the results show that these effects can be obtained while the protein is not unfolded if a protecting agent is used as a salt rather than other salts such as sodium chloride.

[0200] This has the advantage that solid protein preparations such as particulate matter can be advantageously prepared at a lower pressure compared to the pressure required when no protecting agent is added, which can prevent or reduce the degradation of the protein in the protein preparation.

[0201] Accordingly, the present invention preferably relates to a method for preparing a protein preparation, comprising: a) providing a mixture of a protein (preferably an enzyme), a protecting agent, water, a carrier (preferably flour) and an optional excipient (specifically microcrystalline cellulose); b) kneading the mixture obtained in step a) to form a dough; c) molding, shaping, pressing, tableting, granulating or extruding the dough, preferably extruding.

[0202] Preferably, the molding, shaping, pressing, tableting, granulating or extruding is carried out at a temperature below 60 °C, more preferably below 50 °C, below 45 °C, below 40 °C, below 25 °C, most preferably below 20 °C. Specifically, the molding, shaping, tableting, granulating, pressing or extruding is carried out at a temperature of 10 °C to 60 °C, more preferably at 20 to 50 °C, even more preferably at 30 to 45 °C.

[0203] Preferably, the pressing, tableting, granulating or extruding is carried out at a pressure less than 40 bar, more preferably less than 30 bar, less than 20 bar, less than 15 bar, less than 10 bar, most preferably less than 5 bar.

[0204] Preferably, the pressing, tableting, granulating or extruding is carried out at a pressure of about 0.5 to about 40 bar, more preferably at about 1 to about 25 bar, about 2 to about 20 bar, about 3 to about 15 bar, most preferably at about 4 to about 10 bar, specifically about 5 to about 7 bar.

[0205] At such a pressure, it is advantageous that a heat-stable protein preparation can be obtained on a technical or industrial scale. Without being bound by any theory, it is believed that this may be due to the reduction of protein degradation by mechanically processing the protein preparation according to the present invention.

[0206] In another preferred embodiment, the method for preparing a protein preparation further comprises the step of spray-drying a mixture comprising a protein (preferably an enzyme), a protecting agent as defined herein (preferably one or more reducing sugars and / or metal ions (preferably transition metal ions)) and an optional carrier (preferably flour) to obtain a spray-dried protein preparation.

[0207] Preferably, the mixture further comprises water, more preferably comprising at least 20% by weight of water, at least 30% by weight of water, more preferably at least 40% by weight of water, at least 50% by weight of water, at least 60% by weight of water, at least 70% by weight of water, at least 80% by weight of water, most preferably at least 90% by weight of water, based on the total weight of the mixture.

[0208] Preferably, the mixture comprises from about 70% to about 99% by weight of water, specifically from about 80% to about 95% by weight of water, based on the total weight of the mixture.

[0209] In a specific embodiment, the present invention relates to a method for preparing a solid protein preparation, which comprises

[0210] a) providing an aqueous mixture, preferably a solution, of water, a protein and a protecting agent, wherein the protecting agent is preferably a lyophilic salt, more preferably ammonium citrate tribasic;

[0211] b) precipitating the protein from the mixture obtained in step a);

[0212] c) collecting the protein to obtain a solid protein preparation.

[0213] The amount of water present in the aqueous mixture should be sufficient to at least substantially dissolve the protein and the protecting agent. Generally, the amount of water present in the mixture is at least 20% by weight, preferably at least 25% by weight, at least 30% by weight, based on the total weight of the protein preparation. Preferably, the amount of water present in the mixture is from about 20% to about 90% by weight, more preferably from about 25% to about 50% by weight, more preferably from about 30% to about 40% by weight, of the total weight of the protein preparation.

[0214] The concentration of the protecting agent in the aqueous mixture should be sufficient to salt out the non-denatured, folded form of the protein from the aqueous phase of the aqueous mixture.

[0215] Those skilled in the art will understand that the concentration of the protecting agent required to salt out a protein from an aqueous medium depends on the type of protecting agent and protein used. Generally, the concentration of the protecting agent is from about 1.3 mol / L to about 5 mol / L, specifically from about 1.5 mol / L to about 3 mol / L, more specifically from about 1.7 mol / L to about 2.5 mol / L. Here, the concentration is determined based on the total number of moles of the protecting agent present in the protein preparation / total volume (in liters) of the liquid (usually an aqueous medium).

