METHOD FOR INCREASING THE STABILITY OF A PHYTASE IN A SOLID COMPOSITION AND A GRANULATED COMPOSITION COMPRISING PHOSPHATE AND PHYTASE

Abstract

The present compositions and methods relate to the thermostabilization of phytase with inorganic phosphate and to the enhancement of phytase recovery during the production of heat-treated animal feed pellets containing phytase. The phosphate is incorporated into the solid composition. The phosphate and phytase are in functional proximity and at a molar ratio of at least 50:1. Multilayer granules are used.

Description

METHOD FOR INCREASING THE STABILITY OF A PHYTASE IN A SOLID COMPOSITION AND A GRANULATED COMPOSITION COMPRISING PHOSPHATE AND PHYTASE nnccon / nznz / a / Y FIELD OF INVENTION The present compositions and methods relate to the thermostabilization of a phytase with phosphate and to the increased recovery of phytase during the production of heat-treated animal feed pellets or feed containing phytase. BACKGROUND OF THE INVENTION The use of active agents, such as enzymes, is common in animal feed and food. Enzymes are known to improve the digestibility of animal feed and food, reduce antinutritional factors, and enhance animal productivity. When compared to dry feed mixes, feed pellets have properties that are favored by the industry, such as improved feed quality, reduced pathogens, reduced dust levels during manufacturing, convenient handling, and more uniform dosing of ingredients. The pelleting processes preferred in the industry use steam injection, in a process known as Ref. 312724 Conditioning, which adds moisture and raises the temperature before the pelleting stage, which forces the steam-heated feed ingredients or conditioned mass through a die. Pelleting process temperatures are typically around 70°C to 100°C or higher. Due to the steam, temperature, compressive forces, and chemical agents used in pelleting processes, the activity or potency of enzymes is typically significantly reduced during processing. Inactivation is at least partially reversible if the enzyme is reactivated after processing and irreversible if its catalytic activity does not resume after processing. Irreversible enzyme inactivation is absolutely undesirable in processes such as pelleting. The food and feed industries need stable and long-lasting enzyme granules that serve as components in formulations that undergo steam pelleting processes without appreciable loss of enzymatic activity. SUMMARY OF THE INVENTION The present compositions and methods relate to increasing the stability of a phytase in a composition using phosphate and to the recovery of phytase from animal feed pellets produced by steam granulation. 1. In one aspect, a method is provided for increasing the stability of a phytase in a solid composition or for recovering the phytase in a pelleting process comprising the solid composition, the method comprising introducing phosphate into the solid composition, wherein the phosphate and the phytase are in functional proximity and at a molar ratio of at least 50:1 and wherein the solid composition comprises a molar ratio of less than 10:1 of inositol or inositol phosphates to phytase. 2. In some forms of the method according to section 1, phytase and phosphate form, or are incorporated in or on, a solid support. 3. In some forms of the method according to section 1 or 2, the phytase and phosphate form or are incorporated into a granule. In some forms of the method according to section 3, the granule is a matrix granule. 5. In some forms of the method according to section 3, the granule is a multilayer granule. 6. In some forms of the method according to any of the heading 5, the phytase and phosphate are incorporated into a single layer of the multilayer granule. 7. In some forms of the method according to any of the headings 3-6, phytase and phosphate are incorporated into the granule core. 8. In some embodiments of the method according to any of headings 3-7, the granule is included in a pellet of food or animal feed, wherein the percentage of activity recovered after heat treatment is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% or higher, after pelleting with steam. 9. In some embodiments of the method according to any of headings 3-8, the granule is incorporated into a pellet of food or animal feed, wherein the relative improvement in recovered activity is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% or more, after pelleting of the food or animal feed, compared with the activity recovered using, in the same pellet of food or animal feed, a granule having no phosphate and phytase in functional proximity and a molar ratio of at least 50:1. 10. In another aspect, a method is provided for increasing the stability of a phytase in a liquid composition or for recovering the phytase in a pelleting process comprising the liquid composition, the method comprising introducing phosphate into the composition, wherein the phosphate and the phytase are in functional proximity and the phosphate is present at a concentration of 50 mM or more and wherein the liquid composition comprises inositol or inositol phosphates at less than 10 mM. 11. In some forms of the method according to section 10, phytase and phosphate are in solution or suspension. 