Protein preparation

Abstract

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

Description

Technical Field

[0001] The present invention relates to protein formulations, foods containing protein formulations, and methods for preparing protein formulations.

Background Art

[0002] In the food industry, the use of proteins, especially enzymes, as activators is becoming increasingly popular, particularly for the beneficial effects on the health of the animal gastrointestinal tract.

[0003] However, many proteins used in industrial processes have the problem of insufficient stability with respect to the conditions normally used in the production of protein formulations or foods containing such protein formulations.

[0004] For example, many proteins are heat-sensitive, so when heat is applied, such as during extrusion of the protein together with other food ingredients or pelletization with steam, they are at least partially degraded. This is disadvantageous since many types of foods, such as animal feed, are preferably formed in pellet form for reasons of storage efficiency, stability, ease of handling, etc.

[0005] This problem is addressed in European Patent No. 2497372, which describes granules containing a core, an activator such as an enzyme, and at least one protective coating. The coating is intended to prevent the movement of moisture to the core containing the activator. Usually, the coating includes a moisture-proof coating that slows down the rate of moisture movement to the granules and / or a moisture hydration coating that absorbs moisture and thereby impedes or delays the extent or rate at which external moisture moves to the core.

[0006] However, such coated granules further suffer significant deterioration after being subjected to pelletization at 90 °C, and moreover, require an additional step of applying a coating to the core containing the enzyme.

Summary of the Invention

Means for Solving the Problem

[0007] The inventors have developed a protein formulation that overcomes one or more of the above drawbacks. In particular, the inventors have developed a protein formulation having similar or improved stability, in particular similar or improved stability of the protein against heat, and / or similar or improved storage stability as compared to a reference protein formulation. The above protein formulation contains a protective agent, and the protective agent can be mixed with the protein and optionally other components without requiring a coating step to obtain the protein formulation of the present invention. This is advantageous because the protein formulation can be prepared in a cost-effective and efficient manner in terms of time and cost.

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

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0010] As used herein, the term "or" is defined as "and / or" unless otherwise specified.

[0011] As used herein, the term "a" or "an" is defined as "at least one" unless otherwise specified.

[0012] When referring to a noun (such as a compound, additive, etc.) in the singular, it is intended to include the plural.

[0013] The terms "essentially" or "substantially" are generally used herein to indicate having the general characteristics or functions of the specified thing. When referring to a quantifiable characteristic, this term is usually used to indicate that it is 30% or more, particularly 50% or more, particularly 70% or more, more particularly at least 90%, more particularly at least 95%, and still more particularly at least 98% of the maximum value of that characteristic. The term "essentially free of" is generally used herein to indicate that a substance is not present (below the detection limit achievable by available analytical techniques as of the effective filing date) or is present in such a small amount that it does not significantly affect the properties of the product that essentially contains that substance.

[0014] In the context of the present application, the term "about" generally means a deviation of 15% or less, particularly 10% or less, more particularly 5%, 4%, 3%, 2%, 1%, 0.5% or less from the given value.

[0015] As used herein, "protein" refers to a chain of amino acids arranged in a specific order determined by the coding sequence of a polynucleotide encoding a polypeptide. Generally, the chain contains at least 10 or more amino acids, desirably at least 15 or more amino acids such as 20 or more amino acids. Generally speaking, a protein has up to 40,000 amino acid residues, such as, for example, 35,000 amino acid residues, but there is no upper limit to the number of amino acids that may be present in a protein. On average, a protein generally contains about 100 to 1500 amino acid residues.

[0016] As used herein, "enzyme" refers to a protein having the ability to catalyze a biochemical reaction under physiological conditions. Enzymes may be referred to herein by their corresponding enzyme (EC) numbers 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 on June 17, 2022). Examples of enzymes include hydrolytic enzymes such as, for example, phosphatase and peptidase.

[0017] "Reducing sugar" is generally known in the art to refer to an open-form sugar molecule containing an aldehyde group having the activity to act as a reducing agent. Generally, a reducing sugar may exist in both a closed form in which the reducing sugar forms a 5- or 6-membered ring having a hemiacetal or hemiketal functional group, and an open form in which the closed-form hemiacetal is opened to form a linear structure containing an aldehyde group at the end. Examples of reducing sugars include glucose, fructose, lactose, and maltose.

[0018] As used herein, "amine" is generally understood in the art to refer to a functional group having the structural formula NR1R2R3, where the R1, R2, and R3 may be the same or different and may be any group that forms a chemically stable molecule under physiological conditions. An example of an amine is NH3, where each of R1, R2, and R3 represents a hydrogen group ("H").

[0019] As used herein, "ammonium" is generally understood in the art to refer to a functional group having the structural formula N+R1R2R3H, where R1, R2, and R3 may be the same or different and may be any group that forms a chemically stable molecule under physiological conditions. Examples of ammonium include NH4 + ions, where each R1, R2, and R3 represents a hydrogen group ("H").

[0020] As used herein, "metal" generally means an element selected from Groups 1 to 13 of the periodic table, excluding hydrogen and boron, or an element selected from tin (Sn), lead (Pb), bismuth (Bi), and polonium (Po). Examples of metals include transition metals, alkali metals, alkaline earth metals, lanthanoids, and actinoids.

[0021] With respect to the term "transition metal" as used herein, it generally means an element selected from Groups 3, 4 and 5 periods, 4, 4 - 7 periods, 5, 4 - 7 periods, 6, 4 - 7 periods, 7, 4 - 7 periods, 8, 4 - 7 periods, 9, 4 - 6 periods, 10, 4 - 6 periods, 11, 4 - 6 periods, and 12, 4 - 7 periods. The transition metal may take on any oxidation state, but is preferably a divalent or trivalent metal ion.

[0022] A salt is defined as a compound formed by the combination or neutralization of an acid and a base. When ions are bonded by ionic bonds, a salt may be formed. When a salt is dissolved in a solvent such as water, it may dissociate into ions (excluding H + or OH - ). "Inorganic salt" is generally understood in the art to refer to a salt that does not contain a C - H bond in its backbone. Examples of inorganic salts include sodium chloride, ammonium phosphate, etc. "Organic salt" is generally understood in the art to refer to a salt that is not inorganic, i.e., a salt that contains a C - H bond in its backbone. Examples include citrate or tartrate salts.

[0023] The term "nucleophile" or "nucleophilic group" is generally understood in the art to refer to a molecule or functional group that can react with an electrophilic group to donate electrons. Usually, a nucleophile has a high electron density due to, for example, the presence of a lone pair of electrons (also called a non-bonding electron pair) and / or an adjacent electron-donating group. Here, the term "electrophile" or "electrophilic group" is understood in the art to refer to a molecule or group that can accept electrons. Usually, an electrophile has a low electron density due to, for example, 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 through, for example, polarization (induction) or stabilization by delocalization of electrons. An electron-donating group is generally understood in the art to refer to a group that can increase the electron density of an adjacent group through, for example, polarization (induction) or delocalization of electrons.

[0024] In the context of the present application, the terms "chelating agent", "chelating molecule" or "chelator" are used herein with the same meaning and generally refer to a molecule having the ability to bind to a metal ion, preferably a transition metal ion, thereby forming a complex. Usually, a chelating molecule is a polyatomic or bidentate molecule containing at least two functional groups capable of coordinating 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 may be coordinated by a chelating molecule include Cu 2+ , Co 2+ , Ni 2+ , Mn 2+ , Zn 2+ and Fe 2+ . Examples of trivalent metal ions (M 2+ ) include Fe 3+ . Examples of functional groups having the ability to coordinate a transition metal ion include carboxylate groups and amines.

[0025] In the context of the present application, a "metal precipitant" generally refers to an agent that does not dissolve in an aqueous medium or has limited solubility in an aqueous medium at about 25 °C, atmospheric pressure, and physiological pH, preferably a metal ion, more preferably a transition metal ion, and has the ability to form a complex or salt with the metal ion. The solubility in the aqueous medium may be determined visually, for example, by observing precipitates, crystals, cloudiness, turbidity, etc., or detected using analytical means such as high performance liquid chromatography.

[0026] As will be understood by those skilled in the art, in the context of the present application, the above-mentioned metal precipitant may form a salt or complex with a metal ion, preferably a transition metal ion, and limit the solubility in any suitable aqueous medium. Examples of suitable water-soluble media include limited solubility in an aqueous solution (aqueous enzyme solution) and limited solubility in the aqueous phase of an aqueous emulsion or aqueous suspension. An example of an aqueous suspension is a dough containing protein, water, and flour. Here, the limited solubility may refer to the limited solubility in the aqueous phase of the dough.

[0027] Regarding the term "dough" used herein, it generally refers to a mixture containing at least flour, water, protein, and a protecting agent and having a viscoelastic consistency. Dough can usually be kneaded using a suitable tool such as a hand or mechanical mixer. Dough can usually retain its shape substantially without a tool for holding it in a fixed position such as a container.

[0028] In the context of the present application, "food" refers to any product suitable for consumption by animals, including humans, and means those not advised by general health authorities such as the FDA and / or EFSA not to be taken in the diet.

[0029] Protein formulation The instability of a protein, particularly its instability with respect to heat (usually at temperatures of 40 °C or higher) or the passage of time at ambient temperature (usually at least 24 hours), is thought to be at least partially due to degradation caused by the presence of reducing sugars and / or metal ions, which are typically present in protein formulations, for example as residues from the protein manufacturing process.

[0030] Without wishing to be bound by any theory, it is expected that amines present in the amino acid residues of a protein may 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").

[0031] The above Maillard reaction may be promoted by metal ions, particularly transition metal ions (Kato et al., 1981, J. Agric. Food Chem, 29, 540-543).

[0032] Therefore, by introducing into the protein formulation a protective agent that at least partially removes reducing sugars and metal ions as active ingredients from the protein formulation, the proteins present in the formulation are prevented from participating in the Maillard reaction. This significantly improves the resistance of the protein formulation to the presence of reducing sugars and metal ions, particularly transition metal ions, compared to other protein formulations known in the art. Thereby, the stability of the protein over time and / or at high temperatures may be significantly improved.

[0033] Accordingly, the present invention relates to a protein formulation comprising a protein and a protective agent, wherein the protective agent comprises (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitating agent and / or chelating molecule.

[0034] Protein The protein preparation according to the present invention may contain a protein for any purpose, that is, any protein having related commercial, nutritional, pharmaceutical or scientific uses.

[0035] For example, the protein may be a therapeutic peptide such as an antibody, a peptide, preferably an antibacterial peptide or an enzyme.

[0036] Preferably, the protein is a natural protein, more preferably an enzyme. Preferably, the protein is an enzyme that has value as a food ingredient, more preferably in animal feed, in household industries such as detergents, in leather processing or in the fermentation of waste such as fiber waste, paper waste, and wastewater.

[0037] Preferably, the protein in the protein preparation of the present invention is an enzyme belonging to the class of oxidoreductases (EC1), transferases (EC2), hydrolases (EC3), lyases (EC4), isomerases (EC5), ligases (EC6) or translocases (EC7), and most preferably, the enzyme in the enzyme preparation of the present invention belongs to the class of hydrolases (EC3).