[0216] Those skilled in the art will understand that the precipitation of the protein from the aqueous mixture obtained in step a) can be monitored visually, for example by observing the formation of solids, haze or turbidity, or can be monitored using suitable imaging means, especially when the aqueous mixture is substantially a solution. If the aqueous mixture is not substantially a solution, or the precipitation of the protein is not easily monitored, for example due to the presence of solid particles (such as in a suspension), a comparable model solution (for example, lacking the solid particles of the suspension but otherwise the same) can be used to determine the precipitation of the protein from the aqueous phase of the aqueous mixture. Those skilled in the art will be able to determine the precipitation of the protein from an aqueous mixture other than a solution (such as a suspension) based on common general knowledge and the information provided herein.

[0217] The time required in step b) generally depends on the protein and the protective agent used in the method according to the invention. Generally, the time required for the protein to precipitate from the solution obtained in step a) is at least about 60 seconds, preferably at least about 90 seconds, at least about 120 seconds, at least about 150 seconds, at least about 180 seconds, specifically at least about 240 seconds, more specifically at least about 300 seconds, such as at least about 360 seconds. Preferably, the time required for the protein to precipitate from the aqueous phase is from about 60 seconds to about 360 seconds, more preferably from about 120 seconds to about 300 seconds, and most preferably from about 180 seconds to about 240 seconds.

[0218] The method for preparing the solid protein preparation further preferably comprises the following step: providing a carrier (preferably flour) and an optional excipient (preferably microcrystalline cellulose) in the mixture in step a).

[0219] Thus, in a preferred embodiment, the present invention relates to a method for preparing a solid protein preparation, which comprises

[0220] a) providing a suspension comprising water, a protein, a protective agent, a carrier (preferably flour) and an optional excipient;

[0221] b) precipitating the protein from the aqueous phase of the suspension obtained in step a);

[0222] c) collecting the protein to obtain a solid protein preparation.

[0223] Preferably, the mixture containing the carrier and the optional excipient obtained in step a) is subsequently kneaded to form a dough.

[0224] More preferably, the dough is subsequently subjected to the steps of molding, tableting, shaping, granulating, pressing, and extruding to form an extruded dough.

[0225] The present invention further relates to a protein preparation obtainable by the method according to the present invention. Such protein preparations are distinguished from protein preparations of the same kind in that, due to the presence of a protecting agent, the protein folding is more compact. Generally, more compact protein folding provides the protein with higher stability, such as higher thermal stability. Thus, compared to the unfolding temperature of the same protein not obtainable by the method according to the present invention, the protein obtainable by the method according to the present invention generally has a higher unfolding temperature, as determined by circular dichroism spectroscopy.

[0226] The folding of the protein can be determined by any suitable analytical method known in the art. For example, by circular dichroism (CD) spectroscopy or differential scanning calorimetry (DSC) of the protein in solution.

[0227] The method according to the present invention further preferably comprises the step of drying the protein preparation or food according to the present invention. The drying can be achieved using any suitable method known in the art, such as by placing the protein preparation or food in an oven. Preferably, the protein preparation or food is dried at a temperature of at least 40 °C, preferably at least 50 °C, most preferably at least 60 °C. Generally, the protein preparation or food is dried at a temperature of 40 °C to 80 °C, preferably about 60 °C.

[0228] Preferably, the dried protein preparation according to the present invention contains less than 10% by weight of water, preferably less than 5% by weight of water, more preferably less than 4% by weight of water, based on the total weight of the dried protein preparation.

[0229] Preferably, the dried protein preparation according to the present invention contains from about 0.5% to about 10% by weight of water, preferably from about 1% to about 5% by weight of water, more preferably from about 2% to about 4% by weight of water, based on the total weight of the dried protein preparation.

[0230] Preferably, the method according to the present invention does not include the step of coating a protein core with a coating comprising a protecting agent as defined herein or consisting essentially of a protecting agent as defined herein.

[0231] The present invention further relates to a method for preparing a food (preferably animal feed), which comprises mixing the protein preparation according to the present invention with one or more other food ingredients (preferably at least one fat, protein, preferably coagulated protein or carbohydrate).

[0232] Preferably, the mixing comprises the step of molding, shaping, pressing, tableting, granulating or extruding the protein preparation according to the present invention with a mixture of one or more of carbohydrates, fats and proteinaceous substances into the desired form. Preferably, the protein preparation according to the present invention is extruded to form granules.

[0233] Preferably, the molding, shaping, pressing, tablet-making, granulation or extrusion is carried out at an elevated temperature, preferably at a temperature higher than 40 °C, more preferably higher than 50 °C, higher than 60 °C, higher than 70 °C, higher than 75 °C, most preferably higher than 80 °C. Specifically, the molding, shaping, tablet-making, granulation, pressing or extrusion is carried out at a temperature of 50 °C to 150 °C, more preferably at 60 to 130 °C, even more preferably at 80 to 120 °C.