12. In some forms of the method in heading 10 or 11, phytase and phosphate are included in a pellet of food or animal feed, wherein the percentage of activity recovered is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% after pelleting of the food or animal feed. 13. In some embodiments of the method of any of headings 10-12, phytase and phosphate are applied to the surface of a pellet of food or animal feed, wherein the percentage of activity recovered is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% after pelleting of the food or animal feed. 14. In some embodiments of the method in any of headings 10-13, the relative improvement in recovered activity is at least 10%, at least 20%, at least 30%, at least 40% or at least 50% after pelleting of the feed or animal feed, compared with the activity recovered using, in the same feed or animal feed pellet, a pellet having no phosphate and phytase in functional proximity, the phosphate being present at a concentration of 50 mM or more. 15. In some forms of the method in any of the preceding headings, phosphate is not a product of the hydrolysis of phytate present in the solid or liquid composition. 16. In some forms of the method of any of the preceding headings, the solid or liquid composition is essentially free of inositol or inositol phosphates. 17. In some forms of the method in any of the preceding headings, the phosphate is a monophosphate. 18. In some forms of the method in any of the preceding headings, the Tm by DSC (Differential Scanning Calorimetry) of phytase increases by at least 2.7 °C compared to a control phytase not stabilized with phosphate at a molar concentration ratio of phosphate to phytase of at least 50:1 or a phosphate concentration of at least 50 mM. 19. In another aspect, a granulated composition comprising phosphate and phytase is provided, wherein the phosphate and phytase are in functional proximity and a molar ratio of at least 50:1 or the phosphate is present in an amount of at least 50 mM. 20. In some forms of the granule according to heading 19, the granule is a matrix granule. 21. In some forms of the granule according to section 19, the granule is a multilayer granule. 22. In some forms of the granule according to section 20, phytase and phosphate are incorporated into a single layer of the multilayer granule. 23. In some forms of the granule according to any of the headings 19-22, phytase and phosphate are incorporated into the core of the granule. 24. In some forms of the granule according to any of the headings 19-22, the phosphate is not a hydrolysis product of the phytate present in the solid or liquid composition. 25. In some forms of the granule according to any of the headings 19-22, the solid or liquid composition does not contain phytate. 26. In some forms of the granule according to any of the headings 19-22, the phosphate is a monophosphate. 27. In another aspect, a pellet composition is provided comprising the granule of any of the headings 19-26. 28. In another aspect, a pellet composition is provided comprising a phytase and phosphate in functional proximity and a molar ratio of at least 50:1 or wherein the phosphate is present at a concentration of at least 50 mM, wherein the phosphate is not a hydrolysis product of the phytate present in the pellet composition. These and other aspects and modalities of the present compositions and methods will become evident from the description, including the attached figures. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing the effect of adding increasing concentrations of sodium phytate to phytase on the melting temperature of the enzyme in the liquid state. Figure 2 is a graph showing the UFC phytase melting temperature profile with increasing concentrations of sodium phosphate. Figure 3 is a graph showing the effect of adding increasing concentrations of sodium phosphate to phytase on the melting temperature of the enzyme in the liquid state. Figure 4 is a graph showing a comparison of different excipients, normalized to anionic equivalents, at the melting temperature of phytase in the liquid state. Figure 5 is a graph showing a comparison of different excipients, normalized to load equivalents, at the melting temperature of phytase in the liquid state. Figure 6 is a graph showing the profiles of nnccpn / nznz / a / Y thermal inactivation of UFC phytase with increasing levels of sodium phosphate. Figure 7 is a graph showing the thermal inactivation profiles of UFC phytase with increasing levels of sodium citrate. Figure 8 is a graph showing the competitive inhibition of phytase in the presence of phytic acid substrate and increasing levels of sodium phosphate. Figure 9 is a graph showing the competitive inhibition of phytase in the presence of phytic acid substrate and increasing levels of sodium citrate. Figure 10 is a graph showing the vapor simulator recovery of granules that (A) have no receptor or that have (B) phytate or (C) phosphate. Figure 11 is a graph showing the recovery in feed pelleting of pellets that (A) have no receptor or that have (B) phytate or (C) phosphate. DETAILED DESCRIPTION OF THE INVENTION I. Definitions and Abbreviations Before describing the present strains and methods in detail, the following terms are defined for clarity. Undefined terms should be used with their usual meaning as used in the relevant technique. As used herein, the term phytic acid refers to inositol hexaphosphate (IP6), which may include minor amounts (i.e., less than 10%) of inositol phosphates that are catabolites of IP6, including inositol pentaphosphate (IP5), inositol tetraphosphate (IP4), inositol triphosphate (IP3), inositol