[0038] The protein in the protein preparation of the present invention may be an enzyme from the class of oxidoreductases such as enzymes 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, EC1.17, EC1.18, EC1.19, EC1.20, EC1.21, EC1.22, EC1.23 or EC1.97.

[0039] Alternatively, the protein in the protein preparation of the present invention is an enzyme from the class of transferases such as enzymes from subclasses EC2.1, EC2.2, EC2.3, EC2.4, EC2.5, EC2.6, EC2.7, EC2.8, EC2.9 and EC2.10.

[0040] Preferably, the protein in the protein preparation of the present invention is an enzyme from the class of hydrolases such as enzymes from hydrolases acting on ester bonds of subclasses (EC3.1), glycosylases (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).

[0041] Most preferably, the protein is an enzyme from subclasses EC3.1, EC3.2 or EC3.4.

[0042] Examples of enzymes from subclass EC3.1 include carboxylic ester hydrolases (EC3.1.1), thioester hydrolases (EC3.1.2), phosphoric monoester hydrolases (EC3.1.3), phosphoric diester hydrolases (EC3.1.4), triphosphoric monoester hydrolases (EC3.1.5), sulfuric ester hydrolases (EC3.1.6), diphosphoric monoester hydrolases (EC3.1.7), phosphoric triester hydrolases (EC3.1.8), exodeoxyribonucleases that produce 5'-phosphomonoesters (EC3.1.11), exodeoxyribonucleases that produce 5'-phosphomonoesters (EC3.1.12), exoribonucleases that produce 5'-phosphomonoesters (EC3.1.13), exoribonucleases that produce 3'-phosphomonoesters (EC3.1.14), exonucleases that act on ribonucleic or deoxyribonucleic acids and produce 5'-phosphomonoesters (EC3.1.15), exonucleases that act on ribonucleic or deoxyribonucleic acids and produce 3'-phosphomonoesters (EC3.1.16), endodeoxyribonucleases that produce 5'-phosphomonoesters (EC3.1.21), endodeoxyribonucleases that produce 3'-phosphomonoesters (EC3.1.22), site-specific endodeoxyribonucleases specific for modified bases (EC3.1.25), endoribonucleases that produce 5'-phosphomonoesters (EC3.1.26), endoribonucleases that produce 3'-phosphomonoesters (EC3.1.27), endoribonucleases that act on ribonucleic or deoxyribonucleic acids and produce 5'-phosphomonoesters (EC3.1.30), and endoribonucleases that act on ribonucleic or deoxyribonucleic acids and produce 3'-phosphomonoesters (EC3.1.31).

[0043] Examples of enzymes from subclass EC3.2 include glycosidases (EC3.2.1) or hydrolases of N-glycosyl compounds (EC3.2.2).

[0044] Examples of enzymes from subclass EC3.4 include aminopeptidases (EC3.4.11), dipeptidases (EC3.4.13), dipeptidyl peptidases and tripeptidyl peptidases (EC3.4.14), peptidyl dipeptidases (EC3.4.15), serine-type carboxypeptidases (EC3.4.16), metallo-carboxypeptidases (EC3.4.17), cysteine-type carboxypeptidases (EC3.4.18), omega peptidases (3.4.19), serine endopeptidases (3.4.21), cysteine endopeptidases (3.4.22), aspartic endopeptidases (EC3.4.23), metallo endopeptidases (EC3.4.24), threonine endopeptidases (EC3.4.25), and endopeptidases of unknown catalytic mechanism (EC3.4.99).

[0045] Most preferably, the protein in the protein preparation of the present invention is 3-phytase (EC3.1.3.8), 4-phytase, and in the art, it is also called 6-phytase (EC3.1.3.26), 5-phytase (EC3.1.3.72), chitinase (EC3.2.1.14), cellulase (EC3.2.1.4), peptidase (EC3.4), xylanase (EXC3.2.1), amylase (EC3.2.1), lipase (EC3.2.1.6), mannanase (EC3.2.1), pectinase (EC3.2.1.15, β-glucanase (EC3.2.1.6), α-galactosidase (EC3.1.2.22).

[0046] In one aspect, the protein in the protein preparation of the present invention is an enzyme from the class of lyase enzymes such as enzymes from subclasses EC4.1, EC4.2, EC4.3, EC4.4, EC4.5, EC4.6, EC4.7, and EC4.99.

[0047] Alternatively, the protein in the protein preparation of the present invention is an enzyme from the class of isomerase enzymes such as enzymes from subclasses EC5.1, EC5.2, EC5.3, EC5.4, EC5.5, and EC5.99.

[0048] Alternatively, the protein in the protein preparation of the present invention is an enzyme from the class of synthetic enzymes such as enzymes from subclasses EC6.1, EC6.2, EC6.3, EC6.4, EC6.5, and EC6.6.

[0049] The protein in the protein preparation of the present invention may be an enzyme from the class of translocases such as enzymes from subclasses EC7.1, EC7.2, EC7.3, EC7.4, EC7.5, and EC7.6.

[0050] The protein in the protein preparation according to the present invention may be produced by any method known in the art. Preferably, the protein, preferably the 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 the expression of the protein, preferably the enzyme, include filamentous fungi, yeasts, bacteria, and algae. The microorganism may be a wild-type microorganism or may be genetically modified, for example, to improve the yield of the protein present in the protein preparation of the present invention.

[0051] During the protein expression process, the protein, preferably the enzyme, is usually expressed in a culture medium or in cells and then may be processed to obtain the protein from the culture medium or cells. Thus, the obtained protein, preferably the enzyme, may be obtained as a mixture containing one or more additional components derived from the culture medium or cells of the microorganism, such as other proteins, peptides, amino acids, nucleic acids such as DNA or RNA, residues of cells of the protein-producing microorganism, carbohydrates, and lipids.

[0052] The obtained protein, preferably the enzyme, may be at least partially purified to remove one or more of these components, or a mixture of crude proteins may be used directly without substantial purification for the protein preparation of the present invention. Examples of purification include those including filtration, centrifugation, and column chromatography, and combinations thereof.

[0053] Examples of commercially available 6-phytases suitable for use in the present invention include Enzy Phostar, Kaesler Nutrition GmbH.

[0054] Preferably, the protein preparation of the present invention contains residues from protein-producing microorganisms, and more preferably, the residues contain cell substances such as carbohydrates containing reducing sugars, lipids containing phospholipids and glycolipids, or proteins.

[0055] Preferably, the protein preparation of 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.

[0056] In a preferred embodiment, the protein preparation of the present invention contains one or more reducing sugars produced by a microorganism, particularly a microorganism that expresses the protein present in the protein preparation, and optionally one or more reducing sugars are present as residues from the medium.

[0057] The protein preparation preferably contains cell substances of the protein-producing microorganism, such as residues of the microorganism and / or compounds excreted by the microorganism. As used herein, the term "residue" means those that include compounds generated or present during the protein production process, and derivatives of such compounds. The term "protein production process" includes any step related to at least partially isolating the protein from cells or culture media, such as extraction of the protein from the medium or cells.

[0058] Preferably, the protein preparation of the present invention contains compounds present in the cell wall of a microorganism, such as a cell wall of bacteria, yeast, fungi or algae, such as glycoproteins, chitin or glucan, or derivatives of such compounds, and includes degradation products thereof.

[0059] In another preferred embodiment, the protein preparation of the present invention contains compounds present in the cell membranes of microorganisms such as the cell membranes of bacteria, fungi, yeast or algae, and includes phospholipids and proteins, and fragments thereof.

[0060] Even more preferred in the protein preparation of the present invention are compounds present in the cytoplasm of microorganisms, including nucleic acid molecules such as plasmids, DNA and RNA ribosomes, intracellular membranes, proteins containing enzymes, lipids and carbohydrates.

[0061] In another preferred embodiment, the protein preparation of the present invention contains compounds excreted by microorganisms such as proteins, for example host cell proteins and derivatives thereof, such as peptides and amino acids.

[0062] Even more preferably, the protein preparation of the present invention contains residues of the culture medium.

[0063] Examples of such compounds include nitrogen sources such as peptone, glutamine or nitrate, carbon sources such as fermentable carbon sources such as glucose, lactose, molasses, corn steep liquor, 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 degradation products of these compounds.

[0064] Removing fermentation by-products or residues of the culture medium from the protein requires one or more purification steps, and it can be particularly challenging to remove trace amounts of such residues. Furthermore, in the purification process, at least a portion of the protein is typically lost during purification, resulting in a lower yield of the protein. Thus, an advantage of the protein formulations of the present invention is that certain amounts of fermentation residues, particularly metal ions such as transition metal ions and reducing sugars, are tolerated. This allows for the use of crude or partially purified proteins in the protein formulations of the present invention. As a result, it is not necessary to substantially purify the protein before adding it to the protein formulations of the present invention, thereby saving time, cost, and avoiding undesirable losses.

[0065] As will be understood by those skilled in the art, the protein formulations meet the legal requirements set by relevant authorities such as the US Food and Drug Administration (FDA) or the European Food Safety Authority (EFSA) in the case of food products. Thus, even if cell material, fermentation residues or other compounds are present, they are present in relatively small amounts and the protein formulations meet the legal requirements defined by the relevant authorities.

[0066] Alternatively or in addition, the reducing sugar and / or metal ion may be added to the protein in the protein formulation, for example, in the form of a carrier or excipient. Examples of carriers include cereal flours, particularly wheat flour, and lactose.

[0067] The protein may be added to the protein formulations of the present invention in a dry form, such as a powder or granulate, or dissolved and added in any form.

[0068] Preferably, the protein is added to a solution, particularly an aqueous solution. The aqueous solution may contain one or more additional components, for example, to stabilize the protein in the solution. Preferably, the aqueous solution contains a stabilizer, solubilizer, cofactor, or cosolvent. Preferably, the aqueous solution further contains glycerol. Glycerol may advantageously stabilize the protein by preventing aggregation during protein refolding.

[0069] Preferably, the protein is present in the protein formulation of the present invention in an amount between about 0.001 wt% and about 50 wt%, preferably between about 0.01 wt% and about 40 wt%, between about 0.1 wt% and about 25 wt%, and most preferably between about 0.2 wt% and about 10 wt% based on the total dry weight of the protein formulation.

[0070] Protectant The protein formulation of the present invention further comprises a protectant comprising (i) at least one amine and / or ammonium group, and (ii) a metal precipitant and / or chelating molecule.

[0071] Without wishing to be bound by any theory, it is believed that the amine or ammonium group of the protectant may react with reducing sugars in the Maillard reaction or Maillard-type reactions (Sengar and Sharma, J Food Sci Technol. 2014.51(9):1686 - 1696). Thereby, any reducing sugars that may be present in the protein formulation are inactivated, and thus the protein is no longer involved in the Maillard reaction or at least the involvement is reduced, ultimately leading to the degradation of the protein.

[0072] As will be understood by those skilled in the art, the protectant may be present in the protein formulation of the present invention in the form of a molecule containing an amine group. In this form, the protectant may contain a molecule represented by the chemical formula NR1R2R3. In this form, the amine group contains a lone pair of electrons that can react as a nucleophile with the aldehyde of the reducing sugar.