[0234] Preferably, the duration of the molding, shaping, pressing, tablet-making, granulation or extrusion is from about 10 seconds to about 5 hours, preferably from about 20 seconds to about 3 hours, from about 30 seconds to about 2 hours, from about 45 seconds to about 1 hour, from about 1 minute to about 45 minutes, from about 1.5 minutes to about 30 minutes, most preferably from about 2 minutes to about 15 minutes.

[0235] Advantageously, it has been found that the protein preparation according to the invention is substantially stable under these conditions. Preferably, at such a temperature, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, most preferably 100% of the enzyme still has activity.

[0236] The present invention further relates to a method of feeding an animal, which comprises providing a food or a protein preparation, preferably an enzyme preparation according to the present invention, and administering the food or the protein preparation, preferably the enzyme preparation, to an animal in need thereof.

[0237] The food or the protein preparation is administered to any animal that can benefit from the protein preparation. Preferably, the food or the protein preparation is administered to livestock (such as cattle, goats, sheep, lambs, pigs and horses), or to poultry (such as chickens, ducks, geese, turkeys, pheasants and other fowl), or to pet animals (such as cats, dogs, rabbits, guinea pigs, hamsters or rats), or to wild animals (such as deer, elk or wild boars).

[0238] The present invention further relates to the use of a protecting agent in improving the stability of a protein preparation, specifically in improving the tolerance of the protein preparation to reducing sugars and / or metal ions, thermal stability, and / or storage stability and / or stability to mechanical stress, wherein the protecting agent comprises (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule, preferably ammonium citrate tribasic, diammonium hydrogen citrate, ammonium dihydrogen citrate, ammonium dihydrogen phosphate, ammonium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate and ammonium bitartrate and ammonium hydrogen tartrate.

[0239] For the purposes of clarity and brevity, the various features are described in this invention as part of the same or separate embodiments. However, it should be understood that the scope of the invention may include embodiments having combinations of all or some of the said features.

[0240] The present invention is illustrated by the following non-limiting examples.

[0241] Example 1

[0242] 300 g of wheat flour (type 405) was mixed with 168 g of tap water and 0 - 6 wt% of ammonium sulfate, sodium chloride, sodium sulfate, ammonium citrate, and ammonium acetate. The input power during mixing was measured in Farinograph units (FE) by a Farinograph (Farinograph E, Brabender), where 1 FE corresponds to 9.8 ± 0.2 mN·m.

[0243] The results are shown in Figure 1 . From Figure 1 It can be seen that the presence of salts reduces the energy requirement for kneading the dough. This will reduce the energy requirement of the wet granulation process and allow the use of milder manufacturing conditions, thereby reducing the likelihood of enzyme degradation in the enzyme preparation.

[0244] Example 2

[0245] 300 g of various types of flours and starches was mixed with 168 g of tap water and 0 - 6 wt% ammonium sulfate. The input power during mixing was measured in Farinograph units (FE) by a Farinograph (Farinograph E, Brabender), where 1 FE corresponds to 9.8 ± 0.2 mN·m.

[0246] The results are shown in Figure 2 . From Figure 2 It can be seen that the presence of ammonium sulfate reduces the energy requirement for kneading the dough, except for pure starch. This can be explained by the fact that starch does not contain the gluten fraction of flour.

[0247] Example 3: Testing the stability of various enzyme preparations using different potential protectants

[0248] Manufacture of phytase particulate matter

[0249] Homogenize the dry ingredients (wheat flour, microcrystalline cellulose, and salt) for one minute in a food processor (MUM 2, Bosch). Subsequently, add a 6 - phytase solution (>40,000 U / g, EnzyPhostar, Kaesler Nutrition GmbH) to each prepared batch and knead the mixture for approximately 3 - 4 minutes until the dough starts to separate into individual balls. The final content of the dough is shown in Table 1 (ammonium citrate). Repeat these examples using sodium chloride, ammonium acetate, ammonium chloride, ammonium sulfate, diammonium citrate, diammonium hydrogen phosphate, diammonium tartrate, and ammonium acetate instead of ammonium citrate. The content of salt and phytase may vary slightly to obtain the desired dough consistency as described above.

[0250] Table 1; Composition of dough containing ammonium citrate; *Based on the total weight of the dry ingredients.

[0251]

[0252] Then remove the batch from the hopper and attach a mincing machine accessory with a 1 - mm die. Pass the dough through the mincing machine set to level 2 and collect the extruded filaments. Subsequently, round the filaments in a spheronizer (MBS, Caleva) at 1600 rpm with a residence time of 30 seconds. Place the obtained pellets on a plastic tray in a heated oven (Heratherm OMH400, Thermo Scientific) and dry at 60 °C for 4 hours. Measure the residual moisture of the pellets at 103 °C and determine it in the automatic end - mode (Sartorius MA35). Determine the salt content based on the dry weight of the phytase pellets.

[0253] Testing of particulate matter stability

[0254] For each case, place two 0.5 - g samples of phytase pellets into vials. Leave one vial at room temperature as a control (to determine the starting activity of the enzyme), and place the other sample in an autoclave (VX - 150, Systec GmbH) for a thermal stability pressure test and autoclave in the open state according to the set parameters (to determine the residual (relative) enzyme activity). After starting the autoclave, heat to a temperature of 94 °C and maintain this temperature for two minutes. This allows the sample to react with the hot steam to simulate the conditioning step required in the feed pelleting process. Then, open the device after cooling to 74 °C, place the sample directly on ice and further cool for 30 minutes.

[0255] For enzyme extraction, 100 ml of 250 mM HAc-NaAc buffer (pH 5.5, containing 0.1% polysorbate 20 (v / v) (reaction buffer)) was added to all samples, and then the samples were stirred at 200 rpm for 45 minutes. The samples were diluted again with the same extraction buffer so that the absorbance to be measured was within the linear range of the plate photometer. The 6-phytase activity in the samples was determined using ISO method 30024:2009. The activity assay was based on the detection of phosphate released from the substrate sodium phytate (68388, Sigma-Aldrich), which in turn reacted with molybdate and vanadate in nitric acid solution to form a yellow complex, and the complex was detected at an optical density of 415 nm in a plate photometer (FLUOstar Omega, BMG LABTECH GmbH). The samples were measured in replicates ±SD. The relative activity of the heated samples was determined, and the unheated samples were used as 100% reference.

[0256] Results

[0257] The results are shown in Figure 3 . From Figure 3 it can be seen that the activity of 6-phytase did not increase with the increase in the concentration of sodium chloride, ammonium acetate, ammonium chloride or ammonium sulfate, and even decreased in the case of the increase in the concentration of ammonium acetate.

[0258] Without being bound by theory, it is believed that in the 6-phytase preparation containing ammonium acetate, the 6-phytase fraction is at least partially salted out from the preparation. As a result, the 6-phytase is exposed to the mechanical stress of the food processor, which leads to further degradation of the 6-phytase.

[0259] While in the samples supplemented with ammonium citrate tribasic, diammonium hydrogen citrate, diammonium tartrate and Na 2 HPO 4 , the stability of 6-phytase was significantly improved. From Figure 3 it can be seen that there is a linear correlation between the ammonium salt concentration and the relative activity of 6-phytase.

[0260] The results show that the enzyme preparation containing the protectant defined herein exhibits improved activity compared to the enzyme preparation lacking the protectant.

[0261] Example 4: Testing the stability of various enzyme preparations using different enzymes

[0262] Manufacture of different enzyme particulate matters

[0263] In a food processor (MUM 2, Bosch), homogenize the dry components (wheat flour, microcrystalline cellulose, and ammonium citrate tribasic) for one minute. Add 30 ml of a cellulase TXL solution (>30 U / ml) (Trichoderma longibrachiatum, ASA Spezialenzyme GmbH). The final content of the dough is shown in Table 2 (ammonium citrate tribasic) and Table 3 (sodium chloride). Repeat the above example with different enzyme solutions (or add 20 ml of protease SO solution (Aspergillus niger, ASA Spezialenzyme GmbH) or 5 ml of a chitinase solution (>100 U / ml) (Trichoderma harzianum, ASA Spezialenzyme GmbH) to each preparation batch, respectively). To obtain the desired dough consistency, the water content may vary slightly as described above. 2 To obtain the desired dough consistency, the water content may vary slightly as described above.

[0264] Table 2; Composition of dough containing ammonium citrate tribasic; *Based on the total weight of the dry components. One activity unit is defined as the amount of enzyme required to release 1 micromole of glucose reducing sugar equivalent per minute from barley β-glucan (10 mg / mL) at pH 4.5 and 40 °C.

[0265]

[0266] Table 3; Composition of dough containing sodium chloride; *Based on the total weight of the dry components. One activity unit is defined as the amount of enzyme required to release 1 micromole of glucose reducing sugar equivalent per minute from barley β-glucan (10 mg / mL) at pH 4.5 and 40 °C.