[0073] Alternatively or in addition, the protecting agent may be present in the protein formulation of the present invention in the form of an ammonium group. In this form, the protecting agent has the chemical formula NR1R2R3H + X - and may contain a molecule represented by. In this form, the ammonium group may participate in a reversible reaction with an amine group. Generally, such a reversible reaction has the formula: HA + NR1R2R3 ⇔ A - + NR1R2R3H + and may be represented by, where HA represents an acidic group capable of donating H + ions, and A - represents a basic group capable of accepting H + ions. As will be understood by those skilled in the art, the type A - does not necessarily have to carry a net negative charge, but may be represented by any type capable of accepting H + ions (for example, a carbonyl group or an alcohol group present in a reducing sugar). Similarly, the type HA does not necessarily have to be neutral, but may be represented by any type capable of donating H + ions (such as an ammonium ion or an ammonium salt).

[0074] Therefore, without wishing to be bound by any theory, it is considered that when the protecting agent contains an ammonium group, the corresponding amine may be generated in situ and may then be involved in the Maillard-type reaction described above. However, as will be understood by those skilled in the art, the electrons present in the N-H bond may also react directly with a carbonyl group without generating an amine as an intermediate species.

[0075] In the general formulas NR1R2R3 and NR1R2R3H + X - , the substituents R1, R2 and R3 may in principle be any substituents that give a chemically stable molecule.

[0076] For example, the substituents R1, R2 and R3 may each independently be selected from the group consisting of hydrogen (H), an alkyl group, an alkenyl group, an alkynyl group or an aryl group.

[0077] The alkyl group may be a linear alkyl group or a branched alkyl group, and is usually represented by the general formula C n H 2n+1 where n may 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.

[0078] Also, the alkyl group may usually be a cyclic group represented by the general formula C n H 2n where n may be any integer, preferably an integer selected from 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Examples of cyclic alkyl groups include pentyl, hexyl and heptyl groups.

[0079] The alkenyl group refers to any unsaturated aliphatic group containing at least one double bond in its skeleton. Therefore, the alkenyl group may contain one double bond or a plurality of double bonds such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 double bonds. The alkenyl may be linear or branched. Examples of alkenyl groups include ethenyl, propenyl, butenyl and the like.

[0080] The alkynyl group refers to any aliphatic unsaturated hydrocarbon containing at least one triple bond in its skeleton. Therefore, the alkynyl group may contain one triple bond or a plurality of triple bonds such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 triple bonds. Examples of alkynyl groups include ethynyl, propynyl, butynyl and the like.

[0081] The aryl group refers to any group containing an aromatic functional group within its skeleton. As is known in the art, an aromatic group refers to a planar cyclic group containing an unsaturated bond, and the unsaturated bond is completely conjugated, meaning that electrons can move freely through the p-orbitals present in the aromatic ring. When a group or molecule has 4n + 2 Π electrons, the group or molecule is generally aromatic and is also called the "Hückel's rule" in the art. The aryl group may contain one aromatic group or a plurality of aromatic groups such as two, three, four, or five. Examples of aromatic groups include benzyl, phenyl, naphthyl, or imidazole.

[0082] Here, the X - may be represented by any suitable negatively charged ion or molecule capable of forming an ionic bond with the positively charged NR1R2R3H + species. Usually, 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, particularly 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 between about 110 g / mol and about 500 g / mol, even more preferably between about 120 g / mol and 350 g / mol, and most preferably between about 140 g / mol and about 200 g / mol.

[0083] Preferably, X - represents an organic acid, more preferably an organic acid selected from the group consisting of citric acid, malic acid, malonic acid, oxalic acid, tartaric acid, lignosulfonic acid, humic acid, fulvate, pectin, amidated pectin, lactic acid, and urate. Most preferably, X - represents citric acid or tartaric acid.

[0084] Alternatively, X - may represent an inorganic molecule, preferably a metal ion precipitating group, and most preferably a phosphate group.

[0085] Preferably, the substituents R1, R2, and R3 are independently selected from hydrogen or hydrocarbons. Alternatively, the substituents may include one or more functional groups such as a carbonyl, ester, carboxylic acid, halogen, amine, amide, nitrile, nitro group, or ether group.

[0086] As will be appreciated by those skilled in the art, the substituents R1, R2, and R3 at least partially determine the nucleophilicity and basicity of the amine group.

[0087] For example, when R1, R2, and R3 represent electron-donating groups, the nucleophilic properties of the amine group are enhanced by an increase in the electron density of the lone pair of electrons present in the amine group. Examples of electron-donating groups include alkyl groups such as straight-chain alkyl groups containing a methyl, ethyl, propyl, or isobutyl group, cyclic alkyl groups such as a pentyl or hexyl group, and aromatic groups such as a benzyl or phenyl group.

[0088] Furthermore, when R1, R2, and R3 represent electron-withdrawing groups, the nucleophilic properties of the amine group decrease due to a decrease in the electron density of the lone pair of electrons present in the amine group. An example of an electron-withdrawing group is a carbonyl group.

[0089] Furthermore, as will be recognized by those skilled in the art, due to the steric hindrance caused by the presence of one or more substituents R1, R2, and R3, the presence of one or more substituents R1, R2, and R3 increases the basicity of the amine compared to an amine lacking said substituents and decreases the nucleophilicity of the amine group compared to an amine lacking said substituents.

[0090] Accordingly, preferably, at least one of R1, R2, and R3 of the general formula NR1R2R3 is a hydrogen atom, more preferably at least two of R1, R2, and R3 are hydrogen atoms, and most preferably all of R1, R2, and R3 are hydrogen atoms.

[0091] When one of R1, R2 and R3 in the general formula NR1R2R3 is a hydrogen atom, the general formula is NR1R2H, provided that R1 and R2 are as defined above but are not hydrogen. This is called a secondary amine in the art.

[0092] When two of R1, R2 and R3 in the general formula NR1R2R3 are hydrogen atoms, the general formula is NR1H2, provided that R1 is as defined above but is not a hydrogen atom. This is called a primary amine in the art.

[0093] When all three of R1, R2 and R3 in the general formula NR1R2R3 are hydrogen atoms, the general formula is NH3, which is called ammonia in the art. Preferably, the protecting agent contains ammonia.

[0094] In one form, the protecting agent contains an amino acid, particularly an amino acid selected from the group consisting of methionine, phenylalanine, tryptophan, asparagine, glutamine, serine, threonine, cysteine, lysine, arginine and histidine, or its conjugate acid.

[0095] Preferably, the protecting agent in the protein preparation of the present invention contains ammonia (NH3), ammonium ions (NH4 + ), or both. When the protecting agent contains ammonium ions, the ammonium ions generally exist in the form of a salt with a negatively charged counterion or negatively charged molecule (X - ) as described above.

[0096] Preferably, the protecting agent in the protein preparation of the present invention contains ammonium salts of organic acids, preferably citrate, malate, malonate, oxalate, tartrate, lactate, urate, lignosulfonate, humate, fulvate, ammonium salts of pectin, and amidated pectin, or inorganic salts, preferably ammonium salts of phosphate, carbonate and sulfide.

[0097] The protective agent further comprises at least one metal precipitant and / or chelating molecule.

[0098] 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, and even more preferably can bind transition metal ions in the 4th period of the periodic table.

[0099] The transition metal is preferably a divalent or trivalent transition metal ion.

[0100] The transition metal is preferably selected from scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc. Preferably, the transition metal ion is Cu 2+ , Fe 2+ , Fe 3+ , Zn 2+ , Mn 2+ or Mb 2+ selected from the types of. These are transition metal ions that often exist as residues from the medium used to culture microorganisms for producing proteins, especially in protein formulations.

[0101] Preferably, the protective agent is a chelating molecule, more preferably a metal ion chelating molecule, especially a transition metal ion chelating molecule, specifically Fe 3+ , Fe 2+ , Cu 2+ , Mn 2+ , Mb 2+ and / or Co 2+ containing chelating molecules.

[0102] While not wishing to be bound by any theory, it is believed that chelating molecules advantageously form stable soluble complexes with metal ions, preferably transition metal ions. Thus, the metal ions are prevented from accelerating the Maillard reaction and leading to the degradation of proteins in the protein formulation.

[0103] Preferably, the protective agent contains citrate or tartrate, more preferably an ammonium salt of citrate, including diammonium hydrogen citrate, diammonium tartrate, ammonium hydrogen tartrate, ammonium dihydrogen citrate, and triammonium citrate. Most preferably, the protective agent contains triammonium citrate or diammonium hydrogen citrate.

[0104] Alternatively or in addition, the protective agent is a metal precipitant, preferably a metal ion precipitant, most preferably a transition metal ion precipitant, especially Fe 3+ 、Fe 2+ 、Cu 2+ 、Mn 2+ 、Mb 2+ 、Co 2+ precipitant.

[0105] The metal precipitant preferably has the ability to form complexes or salts with transition metal ions having limited solubility in aqueous media such as dough. Without wishing to be bound by any theory, it is believed that the metal precipitant has the ability to form stable complexes or salts with metal ions having limited solubility in aqueous media by preventing the diffusion of metal ions through aqueous media such as dough.

[0106] As a result, the transition metal ions are removed from the reaction, preventing the unwanted effects of transition metal ions on promoting the Maillard reaction.

[0107] Preferably, the metal precipitant-metal ion complex or salt has a solubility in water of 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, and most preferably at most 25 μg / l at 25 °C, atmospheric pressure, and physiological pH.

[0108] Preferably, the metal precipitant and the salt or complex of the transition metal ion have a solubility between about 5 and about 10,000 μg / l, such as between about 10 and about 1000 μg / l, preferably between about 15 and about 100 μg / l, between about 20 and about 50 μg / l, and most preferably between about 21 and about 40 μg / l, at 25°C, atmospheric pressure, and physiological pH.

[0109] Normally, physiological pH here means a pH of about 7.4, preferably between about 4 and 10, preferably between about 5 and about 9, between about 6 and about 8, and most preferably about 7, especially between 7 and 8.

[0110] As will be understood by those skilled in the art, solubility in water is a property of the metal precipitant-metal ion complex, which can usually be found in appropriate substance databases such as the "Gestis-Stoffendatenbank", https: / / gestis.dguv.de / list (accessed on July 20, 2022).

[0111] Experimentally, the solubility in water can be determined using conventional methods known in the art. For example, the solubility can be measured at 25°C, atmospheric pressure, and physiological pH by mixing increasing amounts of the component whose solubility is to be measured in water until precipitation, cloudiness, turbidity, etc. are observed.

[0112] The solubility may also be determined using analytical means such as using high performance liquid chromatography.

[0113] Preferably, the metal precipitant is diammonium hydrogen phosphate, ammonium phosphate, ammonium dihydrogen phosphate, ammonium carbonate, ammonium bicarbonate, ammonium hydrogen sulfide, or ammonium sulfide.

[0114] Preferably, the metal precipitant is not a sulfate or chloride salt.

[0115] The protective agent preferably includes molecules having the ability to increase the surface tension of water. Such molecules (usually salts) are also referred to in the art as "cosmotropic molecules". Unlike chaotropic molecules, cosmotropic molecules generally contribute to the stability and structure of water-water interactions. Thereby, cosmotropic molecules stabilize the intramolecular interactions within molecules such as proteins, thereby providing the protein with a more stable and compact folding. When the concentration of such cosmotropic molecules is high enough, the protein is usually salting out from the aqueous phase in a non-denatured, compact folded form. The protein salted out in a compact form usually includes a hydration layer surrounding the protein, in which molecules of the (cosmotropic) salt are embedded.