[0267]

[0268] Knead the mixture for about 3 - 4 minutes until the finished dough begins to separate into individual balls. Optionally add water to allow the formation of such individual balls. Then remove the batch from the cylinder and attach a shredder attachment with a 1 mm die. Pass the dough through the shredder set to level 2 and collect the extruded filaments. Subsequently, round the filaments in a rounding machine (MBS, Kaleva) at 1600 rpm with a residence time of 30 seconds. Place the obtained particulate matter on a plastic tray in a heating oven (Heratherm OMH400, Thermo Fisher Scientific) and dry at 60 °C for 4 hours. Measure the residual moisture at 103 °C and stop automatically (Sartorius MA35). Finally, the residual moisture content of the particulate matter is <10%. Ideally <5%. Determine the salt content based on the dry weight of the enzyme particulate matter.

[0269] Testing of particulate matter stability

[0270] To analyze each condition and enzyme, two identical 1 g samples were weighed and placed into vials. One vial was placed at room temperature as a control (to determine the starting activity of the enzyme), and the other samples were placed in an autoclave (VX-150, Systec GmbH) for heat stability pressure testing and autoclaved according to the set parameters (to determine the residual (relative) enzyme activity). After starting the autoclave, it was heated to a temperature of 94 °C and held at that temperature for two minutes. This caused the samples to react with the hot steam to simulate the conditioning step required in the feed pelleting process. Then, after cooling to 74 °C, the equipment was opened, and the samples were placed directly on ice and further cooled for 30 minutes.

[0271] For cellulase extraction, 50 ml of 25 mM HAc-NaAc buffer at pH 4.5 was added to all samples, and then the samples were stirred at 200 rpm for 45 minutes. Then, 1.5 ml of the extract was taken and centrifuged at 10,000 xg for 10 minutes. If necessary to keep within the linear measurement range of the photometer, the samples were diluted with sodium acetate buffer. To analyze the activity of the cellulase TXL with endo-1,4-β-glucanase activity, the test manual for β-glucanase tablets was followed according to the manufacturer's instructions (the substrate used was Azurine cross-linked barley β-glucan; T-BGZ-200T; Megazyme, Bray, Ireland). The diluted or undiluted clarified extract was placed in 25 mM HAc-NaAc buffer at pH 4.5, and 500 μl was taken and preheated in a glass test tube at 40 °C for 5 minutes. Then, the enzyme reaction was initiated by adding the β-glucanase tablet. After 10 minutes, the reaction was stopped by adding 10.0 mL of Trizma Base solution (2% w / v, pH 8.5). After the samples were cooled at room temperature for 5 minutes, they were filtered using Whatmann filters, 300 μL of the clarified solution was taken, and analyzed in a plate photometer (FLUOstar Omega, BMG LABTECH GmbH, Germany). The absorbance was measured at 590 nm. The samples were measured in replicates ± SD. The relative activity of the heated samples was determined, with the unheated samples as 100% reference.

[0272] For protease extraction, 100 ml of 25 mM HAc-NaAc buffer (reaction buffer) at pH 4.0 was added to all samples, which were then stirred at 200 rpm for 45 min. Then, 1.0 ml of the extract was taken and centrifuged at 10,000 xg for 10 min. Samples were diluted with sodium acetate buffer if necessary to remain within the linear measurement range of the photometer. To analyze the activity of protease S-02 with acid peptidase activity, the assay manual of the Pierce TM Colorimetric Protease Assay Kit (Thermo Fisher Scientific, 23263) was followed. The assay for acid peptidase was adjusted (according to the manual instructions). For this, 50 μl of succinylated casein (2 mg / ml reaction buffer) in the reaction buffer was pipetted into the wells. Then, 25 μl of the diluted or undiluted sample or control was added to the substrate. The microtiter plate was incubated at 40 °C for 20 min. Then, 75 μl of 50 mM sodium borate buffer (pH 8.5) was pipetted into the samples to stop the enzymatic reaction. Then, 50 μl of TNBSA solution (2,4,6-trinitrobenzenesulfonic acid, 5% (w / v) in methanol) was pipetted into the samples and incubated at room temperature for 20 min. Absorbance at 450 nm was analyzed in a plate photometer (FLUOstar Omega, BMGLABTECH GmbH). Samples were measured in duplicates ± SD. The relative activity of the heated samples was determined, with the unheated samples taken as 100% reference.

[0273] For chitinase extraction, 10 ml of 100 mM HAc-NaAc buffer (reaction buffer) at pH 5.5 was added to all samples, which were then stirred at 200 rpm for 45 minutes. Then, 1.0 ml of the extract was taken and centrifuged at 10,000 xg for 10 minutes. The clarified supernatant was used directly for the enzyme activity assay. Based on the enzymatic hydrolysis of the chitinase substrate (1,4-β-poly-N-acetylglucosaminidase activity towards the specific substrate 4-nitrophenyl-N-acetyl-β-glucosamine (Carl Roth GmbH+Co.KG, Germany)), chitinase activity was detected using a chitinase assay kit (Sigma Aldrich; CS0980) according to the manufacturer's instructions. This hydrolysis releases p-nitrophenol, which can be measured colorimetrically after ionization at alkaline pH and serves as a standard for the linear range of the test plate photometer. 90 μl of the substrate solution (1 mg / ml in double-distilled water) was placed in a 96-well plate. 10 μl of the extract or standard was added thereto. After mixing, it was incubated at 37 °C for 30 minutes, and then 200 μl of 112.5 mg / ml (w / v%) sodium carbonate decahydrate solution was added. The absorbance at 405 nm was analyzed in a plate photometer (FLUOstar Omega, BMG LABTECH GmbH, Germany). Samples were measured in duplicates ± SD. The relative activity of the heated samples was determined, with the unheated samples taken as 100% reference.

[0274] Results

[0275] The results are shown in Figure 4 . It can be seen that for various enzymes, their stability is improved in the presence of ammonium citrate tribasic, but not in the presence of sodium chloride.

[0276] Example 5: Manufacture of the enzyme preparation according to the invention on a technical scale

[0277] Manually place the dry components (440 g wheat flour, 114 g microcrystalline cellulose, and varying amounts of ammonium citrate tribasic) into a bag and homogenize for one minute. Pour the different mixtures separately into a feeder (K-ML-SFS-KT20, Coperion K-Tron). Then feed the dry mixture into an extruder (ZSE18MAXX-HP, Leistritz) at a dose of 1 kg / h. The screw speed is adjusted to 500 rpm. The screw design mainly consists of conveying elements. The dry product is successfully added in zone 6, and the liquid is added in zone 7. The discharge of the extruder is actively cooled with tap water (about 14 °C). Add a phytase solution (>40,000 FTU / g, ENZY Phostar, Kessler Nutrition GmbH) with a 1 mm liquid nozzle through a pump (NEMA 4x IP66, Watson Marlow) at a dose of 7.5 to 13 g / min, depending on the formulation of the dry ingredients, to form a regular extrudate at medium pressure in the extruder. The composition of the dough samples is listed in Table 4. Extrude the dough through a die with a diameter of 0.8 mm, and then round it in a rounding machine (MBS, Kaleva) at 1600 rpm and a residence time of 30 seconds for drying. Place the obtained particulate matter in a drying oven (Heratherm OMH400, Thermo Fisher) to dry, with the product temperature reaching up to 60 °C. Measure the residual moisture at 103 °C and automatically stop (MA35, Sartorius). Determine the ammonium citrate tribasic content based on the dry weight of the phytase particulate matter.

[0278] Table 4; Composition of dough containing sodium chloride; *Based on the total weight of the dry ingredients.

[0279]

[0280] In the second run, repeat the experiment at different pressures (the pressure sensor indicates pressures ranging from 0 to 30 bar). Change the pressure by varying the content of the enzyme solution and / or the salt content. As Figure 5 can be seen from b, by maintaining a low pressure, preferably below 10 bar and even lower, enzyme preparations can be prepared while minimizing enzyme losses due to degradation.

[0281] Example 6: Manufacture of the enzyme preparation according to the invention using spray drying.

[0282] Prepare an enzyme preparation by mixing 30 g wheat flour, 10.5 g ammonium citrate tribasic, 20 g fermentation supernatant (E. coli 6-phytase, 146 FTU / ml), and 139.5 g tap water. Subsequently, spray-dry the homogenized mixture using a Büchi B-290 Advanced with a nozzle diameter of 1.5 mm and a pressure of 8 bar, with the following settings:

[0283] Spraying parameters: Inlet temperature: 110 °C

[0284] Outlet temperature: 70 °C

[0285] Pump capacity: 20

[0286] Flow rate: 40 mm

[0287] Suction capacity: 100

[0288] Collection container temperature: 46 - 48 °C

[0289] Spraying time: 15 minutes.

[0290] Mix 30 g of wheat flour, 20 g of fermentation supernatant (E. coli 6 - phytase, 146 FTU / ml) and 150 g of tap water to prepare a reference enzyme preparation (without ammonium citrate). Subsequently, spray-dry the homogenized mixture using a Büchi B-290 Advanced with a nozzle diameter of 1.5 mm and a pressure of 8 bar, with the settings as follows:

[0291] Spraying parameters: Inlet temperature: 110 °C

[0292] Outlet temperature: 66 °C

[0293] Pump capacity: 20

[0294] Flow rate: 40 mm

[0295] Suction capacity: 100

[0296] Yield: 2.42 g

[0297] Collection container temperature: 40 - 46 °C

[0298] Spraying time: 15 minutes.