[0116] As used herein, the term "salting out" means the salt-induced precipitation of a protein from an aqueous medium. It is possible to distinguish between the natural salting out of a protein in a non-denatured form and the salting out of a protein in a denatured form. The latter usually occurs in the presence of salts that promote the unfolding of the protein. Unfolded proteins are generally less soluble in water because the hydrophobic portions of the protein are exposed to water and the solubility decreases.

[0117] Without wishing to be bound by any theory, the inventors believe that by using a cosmotropic salt to increase the surface tension of water, the hydrophobic interaction between the protein and water increases. As expected, the protein correspondingly reduces its surface area and adopts a more compact protein folding to minimize contact with the protective agent. The inventors believe that this compact folding increases the stability of the protein, thereby making the protein more resistant to external stresses such as mechanical and thermal stresses.

[0118] Accordingly, the protective agent preferably comprises a cosmotropic molecule, in particular a cosmotropic salt. This is particularly advantageous when the protein formulation is solid, since solid protein formulations are often subjected to mechanical stress, for example during the preparation of a food or feed product comprising said protein formulation or during downstream processing steps.

[0119] 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 protective agent capable of increasing the surface tension of water based on general knowledge and the information provided herein.

[0120] As will be understood by a person skilled in the art, said at least one amine and / or ammonium group may be contained within the same molecule as said at least one metal precipitant and / or chelating molecule.

[0121] Accordingly, the present invention preferably relates to a protein formulation comprising a protein, preferably an enzyme, and a protective agent, said protective agent being 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.

[0122] Preferably, the protective agent is a small molecule. In the art, a molecule is usually referred to as a small molecule when it is an organic molecule having a molecular weight of less than 1000 g / mol, preferably less than 900 g / mol, more preferably less than 700 g / mol, less than 500 g / mol, less than 350 g / mol, in particular less than 200 g / mol. Preferably, the molecular weight of the small molecule is between about 80 g / mol and about 700 g / mol, more preferably between about 90 g / mol and about 500 g / mol, for example between about 100 g / mol and about 350 g / mol, in particular between about 110 g / mol and about 200 g / mol.

[0123] Preferably, the molecule is a salt containing a cation and an anion, the cation contains ammonium, and the anion is a chelating agent or a metal precipitant.

[0124] Preferred examples of such molecules include ammonium citrate tribasic, ammonium citrate dibasic, ammonium dihydrogen citrate, ammonium tartrate dibasic, ammonium hydrogen tartrate, ammonium phosphate, ammonium dihydrogen phosphate, and diammonium hydrogen phosphate.

[0125] Alternatively, the protective agent contains at least two molecules, the first molecule contains at least one amine and / or ammonium group, and the second molecule contains at least one metal precipitant and / or chelating molecule. For example, the protective agent may contain a first molecule containing an amine or ammonium group and a second chelating molecule such as citric acid or a salt thereof. Here, the salt of citric acid may be a different salt such as an ammonium salt or a sodium salt.

[0126] As will be understood by those skilled in the art, protective agents formed in situ are also encompassed by the present invention.

[0127] In the protein preparation of the present invention, the weight ratio (w / w ratio) between the protein, preferably an enzyme, and the protective agent is preferably about 1:5, preferably about 1:10, about 1:20, preferably about 1:30, preferably 1:50, more preferably 1:100.

[0128] The present invention further relates to a protein formulation that is preferably at least partially resistant to reducing sugars and / or metal ions and has thermal stability and / or storage stability. Preferably, at least 20% of the protein present in the protein formulation of the present invention is still active after the protein formulation has been exposed to a temperature of at least 50°C. Most preferably, after the protein formulation of the present invention has been exposed 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%, most preferably 100% of the protein is still active.

[0129] The activity of the protein can be determined using an appropriate activity assay, as shown in Examples 3 and 4, for example. To test the activity of 6-phytase, a protocol compliant with ISO method 30024:2009 may be used, for example.

[0130] Preferably, the protein formulation of 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, most preferably at least 120°C. These are usually the conditions used during an extrusion or steam pellet process.

[0131] Preferably, the protein formulation of the present invention is stable at a temperature between 55°C and 150°C, more preferably between 60°C and 140°C, between 65°C and 130°C, between 70°C and 125°C, between 75°C and 120°C, between 80°C and 115°C, between 85°C and 110°C, between 90°C and 105°C, between 95°C and 100°C.

[0132] At such temperatures, at least 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 is still active. In certain embodiments, after the protein formulation of the present invention has been exposed 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 is still active.

[0133] Typically, the protein formulation of the present invention is exposed to heat, for example at least 50°C, preferably at least 90°C, for a period of about 10 seconds to about 5 hours, preferably about 20 seconds to about 3 hours, 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, most preferably about 2 minutes to about 15 minutes.

[0134] The protective agent may be present in the protein protective agent of the present invention in any amount sufficient to enhance the thermal stability of the protein.

[0135] Preferably, the protective agent is present in an amount between about 5 wt% and about 50 wt%, preferably between about 10 wt% and about 40 wt%, most preferably between about 15 wt% and about 30 wt% based on the dry weight of the protein formulation.

[0136] Preferably, the protective agent is present in an amount of at least 5 wt% based on the dry weight of the protein formulation, and more preferably in an amount of at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, most preferably at least 50 wt% based on the dry weight of the protein formulation.

[0137] Preferably, the protecting agent is present in an amount of up to 50 wt%, more preferably up to 45 wt%, up to 40 wt%, up to 35 wt%, up to 30 wt%, up to 25 wt% based on the dry weight of the protein formulation.

[0138] In a protein formulation containing a liquid medium, the protecting agent 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.

[0139] Preferably, in a protein formulation containing a liquid medium, the protecting agent is present in an amount of up to 5 mol / L, more preferably up to 4 mol / L, up to 3.5 mol / L, for example up to 3 mol / L, particularly up to 2.5 mol / L.

[0140] The normal amount of the protecting agent in such a protein formulation is between about 1.3 mol / L and about 5 mol / L, particularly between about 1.5 mol / L and about 3 mol / L. Here, the concentration is determined by calculating the total number of moles of the protecting agent per total volume (in liters) of the liquid present in the protein formulation.

[0141] At such a concentration of the protecting agent in the protein formulation, usually, the folding of the protein becomes more compact, and the protein is usually salted out from the aqueous medium in such a compact fold and in a non-denatured state.

[0142] The protein formulation of the present invention may further contain one or more carriers. In principle, any carrier that conforms to the relevant rules regarding protein formulations is suitable for use in the protein formulation of the present invention. Preferably, the protein formulation of the present invention contains flour, most preferably flour containing gluten, such as wheat flour, barley flour, rye flour, spelt wheat flour, emmer wheat flour, and combinations thereof. Most preferably, the protein formulation of the present invention contains wheat flour.

[0143] Such protein formulations are particularly suitable for extrusion because they form dough, at least in part, due to the presence of gluten in the flour. Such dough may advantageously be subjected to one or more steps of an extrusion, (steam) pelletization or tableting process, as described below.

[0144] Preferably, the protein formulation containing flour contains at least 10 wt% flour, more preferably at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt% flour, based on the total dry weight of the protein formulation.

[0145] Preferably, the protein formulation containing flour contains flour in the range of about 10 to about 90 wt%, more preferably in the range of about 20 wt% to about 80 wt%, in the range of about 30 wt% to about 60 wt%, in the range of about 40 wt% to about 50 wt%, based on the total dry weight of the protein formulation.

[0146] Preferably, the protein formulation of the present invention further contains an excipient. In principle, any excipient that complies with the relevant regulations regarding protein formulations is suitable for use in the protein formulation of the present invention. Preferably, the protein formulation of the present invention further contains microcrystalline cellulose.

[0147] Preferably, the protein formulation containing flour contains at least 1 wt% microcrystalline cellulose, more preferably at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt% microcrystalline cellulose, based on the total dry weight of the protein formulation.

[0148] Preferably, the protein formulation containing flour contains at most 30 wt% microcrystalline cellulose, more preferably at most 25 wt%, at most 20 wt%, at most 18 wt%, at most 15 wt% microcrystalline cellulose, based on the total dry weight of the protein formulation.

[0149] Preferably, the protein preparation containing flour contains microcrystalline cellulose between about 1% and about 30% by weight, more preferably between about 2% and about 25% by weight, based on the total weight of the protein preparation, and contains flour between about 5% and about 20% by weight.

[0150] Preferably, the protein preparation of the present invention contains a protecting agent between about 3% and about 25% by weight, more preferably between about 5% and about 20% by weight, most preferably between about 10% and about 15% by weight, based on the total dry weight, and contains flour between about 30% and about 70% by weight, preferably wheat flour, preferably between about 40% and about 60% by weight, preferably wheat flour, and microcrystalline cellulose between about 5% and about 25% by weight, preferably between about 10% and about 20% by weight, more preferably between about 15% and about 18% by weight, and protein between about 0.001% and about 50% by weight, more preferably between 0.1% and 40% by weight, between about 1% and about 30% by weight, between about 5% and about 20% by weight. The protein is preferably an enzyme, more preferably phytase, chitinase, cellulase or protease.

[0151] The protein preparation of the present invention may be in any outer shape form. For example, the protein preparation may be formulated as a solid, liquid, emulsion or suspension. Preferably, the protein preparation of the present invention is formulated as a solid because this is the most dense form, can be stored efficiently and can be easily handled. Furthermore, a solid protein preparation usually has longer storage stability compared to an equivalent liquid protein preparation of the same protein which is the same except that it is liquid.

[0152] Preferably, the protein preparation of the present invention is formulated as (compressed) tablets, granules, powders, capsules or pellets.

[0153] Preferably, the protein preparation of the present invention is formulated as a mixture of a protein and a protecting agent as defined herein. More preferably, the protein preparation is formulated as a solid mixture forming a powder or granules.

[0154] Preferably, the particle size of such powder or granules ranges from about 10 μm to about 3 mm, preferably between about 50 μm and about 2 mm, more preferably between about 100 μm and about 1 mm, between about 200 μm and about 900 μm, between about 350 μm and about 800 μm, and most preferably between about 500 μm and about 75000 μm.

[0155] In the protein preparation of the present invention, the protein, preferably an enzyme, and the protecting agent as defined herein are preferably at least substantially uniformly dispersed in the protein preparation of the present invention. As used herein, the term "uniform" is generally known to mean that the components are substantially uniformly dispersed throughout the protein preparation of the present invention. This may be tested by obtaining at least two samples from the protein preparation of the present invention, which should have substantially the same, for example, substantially the same composition and / or properties.

[0156] Preferably, the protecting agent as defined herein does not exist as a coating, or the protein does not exist as a core containing a coating in the protein preparation of the present invention. Most preferably, the protein preparation of the present invention is not formulated as a core containing a protein at least partially coated with the protecting agent as defined herein.

[0157] The solid protein preparation of the present invention may further optionally, preferably, contain a humidity barrier and / or a gas barrier coating containing sugar.

[0158] Preferably, the protein preparation is an additive for food or animal feed.

[0159] (Food) product The present invention further relates to a food product of the invention, and a food, preferably an animal feed, comprising one or more additional food ingredients such as carbohydrates, fats and proteinaceous substances. In principle, the food may be any food that may benefit from the presence of the protein preparation of the invention.

[0160] Any fat suitable for consumption by animals, including humans, is suitable for use in the food products of the invention. Such fats include animal fats such as lard or butter, or vegetable oils. Said fats may include saturated fatty acids, unsaturated fatty acids and polyunsaturated fatty acids and combinations thereof.

[0161] 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.

[0162] The fat may further contain one or more of omega 3 fatty acids such as eicosapentaenoic acid, docosahexaenoic acid and docosapentaenoic acid.

[0163] Any carbohydrate suitable for consumption by animals, including humans, including easily digestible and indigestible carbohydrates, is suitable for use in the food products of the invention.

[0164] Any proteinaceous substance suitable for consumption by animals, including humans, is suitable for use in the food products of the invention. For example, non-hydrolyzed proteins, natural proteins, hydrolyzed proteins, oligopeptides, i.e., peptides containing 2 to 50 amino acids, and proteins or any part of a protein such as free amino acids from any source may be used in the food products of the invention. Preferably, said protein is a coagulated protein. The total proteinaceous substance content of the food products of the invention can be determined by the Kjeldahl method known in the art. As will be understood by those skilled in the art, the Kjeldahl method measures the total nitrogen content, and to determine the protein content, the total content of the protective agent needs to be subtracted.

[0165] The food may further contain other food ingredients beneficial to humans including animals, such as vitamins, minerals and / or probiotics.

[0166] Examples of minerals suitable for addition to the food of the present invention are sodium, chloride, potassium, calcium, phosphorus, magnesium, sulfur, iron, zinc, iodine, selenium, copper, manganese, fluoride, chromium and / or molybdenum.

[0167] Examples of essential vitamins suitable for addition to the food preparation of 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.

[0168] Preferably, the food is animal feed. Most preferably, the food relates to food for pet animals such as dog food, cat food, hamster food, guinea pig food, rodent food, mouse food and rabbit food, or food for livestock such as horse food, cattle food, pig food, goat food and sheep food, and food for poultry such as chicken, duck, goose and turkey.

[0169] The food may be formulated in any form or shape, such as solids such as pellets, tablets, capsules, powders, or liquids such as gels, suspensions, emulsions or beverages. Preferably, the food is solid, and most preferably it is a pellet.

[0170] The present invention further relates to household products, preferably to detergents containing the protein preparation of the present invention and one or more of a surfactant, a chelating agent or a bleaching agent.

[0171] The surfactant is preferably an anionic surfactant such as a branched or linear anionic surfactant, or a nonionic surfactant such as a polyalkylene oxide condensate of an alkylphenol.

[0172] The chelating agent may be any suitable chelating agent such as an aluminosilicate material, a silicate, a polycarboxylate or a fatty acid.

[0173] The bleaching agent may be any suitable bleaching agent such as an oxygen bleaching agent or a halogen bleaching agent. In particular, the bleaching agent is a bleaching agent that releases hydrogen peroxide such as perborate, or a hypochlorite bleaching agent such as trichloroisocyanuric acid.

[0174] The household product, preferably the detergent, may optionally contain one or more further components such as a foaming agent, a stabilizer, a solubilizing agent, a corrosion inhibitor, a dye transfer inhibitor, an aroma, a brightening agent or a softening agent.

[0175] Methods and Uses The present invention further relates to a method for preparing a protein preparation of the present invention, comprising a protein and (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, particularly one or more transition metal ions.

[0176] The protein and the protective agent containing (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitant and / or chelating molecule are as described above in this specification.

[0177] The mixing of the protein and the protective agent as defined herein may be carried out using any method in the art such as whipping, tapping or stirring. Preferably, at least a substantially homogeneous mixture of the protein and the protective agent containing (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitant and / or chelating molecule is obtained.

[0178] Preferably, the method further includes the step of mixing water to form an aqueous mixture containing a protein, a protective agent, and preferably one or more reducing sugars and / or metal ions. Preferably, the aqueous mixture contains at least 25 wt% water, more preferably at least 40 wt% water, at least 55 wt% water, and most preferably at least 70 wt% water, based on the total weight of the aqueous mixture. Preferably, the aqueous mixture contains between about 25 wt% and about 70 wt% water, particularly between about 30 wt% and about 50 wt% water, based on the total weight of the aqueous mixture.

[0179] Preferably, the concentration of the protective agent in the aqueous mixture is between about 1.3 mol / L and about 5 mol / L, particularly between about 1.5 mol / L and about 3 mol / L.

[0180] Furthermore, the method includes mixing a carrier, preferably a flour selected from the group consisting of wheat flour, barley flour, rye flour, spelt wheat flour, emmer wheat flour, and combinations thereof or derivatives such as starch, to obtain a protein formulation containing the flour.

[0181] Preferably, the method further includes the step of mixing one or more excipients, particularly microcrystalline cellulose.

[0182] Typically, the protein formulation containing the flour and optionally microcrystalline cellulose is in the form of a dough and is suitable for further processing such as extrusion, pelletization, and tableting.

[0183] Preferably, the dough contains a protein, preferably an enzyme, a protective agent, a flour, water, and microcrystalline cellulose.

[0184] In a preferred method of the present invention, the protein, the protective 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 the protein, the protective agent, water, the carrier, preferably wheat flour, and optionally the excipient, preferably microcrystalline cellulose, is mixed to form a dough, which is then extruded with a suitable extruder to form extruded dough particles called pellets.

[0185] Conventional wet granulation processes usually require a large amount of energy to prepare a dough containing protein and flour, which is undesirable from the viewpoints of durability and economy. The inventors have surprisingly noticed that the energy required to make a dough containing protein and flour can be significantly reduced in the presence of salt. This is shown in Examples 1 and 2 of the present application, and FIGS. 1 and 2. Furthermore, it is shown that when the protective agent is used as a salt against other salts such as sodium chloride, these effects may be obtained without unfolding the protein.

[0186] This is advantageous because solid protein formulations such as pellets can be advantageously made at a lower pressure compared to the pressure required in the absence of the protective agent, preventing or reducing the degradation of the protein from the protein formulation.

[0187] Accordingly, the present invention preferably relates to a method for preparing a protein formulation, comprising: a) providing a mixture of a protein, preferably an enzyme, a protective agent, water, a carrier, preferably a flour, and optionally an excipient, particularly microcrystalline cellulose; b) kneading the mixture obtained in step a) to form a dough; c) shaping, sizing, compressing, tableting, granulating, or extruding the dough, preferably extruding.

[0188] Preferably, the shaping, sizing, compression, tableting, granulation, or extrusion process occurs at a temperature below 60°C, more preferably below 50°C, below 45°C, below 40°C, below 25°C, and most preferably below 20°C. In particular, the shaping, sizing, tableting, granulation, compression, or extrusion process occurs at a temperature between 10°C and 60°C, more preferably between 20°C and 50°C, and even more preferably between 30°C and 45°C.

[0189] Preferably, the compression, tableting, granulation, or extrusion process occurs at a pressure below 40 bar, more preferably below 30 bar, below 20 bar, below 15 bar, below 10 bar, and most preferably below 5 bar.

[0190] Preferably, the compression, tableting, granulation, or extrusion process occurs at a pressure between about 0.5 and about 40 bar, more preferably between about 1 and about 25 bar, between about 2 and about 20 bar, between about 3 and about 15 bar, most preferably between about 4 and about 10 bar, and particularly between about 5 and about 7 bar.

[0191] It has been found that such pressures have the advantage of being able to obtain heat-stable protein formulations on a technical or industrial scale. Without wishing to be bound by any theory, it is believed that this may be due to a reduction in protein degradation by mechanical processing of the protein formulations of the present invention.

[0192] In another preferred embodiment, the method of preparing a protein formulation further comprises spray-drying a mixture comprising a protein, preferably an enzyme, a protective agent as defined herein, preferably one or more reducing sugars and / or metal ions, preferably transition metal ions, and optionally a carrier, preferably a cereal flour, to obtain a spray-dried protein formulation.

[0193] Preferably, the mixture further contains water, and based on the total weight of the mixture, more preferably at least 20 wt%, at least 30 wt% of water, more preferably at least 40 wt% of water, at least 50 wt% of water, at least 60 wt%, at least 70 wt%, at least 80 wt%, most preferably at least 90 wt% of water.

[0194] Preferably, the mixture contains water between about 70 wt% and about 99 wt%, particularly between about 80 wt% and about 95 wt% based on the total weight of the mixture.

[0195] In certain embodiments, the present invention a) provides an aqueous mixture, preferably a solution of water, protein and a protecting agent, and the protecting agent is preferably a kosmotropic salt, more preferably ammonium citrate tribasic, b) precipitating the protein from the mixture obtained in step a), c) collecting the protein to obtain a solid protein preparation, including relates to a method for preparing a solid protein preparation.

[0196] The amount of water present in the aqueous mixture should be sufficient to substantially solubilize at least the protein and the protecting agent. Usually, the amount of water present in the mixture is at least 20 wt% based on the total weight of the protein preparation, preferably at least 25 wt%, at least 30 wt% of water. Preferably, the amount of water present in the mixture is between about 20 wt% and about 90 wt% of the total weight of the protein preparation, more preferably between about 25 wt% and about 50 wt%, more preferably between about 30 wt% and about 40 wt%.

[0197] The concentration of the protecting agent in the aqueous mixture should be sufficient to salting-out the protein in the folded form that has not been denatured from the aqueous phase of the aqueous mixture.

[0198] As will be understood by those skilled in the art, the concentration of the protective agent required to salting out the protein from the aqueous medium is determined by the type of the protective agent and the protein used. Usually, the concentration of the protective agent is between about 1.3 mol / L and about 5 mol / L, particularly between about 1.5 mol / L and about 3 mol / L, more specifically between about 1.7 mol / L and about 2.5 mol / L. Here, the concentration is determined based on the total number of moles of the protective agent per unit volume (in liters) of the liquid, usually the aqueous medium, present in the protein preparation.

[0199] As will be understood by those skilled in the art, the precipitation of the protein from the aqueous mixture obtained in step a) may be visually monitored by observing, for example, the formation of solids, cloudiness or turbidity, or, particularly when the aqueous mixture is substantially a solution, may be monitored using appropriate imaging means. When the aqueous mixture is not substantially a solution, or when the precipitation of the protein cannot be easily monitored due to the presence of solid particles, such as when it is in a suspension, for example, the precipitation of the protein from the aqueous phase of the aqueous mixture may be determined using a comparable model solution (for example, a solution that lacks the solid particles of the suspension but is otherwise the same). Those skilled in the art will be able to determine the precipitation of the protein from aqueous mixtures other than solutions, such as suspensions, based on common general knowledge and the information provided herein.