[0299] Subsequently, heat the obtained powder and determine the relative activity according to the steps described in Example 3. The results are shown in Figure 6 .

[0300] From Figure 6 it can be seen that, compared with the reference enzyme preparation, the powdered enzyme preparation according to the present invention is more thermally stable. These results indicate that the enzyme preparation according to the present invention can also be prepared by spray-drying means.

[0301] Example 7: Stability of the enzyme preparation according to the invention in the presence of glucose.

[0302] In a food processor (MUM 2, Bosch), dry components (79.5 g wheat flour, 20.6 g microcrystalline cellulose, 35 g ammonium citrate tribasic, and 0 - 4 wt% glucose) were homogenized for one minute. To each prepared batch, 50 - 65 g of a 6 - phytase solution (>40,000 U / g, Enzy Phostar, Kessler Nutrition GmbH) was added, and the mixture was kneaded for about 3 - 4 minutes until the finished dough began to separate into individual balls. The finished batches were removed from the cartridge, and a meat grinder attachment with a 1 mm die was installed. The dough was passed through the meat grinder set to level 2, and the extruded filaments were collected. Subsequently, the filaments were rounded in a rounding machine at about 1600 rpm and a residence time of about 30 seconds. The obtained particulate matter was placed on a plastic tray in a heated oven and dried at 60 °C for 4 hours. The residual moisture was measured at 103 °C and automatically stopped (Sartorius MA35).

[0303] Subsequently, the obtained particulate matter was heated, and the relative activity was determined according to the procedure described in Example 3. The results are shown in Figure 7 .

[0304] From Figure 7 it can be seen that in the presence of additional glucose, the relative activity of phytase decreased. It is conceivable that in the presence of glucose, more amino acids in the enzyme are involved in the Maillard reaction, and thus more degradation is observed. These results indicate that the Maillard reaction is the root cause of the lack of stability observed upon heating.

[0305] Example 8: Stability of the enzyme preparation according to the invention in the presence of iron(III) chloride.

[0306] In a food processor (MUM 2, Bosch), dry ingredients (79.5 g wheat flour, 20.6 g microcrystalline cellulose, 35 g ammonium citrate tribasic, and 0.0005 to 0.7 wt% iron(III) chloride) were homogenized for one minute. Added > 50 - 65 g of a 6 - phytase solution (40,000 U / g, Enzy Phostar, Kessler Nutrition GmbH), and the mixture was kneaded for about 3 - 4 minutes until the finished dough began to separate into individual balls. Additionally, a control preparation without ammonium citrate tribasic and 0.0005 to 0.7 wt% iron(III) chloride was prepared using the same protocol. The control preparation contained 79.5 g wheat flour, 20.6 g microcrystalline cellulose, and > 50 - 65 g of a 6 - phytase solution (40,000 U / g, Enzy Phostar, Kessler Nutrition GmbH).

[0307] Remove the completed batch from the barrel and install the grinder attachment with a 1 mm die. Pass the dough through the grinder set at level 2 and collect the extruded filaments. Subsequently, round the filaments in a rounding machine at approximately 1600 rpm and a residence time of approximately 30 seconds. Place the obtained particulate matter on a plastic tray in a heating oven and dry at 60 °C for 4 hours. Measure the residual moisture at 103 °C and automatically stop (Sartorius MA35).

[0308] Subsequently, heat the obtained particulate matter and determine the relative enzyme activity according to the procedure described in Example 3. The results are shown in Figure 8 .

[0309] From Figure 8 it can be seen that in the presence of additional iron(III) ions, the relative activity of phytase decreases. It is conceivable that in the presence of Fe 3+ , more amino acid residues present in the enzyme participate in the Maillard reaction, and thus more degradation is observed. These results indicate that the Maillard reaction is the root cause of the lack of stability observed upon heating. In the presence of ammonium citrate, the slope of the graph is less steep and less degradation is observed. The results indicate that ammonium citrate acts as a protective agent by chelating metal ions.

[0310] Example 9: Stability of the enzyme preparation after storage

[0311] Store three types of phytase particulate matter containing sodium chloride and ammonium citrate respectively, prepared according to the method described in Example 3, at 22 °C and 50% humidity. After 15 months (sodium chloride and ammonium chloride) and 20 months (ammonium citrate), determine the phytase activity of the particulate matter using the method described in Example 3.

[0312] The results are shown in Figure 9 . From Figure 9 it can be seen that compared to the controls containing ammonium chloride (squares) and sodium chloride (circles) and stored for 15 months, the phytase granules containing ammonium citrate (triangles) exhibit higher residual phytase activity after 20 months of storage.