[0200] The total time required in step b) is usually determined by the protein and the protective agent used in the process of the present invention. Usually, the time required to precipitate the protein 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, particularly at least about 240 seconds, more specifically at least about 360 seconds, such as at least about 300 seconds. Preferably, the time required for the protein to precipitate from the aqueous phase is between about 60 seconds and about 360 seconds, more preferably between about 120 seconds and about 300 seconds, most preferably between about 180 seconds and about 240 seconds.

[0201] The method for preparing the solid protein preparation preferably further comprises, in step a), providing a carrier, preferably a flour, and optionally an excipient, preferably microcrystalline cellulose, in the mixture.

[0202] Accordingly, in a preferred embodiment, the present invention a) providing a suspension comprising water, a protein, a protecting agent, a carrier, preferably a flour and optionally an excipient; b) precipitating the protein from the aqueous phase of the suspension obtained in step a); c) collecting the protein to obtain a solid protein preparation; comprises a method for preparing a solid protein preparation.

[0203] The mixture comprising the carrier and optionally the excipient obtained in step a) is preferably then kneaded to form a dough.

[0204] More preferably, the dough is then subjected to processes of shaping, tableting, sizing, granulating, compressing, extrusion molding to form an extruded dough.

[0205] The present invention further relates to a protein preparation obtained by the process of the present invention. Such a protein preparation is distinguished from equivalent protein preparations in that the presence of the protecting agent results in a more compact folding of the protein. Usually, a more compact protein folding confers higher stability to the protein, such as higher thermal stability. Thus, the protein obtained by the process of the present invention usually has a higher denaturation temperature determinable by circular dichroism spectroscopy as compared to the denaturation temperature of the same protein not obtained by the process of the present invention.

[0206] The folding of the protein may be determined using any suitable analytical means known in the art. For example, by subjecting the protein in solution to circular dichroism (CD) spectroscopy or differential scanning calorimetry (DSC).

[0207] The method of the present invention further preferably includes a step of drying the protein preparation or food of the present invention. The drying may be achieved using any suitable method known in the art, such as putting the protein preparation or food into an oven. Preferably, the protein preparation or food is dried at a temperature of at least 40°C, preferably at least 50°C, and most preferably at least 60°C. Usually, the protein preparation or food is dried at a temperature between 40°C and 80°C, preferably at a temperature of about 60°C.

[0208] Preferably, the dried protein preparation of the present invention contains less than 10 wt% water, preferably less than 5 wt% water, more preferably less than 4 wt% water, based on the total weight of the dried protein preparation.

[0209] Preferably, the dried protein preparation of the present invention contains between about 0.5 wt% and about 10 wt% water, preferably between about 01 wt% and about 5 wt% water, more preferably between about 2 wt% and about 4 wt% water, based on the total weight of the dried protein preparation.

[0210] Preferably, the method of the present invention does not include a step of coating a core containing a protein with a coating containing a protective agent defined herein or consisting essentially of a protective agent.

[0211] The present invention further relates to a method for preparing a food, preferably an animal feed, which includes mixing the protein preparation of the present invention with one or more additional food ingredients, preferably at least one fat, protein, preferably a coagulated protein or carbohydrate.

[0212] Preferably, the mixing includes a step of shaping, sizing, compressing, tableting, granulating or extruding a mixture of the protein preparation of the present invention and one or more of carbohydrates, fats and proteinaceous substances into a desired shape. Preferably, the protein preparation of the present invention is extruded to form granules.

[0213] Preferably, the shaping, sizing, compression, tableting, granulation or extrusion is carried out at a high temperature, preferably at a temperature exceeding 40°C, more preferably at a temperature exceeding 50°C, 60°C, 70°C, 75°C, and most preferably at a temperature exceeding 80°C. In particular, the shaping, sizing, tableting, granulation, compression or extrusion is carried out at a temperature between 50°C and 150°C, more preferably between 60°C and 130°C, and even more preferably between 80°C and 120°C.

[0214] Preferably, the shaping, sizing, compression, tableting, granulation or extrusion is carried out during a period of about 10 seconds to about 5 hours, preferably between about 20 seconds and about 3 hours, between about 30 seconds and about 2 hours, between about 45 seconds and about 1 hour, between about 1 minute and about 45 minutes, between about 1.5 minutes and about 30 minutes, and most preferably between about 2 minutes and about 15 minutes.

[0215] It is advantageous that the protein preparation of the present invention is substantially stable under these conditions. Preferably, at such temperatures, at least 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 remains active.

[0216] The present invention further relates to a method of feeding an animal, comprising providing a food or protein preparation according to the present invention, preferably an enzyme preparation, and administering said food or protein preparation, preferably an enzyme preparation, to an animal in need thereof.

[0217] The food or protein preparation may be administered to any animal that benefits from the administration of the protein preparation. Preferably, the food or protein preparation is administered to livestock such as cows, goats, sheep, lambs, pigs and horses, or poultry such as chickens, ducks, geese, turkeys, pheasants and birds, or pet animals such as cats, dogs, rabbits, guinea pigs, hamsters or mice, or wild animals such as deer, haze or wild boar.

[0218] The present invention further relates to the use of a protective agent, said protective agent comprising (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 diammonium tartrate, and ammonium hydrogen tartrate, and relates to the improvement of the stability of the protein preparation, in particular the improvement of the resistance of the protein preparation to reducing sugars and / or metal ions, thermal stability, and / or storage stability, and / or stability to mechanical stress.

[0219] For the purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, but it is understood that the scope of the present invention may include embodiments that combine all or some of the described features.

[0220] The present invention is illustrated by the following non-limiting examples.

[0221] (Example 1) 300 g of wheat flour (type 405) was mixed with 168 g of tap water, 0 - 6 wt% of ammonium sulfate, sodium chloride, sodium sulfate, ammonium citrate and ammonium acetate. The power input to the mixing process was measured in Farinograph units (FE) by a Farinograph E (Brabender). 1 FE corresponds to 9.8 ± 0.2 mN·m.

[0222] The results are shown in Figure 1. From Figure 1, it can be derived that the presence of salt reduces the energy requirement necessary for kneading the dough. This reduces the energy requirement of the wet granulation process and enables use under less severe manufacturing conditions, thereby reducing the possibility of degradation of the enzymes present in the enzyme preparation.

[0223] (Example 2) 300 g of various types of flour and starch were mixed with 168 g of tap water and 0 - 6 wt% ammonium sulfate. The power input to the mixing process was measured in Farinograph units (FE) by a Farinograph (Farinograph E, Brabender). 1 FE corresponds to 9.8 ± 0.2 mN·m.

[0224] The results are shown in Figure 2. From Figure 2, it can be derived that the presence of ammonium sulfate reduces the energy requirement necessary for kneading the dough, except for pure starch. This may be explained by the fact that starch does not contain the gluten fraction of the flour.

[0225] (Example 3) Testing the stability of various enzyme preparations using various potential protectants Manufacture of phytase pellets In a food processor (MUM2, Bosch), the dry materials (wheat flour, microcrystalline cellulose and salt) were homogenized for 1 minute. Thereafter, a 6 - phytase solution (Enzy Phostar, Kaesler Nutrition GmbH) of 40,000 U / g or more was added to each preparation batch, and the mixture was kneaded for about 3 - 4 minutes until the dough began to separate into individual small balls. The final content of the dough is shown in Table 1 for triammonium citrate. In the examples, instead of triammonium citrate, sodium chloride, ammonium acetate, ammonium chloride, ammonium sulfate, diammonium citrate, diammonium hydrogen phosphate, diammonium tartrate and ammonium acetate were used and repeated. The content of the salt and phytase may be slightly changed to obtain the desired viscosity of the dough as described above in this specification.

[0226]

Table 1

[0227] Then, the batch was removed from the ball, and the attachment of the mincer was attached with a 1 mm die. The dough passed through the mincer at level 2, and the extruded filamentous minced meat was collected. Thereafter, the thread was rounded in a spheronizer (MBS, Caleva) at 1600 rpm and a residence time of 30 seconds. The obtained pellets were dried at 60 °C for 4 hours in a heating oven (Heratherm OMH400, Thermo Scientific) on a plastic tray. The measurement of the residual moisture of the pellets was measured at 103 °C by an automatic stop (Sartorius MA35). The salt content was determined based on the dry weight of the phytase pellets.

[0228] Stability test of pellets Under each condition, two samples of 0.5 grams of phytase pellets were prepared in vials. One vial was left at room temperature as a control (for determination of the initial enzyme activity), and the other sample was placed in an autoclave (VX-150, Systec) for the heat resistance stress test and treated with an open pressure sterilizer with specified parameters (for determination of the residual (relative) enzyme activity). After starting the autoclave, it was heated to a temperature of 94 °C and this temperature was maintained for 2 minutes. This allows the sample to react with the hot steam and simulate the necessary conditioning means for the feed granulation process. Thereafter, after cooling to 74 °C, the apparatus was opened, the sample was placed directly on ice and cooled for an additional 30 minutes.

[0229] 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 stirred at 200 rpm for 45 minutes. Samples were diluted again with the same extraction buffer so that the measured absorbance was within the linear range of the plate photometer. The activity of 6-phytase in the samples was determined using ISO method 30024:2009. The determination of activity was based on the detection of phosphate released from the substrate sodium phytate (68388, Sigma-Aldrich), which reacted successively with molybdate and vanadate in nitric acid solution to form a yellow complex and was detected at an optical density of 415 nm with a plate photometer (FLUOstar Omega, BMG LABTECH GmbH). Samples were measured as 2 replicates ± SD. The relative activity of the heated samples was determined and the non-heated samples were adopted as 100% reference.

[0230] Results The results are shown in Figure 3. From Figure 3, it can be deduced that the activity of 6-phytase is not improved in the presence of increasing concentrations of sodium chloride, ammonium acetate, ammonium chloride or ammonium sulfate, and even decreases in the presence of increasing concentrations of ammonium acetate.

[0231] Without being bound by theory, in 6-phytase preparations containing ammonium acetate, it is considered that the 6-phytase fraction is at least partially salting out from the preparation. As a result, 6-phytase is exposed to the mechanical stress of the food processor and leads to further degradation of 6-phytase.

[0232] However, in samples containing ammonium citrate tribasic, diammonium hydrogen citrate, diammonium tartrate and Na3HPO4, the stability of 6-phytase was significantly improved. As can be deduced from Figure 3, there is a linear correlation between the concentration of ammonium salt and the relative activity of 6-phytase.

[0233] The results demonstrate that enzyme preparations containing a protective agent as defined herein exhibit improved activity compared to enzyme preparations that do not contain a protective agent.

[0234] (Example 4) Testing the stability of various enzyme preparations using various enzymes Manufacture of various enzyme pellets In a hood processor (MUM2, Bosch), the dry ingredients (wheat flour, microcrystalline cellulose and ammonium citrate) were homogenized for 1 minute. The cellulase TXL solution (Trichoderma longibrachiatum, ASA Spezialenzyme GmbH) was greater than 30 U / ml in 30 ml. The final content of the dough is shown in Table 2 for ammonium citrate and in Table 3 for sodium chloride. The examples were repeated using various enzyme solutions (i.e., 20 ml of protease SO2 solution (Aspergillus niger, ASA Spezialenzyme GmbH) or 5 ml of chitinase solution (Trichoderma harzianum, ASA Spezialenzyme GmbH), with more than 100 U / ml added to each prepared batch). As described above herein, the water content may vary slightly to obtain the desired viscosity of the dough.

[0235]

Table 2

[0236]

Table 3

[0237] The mixture was kneaded for about 3 - 4 minutes until the completed dough began to separate into individual small balls. Water was optionally added to enable the formation of such individual balls. Then the batch was removed from the bowl and the mincer attachment was fitted with a 1 mm die. The dough passed through the mincer at level 2 and the extruded thread - like minced matter was collected. Thereafter, the thread - like minced matter was rounded in a spheronizer (MBS, Caleva) at 1600 rpm and a residence time of 30 seconds. The resulting pellets were dried on a plastic tray in a heating oven (Heratherm OMH400, Thermo Scientific) at 60 °C for 4 hours. The measurement of residual moisture was measured at 103 °C by automatic stop (Sartorius MA35). Finally, the pellets had a residual moisture content of less than 10%. Ideally less than 5%. The salt content was determined based on the dry weight of the enzyme pellets.

[0238] Stability test of pellets For each condition and enzyme analysis, two identical samples of 1 g were weighed into vials. One vial was left at room temperature as a control (for determination of the initial enzyme activity), while the other was placed in an autoclave (VX - 150, Systec GmbH) for the heat - resistance stress test and treated with an open autoclave at defined parameters (for determination of the residual (relative) enzyme activity). After starting the autoclave, it was heated to a temperature of 94 °C and this temperature was maintained for 2 minutes. This allowed the samples to react with the hot steam and simulate the conditions required for the feed pelleting process. Then, after cooling to 74 °C, the apparatus was opened and the samples were placed directly on ice and cooled for a further 30 minutes.

[0239] For the enzymatic extraction of cellulose, 50 ml of 25 mM HAc-NaAc buffer pH 4.5 was added to all samples and stirred at 200 rpm for 45 minutes. Next, 1.5 ml of the extract was taken and centrifuged at 10,000×g for 10 minutes. If it was necessary to keep the sample within the linear measurement range of the photometer, the sample was diluted with sodium acetate buffer. To analyze the activity of cellulase TXL having endo-1,4-beta-glucanase activity, according to the manufacturer's instructions, the assay manual of beta-glucazyme tablets was followed (the substrate used was azurine-crosslinked barley beta-glucan; T-BGZ-200T; Megazyme). The extracts with or without impurities removed by dilution with 25 mM HAc-NaAc buffer pH 4.5 were preheated at 40 °C for 5 minutes in glass test tubes at 500 μl each. Next, beta-glucazyme tablets were added to initiate the enzymatic reaction. After 10 minutes, 10.0 mL of Trizma Base solution (2% w / v, pH 8.5) was added to stop the reaction. After cooling the sample at room temperature for 5 minutes, it was filtered using a Whatman filter, and 300 μL of the purified solution was analyzed with a plate photometer (FLUOstar Omega, BMG LABTECH GmbH, Germany). The absorbance was measured at 590 nm. The samples were measured as 2 replicates ± SD. The relative activity of the heated samples was determined, and the non-heated samples were adopted as 100% reference.

[0240] For protease extraction, 10 ml of 25 mM HAc-NaAc buffer pH 4.0 (reaction buffer) was added to all samples and stirred at 200 rpm for 45 minutes. Next, 1.0 ml of the extract was taken and centrifuged at 10,000×g for 10 minutes. If it was necessary to keep the sample within the linear measurement range of the photometer, the sample was diluted with sodium acetate buffer. To analyze the activity of protease S-02 having acidic peptidase activity, the assay manual of Pierce™ Colorimetric Protease Assay Kit (Thermo Scientific, 23263) was followed. The assay of acidic 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, and then 25 μl of diluted or undiluted sample or control was added to the substrate. The microtiter plate was incubated at 40 °C for 20 minutes. Next, 75 μl of 50 mM sodium borate buffer (pH 8.5) was pipetted into the sample to stop the enzyme reaction. Next, 50 μl of TNBSA solution (2,4,6-trinitrobenzenesulfonic acid, 5% (w / v) in methanol) was pipetted into the sample and incubated at room temperature for 20 minutes. The absorbance was analyzed at 450 nm with a plate photometer (FLUOstar Omega, BMG LABTECH GmbH). The samples were measured as 2 replicates ± SD. The relative activity of the heated sample was determined and the unheated sample was taken as 100% reference.

[0241] For chitinase extraction, 10 ml of 100 mM HAc-NaAc buffer pH 5.5 (reaction buffer) was added to all samples and stirred at 200 rpm for 45 minutes. Next, 1.0 ml of the extract was taken and centrifuged at 10,000×g for 10 minutes. The clear supernatant was used directly for the enzyme activity assay. Detection of chitinase activity was based on the enzymatic hydrolysis of a chitinase substrate (1,4-β-poly-N-acetylglucosaminidase activity against a specific substrate 4-nitrophenyl-N-acetyl-β-glucosaminide (Carl Roth GmbH + Co. KG, Germany)) using a chitinase assay kit (Sigma Aldrich, CS0980) according to the manufacturer's instructions. This hydrolysis releases p-nitrophenol (4-nitrophenol), which can be measured by colorimetry after ionization at basic pH values at 405 nm and was used as a reference to test the linear range of the plate photometer. 90 μl of the substrate solution (1 mg / ml in double-distilled water) was placed in a 96-well plate. To this, 10 μl of the extract or reference substance was added. After mixing, it was incubated at 37 °C for 30 minutes, followed by the addition of 200 μl of a 112.5 mg / ml (w / v%) sodium carbonate decahydrate solution. The absorbance was analyzed at 450 nm using a plate photometer (FLUOstar Omega, BMG LABTECH GmbH, Germany). Samples were measured in duplicate ±SD. The relative activity of the heated samples was determined, and the non-heated samples were taken as 100% reference.

[0242] Results The results are shown in Figure 4. As can be seen, for each enzyme, the stability was improved in the presence of tribasic ammonium citrate but not in the presence of sodium chloride.

[0243] (Example 5) Manufacture of the enzyme preparation of the present invention on a technical scale The dry ingredients (440 g of wheat flour, 114 g of microcrystalline cellulose, and various amounts of ammonium citrate tribasic) were homogenized by hand in a bag for 1 minute. The various mixtures were separately poured into a feeder (K-ML-SFS-KT20, Coperion K-Tron). Next, the dry mixture was fed to an extruder (ZSE18MAXX-HP, Leistritz) at a feed rate of 1 kg / h. The screw speed was adjusted to 500 rpm. The screw design mainly consisted of a conveying element, the result of adding the dried product in zone 6, and the addition of liquid in zone 7. The extruder discharge was actively cooled with tap water (approx. 14 °C). A phytase solution (ENZY Phostar, Kaesler Nutrition GmbH) of 40,000 FTU / g or more was added via a pump (NEMA 4x IP66, Watson Marlow) with a 1 mm liquid nozzle at a dosage of 7.5 - 13 g / min according to the formulation composition of the dry ingredients, forming a normal extrudate at moderate pressure within the extruder. The composition of the dough samples is given in Table 4. The dough was extruded through a die with a diameter of 0.8 mm and then rounded in a spheronizer (MBS, Caleva) at 1600 rpm and a residence time of 30 seconds. The resulting pellets were dried in a drying oven (Heratherm OMH400, Thermo Scientific) at a maximum product temperature of 60 °C. The measurement of residual moisture was measured at 103 °C with an automatic stop (MA35, Sartorius). The content of ammonium citrate tribasic was determined based on the dry weight of the phytase pellets.

[0244]

Table 4

[0245] In the second run, the experiment was repeated at different pressures (ranging from 0 to 30 bar as indicated by the pressure sensor). The pressure was varied by changing the content of the enzyme solution and / or the content of the salt. As can be derived from Figure 5b, by keeping the pressure low, preferably 10 bar or less, an enzyme preparation can be prepared while minimizing the loss due to enzyme degradation.

[0246] (Example 6) Production of the enzyme preparation of the present invention using spray drying The enzyme preparation was prepared by mixing 30 g of wheat flour, 10.5 g of ammonium citrate, 20 g of fermentation supernatant (E. coli 6 - phytase at 146 FTU / ml), and 139.5 g of tap water. Subsequently, the homogenized mixture was spray - dried at a pressure of 8 bar using a Büchi B - 290 Advanced equipped with a nozzle with a diameter of 1.5 mm and the following settings. Spray parameters: Inlet temperature: 110 °C Outlet temperature: 70 °C Pump capacity: 20 Flow rate: 40 mm Suction capacity: 100 Temperature of the collection container: 46 - 48 °C Spray time: 15 minutes

[0247] The reference enzyme preparation (without ammonium citrate) was prepared by mixing 30 g of wheat flour, 20 g of fermentation supernatant (E. coli 6 - phytase at 146 FTU / ml), and 150 g of tap water. Subsequently, the homogenized mixture was spray - dried at a pressure of 8 bar using a Büchi B - 290 Advanced equipped with a nozzle with a diameter of 1.5 mm and the following settings. Spray parameters: Inlet temperature: 110 °C Outlet temperature: 66 °C Pump capacity: 20 Flow rate: 40 mm Suction capacity: 100 Yield: 2.42 g Temperature of the collection container: 40 - 46 °C Spray time: 15 minutes

[0248] The obtained powder was then heated, and the relative activity was determined according to the procedure described in Example 3. The results are shown in Figure 6.

[0249] As can be derived from Figure 6, the powdered enzyme preparation of the present invention was more stable to heat compared to the reference enzyme preparation. These results indicate that the enzyme preparation of the present invention may also be prepared by means of spray drying.

[0250] (Example 7) Stability of the enzyme preparation of the present invention in the presence of glucose In a food processor (MUM2, Bosch), the dry ingredients (79.5 g of wheat flour, 20.6 g of microcrystalline cellulose, 35 g of ammonium tribasic citrate, and 0 - 4 wt% of glucose) were homogenized for 1 minute. 50 - 65 grams of a 6 - phytase solution (Enzy Phostar, Kaesler Nutrition GmbH) with more than 40,000 U / g was added to each preparation batch, and the mixture was kneaded for about 3 - 4 minutes until the finished dough began to separate into individual small balls. The finished batch was removed from the bowl, and a mincer attachment was fitted with a 1 mm die. The dough was passed through the mincer at level 2, and the extruded thread - like minced meat was collected. Then, the thread - like minced meat was rounded in a spheronizer at about 1600 rpm and a residence time of ~30 seconds. The obtained pellets were dried at 60 °C for 4 hours in a heating oven on a plastic tray. The measurement of residual moisture was measured at 103 °C by automatic stop (Sartorius MA35).

[0251] The obtained pellets were then heated, and the relative enzyme activity was determined according to the procedure described in Example 3. The results are shown in Figure 7.

[0252] As can be derived from Figure 7, in the presence of additional glucose, the relative phytase activity decreased. Due to the presence of glucose, more amino acids present in the enzyme are involved in the Maillard reaction, and therefore, further degradation is expected to be observed. These results suggest that the fundamental cause of the lack of stability observed when heat is applied is the Maillard reaction.