[0313] It can be concluded that compared to the control preparation, the protein preparation according to the present invention exhibits extended storage stability.

Claims

1. A protein preparation comprising a protein and a protective agent, the protective agent comprising (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitant and / or chelating molecule.

2. The protein preparation according to claim 1, wherein the protective agent comprises an ammonium salt.

3. The protein preparation according to any one of the preceding claims, wherein the protective agent is a small molecule, preferably having a molecular weight below 1000 g / mol, specifically in the range of about 80 to about 250 g / mol.

4. The protein preparation according to any one of the preceding claims, wherein the protective agent is a lyophilic salt.

5. The protein preparation according to any one of the preceding claims, wherein the protective agent comprises a chelating molecule.

6. The protein preparation according to claim 5, wherein the chelating molecule is selected from citrates and tartrates, preferably ammonium citrate tribasic, diammonium hydrogen citrate and diammonium tartrate.

7. The protein preparation according to any one of the preceding claims, wherein the protective agent comprises a metal precipitant, preferably a phosphate, carbonate or sulfide, more preferably ammonium phosphate tribasic, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium carbonate, ammonium bicarbonate or ammonium hydrogen sulfide.

8. The protein preparation according to any one of the preceding claims, which further comprises one or more reducing sugars, preferably one or more reducing sugars at least partially produced by microorganisms, even more preferably wherein the microorganisms are enzyme-expressing microorganisms or wherein the one or more reducing sugars are present as residues from the culture medium.

9. The protein preparation according to any one of the preceding claims, which further comprises metal ions, preferably transition metal ions, more preferably zinc ions, iron ions, cobalt ions, manganese ions or molybdenum ions, even more preferably wherein the transition metal ions are present as residues from the culture medium.

10. The protein preparation according to any one of the preceding claims, which comprises a carrier, preferably a flour selected from wheat flour, barley flour, rye flour, spelt wheat flour, triticale flour and combinations thereof, preferably wherein the preparation comprises wheat flour.

11. The protein preparation according to any one of the preceding claims, wherein the protein is an enzyme, preferably wherein the enzyme is selected from phytase, chitinase, peptidase and cellulase.

12. The protein preparation according to any one of the preceding claims, wherein the protective agent is present in an amount of about 5 wt% to about 50 wt%, preferably about 10 wt% to about 40 wt%, most preferably about 15 wt% to about 30 wt% based on the dry weight of the protein preparation.

13. The protein preparation according to any one of the preceding claims, wherein the protein preparation is a solid protein preparation.

14. A food product comprising the protein preparation according to any one of the preceding claims and at least one fat and / or other carbohydrate and / or other protein, preferably coagulated protein.

15. A method for preparing a protein preparation according to any one of claims 1 to 14, which comprises mixing a protein with a protective agent comprising (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule to obtain a protein preparation according to any one of claims 1 to 14.

16. The method according to claim 15, which comprises: a) providing a mixture of water, a protein and a protective agent, wherein the protective agent is preferably a lyophilic salt, more preferably ammonium citrate tribasic; b) precipitating the protein from the aqueous phase of the mixture obtained in step a); c) collecting the protein to obtain a solid protein preparation.

17. The method according to claim 16, wherein the concentration of the protective agent is from about 1.3 mol / L to about 5 mol / L, specifically from about 1.5 mol / L to about 3 mol / L.

18. The method according to any one of claims 14 to 17, which further comprises mixing a carrier, preferably a flour selected from wheat flour, barley flour, rye flour, spelt flour, triticale flour and combinations thereof, to obtain a protein preparation comprising a carrier, preferably a flour.

19. The method according to any one of claims 15 to 18, which further comprises spray-drying the protein preparation to obtain a spray-dried protein preparation.

20. The method according to claim 18, which further comprises extruding the protein preparation comprising flour to obtain an extruded protein preparation, preferably wherein the pressure during the extrusion is from 0.5 to 40 bar, preferably from 1 to 20 bar, specifically from 2 to 5 bar.

21. A protein preparation obtained by the method according to any one of claims 15 to 20, preferably wherein the protein preparation is a solid protein preparation.

22. Use of a protective agent, wherein the protective agent comprises (i) at least one amine and / or ammonium group and (ii) at least one metal precipitant and / or chelating molecule, preferably wherein the protective agent is ammonium citrate tribasic, diammonium hydrogen citrate, ammonium dihydrogen citrate, ammonium phosphate tribasic, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium hydrogen tartrate and diammonium tartrate, for improving the stability of a protein preparation, specifically for improving the thermal stability of the protein, the storage stability of the protein and the stability against mechanical stress.

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