[0253] (Example 8) Stability of the enzyme preparation of the present invention in the presence of iron(III) chloride In a food processor (MUM2, Bosch), the dry ingredients (79.5 g of wheat flour, 20.6 g of microcrystalline cellulose, 35 g of tribasic ammonium citrate, and 0.0005 - 0.7 wt% of iron(III) chloride) were homogenized for 1 minute. 50 - 65 grams of a 6-phytase solution (Enzy Phostar, Kaesler Nutrition GmbH) with a phytase activity of 40,000 U / g or more was added, and the mixture was kneaded for about 3 - 4 minutes until the finished dough began to separate into individual small balls. Additionally, a control preparation without tribasic ammonium citrate and 0.0005 - 0.7 wt% of iron(III) chloride was prepared using the same protocol. The control preparation contained 79.5 g of wheat flour, 20.6 g of microcrystalline cellulose, and 50 - 65 g of a 6-phytase solution (Enzy Phostar, Kaesler Nutrition GmbH) with a phytase activity of 40,000 U / g or more.

[0254] The finished batch was transferred from the bowl, and a mincer attachment was installed with a 1 mm die. The dough passed through the mincer at level 2, and the extruded filamentous minced meat was collected. Subsequently, the filamentous minced meat was rounded in a spheronizer at approximately 1600 rpm and a residence time of approximately 30 seconds. The resulting pellets were dried at 60 °C for 4 hours in a heating oven on a plastic tray. The measurement of residual moisture was measured at 103 °C by automatic stop (Sartorius MA35).

[0255] The obtained pellets were then heated, and the relative enzyme activity was determined according to the procedure described in Example 3. The results are shown in Figure 8.

[0256] As can be derived from Figure 8, in the presence of additional iron(III) ions, the relative phytase activity decreased. In the presence of Fe 3+ ions, more amino acid residues are present on the enzyme particles in the Maillard reaction, and therefore, further degradation is expected to be observed. These results suggest that the fundamental cause of the lack of stability observed when heat is applied is the Maillard reaction. In the presence of ammonium citrate, the slope of the graph becomes gentler, and less degradation is observed. The results indicate that ammonium citrate acts as a protective agent by chelating metal ions.

[0257] (Example 9) Stability of the enzyme preparation after storage Three types of phytase pellets containing sodium chloride and ammonium citrate prepared by the method of Example 3, respectively, were stored at 22 °C and 50% humidity. After 15 months (sodium chloride and ammonium chloride) and 20 months (ammonium citrate), the phytase activity of the pellets was determined using the method described in Example 3.

[0258] The results are shown in Figure 9. From Figure 9, it can be derived that the phytase pellets containing ammonium citrate (triangles) after being stored for 20 months show higher residual phytase activity compared to the control groups containing ammonium chloride (squares) and sodium chloride (circles) stored for 15 months, respectively.

[0259] It can be concluded that the protein preparation of the present invention shows long-term storage stability compared to the control preparation.

[0260] (Supplementary Note) (Supplementary Note 1) containing a protein and a protective agent, The protective agent contains (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitant and / or chelating molecule. Protein preparation.

[0261] (Appendix 2) The protective agent contains an ammonium salt. The protein preparation according to Appendix 1.

[0262] (Appendix 3) The protective agent is a small molecule, preferably having a molecular weight of less than 1000 g / mol, particularly having a molecular weight between about 80 and about 250 g / mol. The protein preparation according to Appendix 1 or 2.

[0263] (Appendix 4) The protective agent is a kosmotropic salt. The protein preparation according to any one of Appendices 1 to 3.

[0264] (Appendix 5) The protective agent contains a chelating molecule. The protein preparation according to any one of Appendices 1 to 4.

[0265] (Appendix 6) The chelating molecule is selected from citrate and tartrate, preferably selected from ammonium citrate tribasic, diammonium hydrogen citrate and diammonium tartrate. The protein preparation according to Appendix 5.

[0266] (Appendix 7) The protective agent contains a metal precipitant, preferably contains phosphate, carbonate or sulfide, more preferably contains ammonium phosphate tribasic, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium carbonate, ammonium bicarbonate or ammonium hydrogen sulfide. The protein preparation according to any one of Appendices 1 to 6.

[0267] (Appendix 8) Furthermore, it contains one or more reducing sugars, preferably one or more reducing sugars that are at least partially produced by microorganisms, and even more preferably, the microorganisms are those expressing enzymes, or the one or more reducing sugars are present as residues from the culture medium. The protein preparation according to any one of Appendices 1 to 7.

[0268] (Appendix 9) Furthermore, it contains metal ions, preferably transition metal ions, more preferably zinc ions, iron ions, cobalt ions, manganese ions or molybdenum ions, and even more preferably, the transition metal ions are present as residues from the culture medium. The protein preparation according to any one of Appendices 1 to 8.

[0269] (Appendix 10) It contains a carrier, preferably a cereal flour selected from wheat flour, barley flour, rye flour, spelt wheat flour, emmer wheat flour and combinations thereof, and preferably, the preparation contains wheat flour. The protein preparation according to any one of Appendices 1 to 9.

[0270] (Appendix 11) The protein is an enzyme, preferably, the enzyme is selected from phytase, chitinase, peptidase and cellulase. The protein preparation according to any one of Appendices 1 to 10.

[0271] (Appendix 12) The protective agent is present in an amount between about 5 wt% and about 50 wt%, preferably between about 10 wt% and about 40 wt%, and most preferably between about 15 wt% and about 30 wt%, based on the dry weight of the protein preparation. The protein preparation according to any one of Appendices 1 to 11.

[0272] (Appendix 13) The protein preparation is a solid protein preparation. The protein preparation according to any one of Appendices 1 to 12.

[0273] (Appendix 14) The protein preparation described in any one of Appendices 1 to 13, and at least one fat, and / or further carbohydrates, and / or further proteins, preferably coagulated proteins, A food product comprising

[0274] (Appendix 15) To obtain the protein preparation described in any one of Appendices 1 to 14, a protein, and (i) at least one amine and / or ammonium group, and (ii) at least one metal precipitant and / or chelating molecule, with a protective agent comprising mixing A method for preparing the protein preparation described in any one of Appendices 1 to 14.

[0275] (Appendix 16) a) Providing a mixture of water, a protein, and a protective agent which is preferably a cosmotropic salt, more preferably ammonium citrate 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 The method according to Appendix 15, comprising

[0276] (Appendix 17) The concentration of the protective agent is between about 1.3 mol / L and about 5 mol / L, particularly between about 1.5 mol / L and about 3 mol / L The method according to Appendix 16.

[0277] (Appendix 18) To obtain a protein preparation comprising a carrier which is preferably a cereal flour, further comprising mixing a carrier, preferably a cereal flour selected from wheat flour, barley flour, rye flour, spelt wheat flour, emmer wheat flour and combinations thereof The method according to any one of Supplementary Notes 14 to 17.

[0278] (Supplementary Note 19) In order to obtain a spray-dried protein preparation, further comprising spray-drying the protein preparation, The method according to any one of Supplementary Notes 15 to 18.

[0279] (Supplementary Note 20) In order to obtain an extruded protein preparation, further comprising extruding the protein preparation containing cereal flour, preferably, the pressure during the extrusion is between 0.5 bar and 40 bar, preferably between 1 and 20 bar, particularly between 2 and 5 bar, The method according to Supplementary Note 18.

[0280] (Supplementary Note 21) Preferably, the protein preparation is a solid protein preparation. A protein preparation obtainable by the method according to any one of Supplementary Notes 15 to 20.

[0281] (Supplementary Note 22) Use of a protective agent in improving the stability of a protein preparation, particularly in improving the thermal stability of the protein, the storage stability of the protein, and the stability against mechanical stress, 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, the protective agent is ammonium citrate tribasic, ammonium citrate dibasic, ammonium dihydrogen citrate, ammonium phosphate tribasic, ammonium phosphate dibasic, ammonium dihydrogen phosphate, ammonium hydrogen tartrate, and ammonium bitartrate. Use.

Claims

1. A protein preparation comprising a protein and a protecting agent, 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.

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

3. The protein preparation according to claim 1 or 2, wherein the protecting agent is a small molecule, preferably having a molecular weight of less than 1000 g / mol, particularly having a molecular weight between about 80 and about 250 g / mol.

4. The protein preparation according to any one of claims 1 to 3, wherein the protecting agent is a kosmotropic salt.

5. The protein preparation according to any one of claims 1 to 4, wherein the protecting agent comprises a chelating molecule.

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

7. The protein preparation according to any one of claims 1 to 6, wherein the protecting agent comprises a metal precipitant, preferably comprising phosphate, carbonate or sulfide, more preferably comprising ammonium phosphate tribasic, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium carbonate, ammonium bicarbonate or ammonium hydrogen sulfide.

8. Furthermore, it comprises one or more reducing sugars, preferably one or more reducing sugars at least partially produced by microorganisms, and even more preferably, the microorganisms are microorganisms expressing enzymes, or one or more reducing sugars are present as residues from the culture medium. The protein preparation according to any one of claims 1 to 7.

9. Furthermore, it comprises metal ions, preferably transition metal ions, more preferably zinc ions, iron ions, cobalt ions, manganese ions or molybdenum ions, and even more preferably, the transition metal ions are present as residues from the culture medium. The protein preparation according to any one of claims 1 to 8.

10. It comprises a carrier, preferably comprising a cereal flour selected from wheat flour, barley flour, rye flour, spelt wheat flour, emmer wheat flour and combinations thereof, preferably, the preparation comprises wheat flour. The protein preparation according to any one of claims 1 to 9.

11. ​ ​ ​ ​ ​ ​ ​ The protein is an enzyme, preferably, the enzyme is selected from phytase, chitinase, peptidase and cellulase. The protein preparation according to any one of claims 1 to 10.

12. The protective agent is present in an amount between about 5 wt% and about 50 wt%, preferably between about 10 wt% and about 40 wt%, most preferably between about 15 wt% and about 30 wt%, based on the dry weight of the protein preparation. The protein preparation according to any one of claims 1 to 11.

13. The protein preparation is a solid protein preparation. The protein preparation according to any one of claims 1 to 12.

14. The protein preparation according to any one of claims 1 to 13, and at least one fat, and / or further carbohydrates, and / or further proteins, preferably coagulated proteins, A food product comprising.

15. To obtain the protein preparation according to any one of claims 1 to 14, 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, Mixing, A method for preparing the protein preparation according to any one of claims 1 to 14.

16. a) Providing a mixture of water, a protein, and a protective agent which is preferably a cosmotrophic 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, The method according to claim 15, comprising.

17. The concentration of the protective agent is between about 1.3 mol / L and about 5 mol / L, especially between about 1.5 mol / L and about 3 mol / L. The method according to claim 16.

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

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

20. To obtain the extruded protein preparation, further comprising extruding the protein preparation comprising the flour, preferably, the pressure during the extrusion is between 0.5 bar and 40 bar, preferably between 1 and 20 bar, particularly between 2 and 5 bar, The method according to claim 18.

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

22. Use of a protective agent in improving the stability of a protein preparation, particularly in improving the thermal stability of the protein, the storage stability of the protein and the stability against mechanical stress, 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, 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. Use.

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