diphosphate (IP2), and inositol monophosphate (IP1), salts thereof, and mixtures thereof. The term phytic acid does not encompass inositol (IPO) or inorganic phosphate. Note that IP1, IP2, IP3, IP4, IP5, and IP6 are all referred to as inositol phosphates. As used herein, the term phosphate (PO43-) refers to an inorganic chemical compound and a salt-forming anion of phosphoric acid. The term phosphate does not include IP6, IP5, IP4, IP3, IP2, IP1, or IPO. As used herein, the term food is used in a broad sense and covers food and food products for humans as well as food for non-human animals (i.e., animal feed). As used herein, the term feed refers to products given to animals in livestock farming. The terms feed and animal feed are used interchangeably. Animals can be ruminants or non-ruminants. Examples of ruminants include cows, sheep, goats, and horses. Examples of non-ruminant animals include monogastric animals such as pigs, poultry (such as chickens and turkeys), fish (such as salmon), dogs, cats, and humans. nnccon / nznz / a / Y As used herein, the term "feed ingredient" includes any formulation that is added or may be added to feed materials or food products and includes formulations that may be used at low levels in a wide variety of products. A feed ingredient may be in the form of a solution or a solid, depending on its use, application, and / or administration. The enzymes described herein may be used as feed or a feed ingredient, or in its preparation or production. Enzymes may be, or may be added to, food supplements. As used herein, the expression amino acid sequence is synonymous with the terms polypeptide, protein, and peptide, and they are used interchangeably. When amino acid sequences exhibit activity, they may be referred to as enzymes. Conventional one-letter or three-letter codes are used for amino acid residues, and amino acid sequences are presented in the standard amino-terminal to carboxy-terminal (i.e., N≡C) orientation. As used herein, the expressions substantially similar and substantially identical in the context of at least two nucleic acids or polypeptides, usually refer to a polynucleotide or polypeptide comprising a sequence that has at least approximately 1% identity, at least approximately 75% identity, at least approximately 80% identity, at least approximately 85% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, or even at least approximately 99% identity or more.compared to the reference sequence (i.e., of natural type). The percentage of sequence identity is calculated using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are: Penalty for opening a gap: 10.0 Penalty for gap extension: 0.05 Protein weight matrix: BLOSUM series DNA weight matrix: IUB % of divergent sequence delay: 40 Gap separation distance: 8 DNA transition weight: 0.50 List of hydrophilic residues: GPSNDQEKR nnccon / nznz / a / Y Negative matrix usage: INACTIVATED Alternation of specific residue penalties: ON Alternating hydrophilic penalties: ON Final gap separation penalty switching INACTIVATED As used herein, the term phytase means a protein or polypeptide that is capable of catalyzing the hydrolysis of phosphoric acid esters, including phytate / phytic acid, and releasing inorganic phosphate. As used herein, the terms natural, precursor, or reference, with respect to a polypeptide, refer to a naturally occurring polypeptide that does not include any artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, the terms natural, precursor, or reference, with respect to a polynucleotide, refer to a naturally occurring polynucleotide that does not include an artificial nucleoside change. However, it should be noted that a polynucleotide encoding a natural, precursor, or reference polypeptide is not limited to a naturally occurring polynucleotide and encompasses any polynucleotide that encodes the natural, precursor, or reference polypeptide. As used herein, the term variant, with respect to a polypeptide, refers to a polypeptide that differs from a specified natural, precursor, or reference polypeptide in that it includes an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, the term variant, with respect to a polynucleotide, refers to a polynucleotide that differs in nucleotide sequence from a specified natural, precursor, or reference polynucleotide. The identity of the natural, precursor, or reference polypeptide or polynucleotide will be evident from the context. As used herein, the term purified refers to a material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g. at least approximately 90% pure, at least approximately 95% pure, at least approximately 98% pure, or even at least approximately 99% pure. As used herein, the terms modification and alteration are used interchangeably and mean to change or vary. In the context of modifying or altering a polypeptide, these terms may mean changing the amino acid sequence, either directly or by changing the coding nucleic acid, or changing the polypeptide structure, such as by glycosylating the enzyme nnccon / nznz / a / Y. As used herein, the term solid support refers to an inert solid material in or onto which phytase and phosphate can be incorporated, for example, by spraying, mixing, absorption, or otherwise forming particles such as granules or powders. Examples of solid supports include, but are not limited to, sodium sulfate, magnesium sulfate, granulated sucrose, starch-sucrose pellets (ASNP), and maltodextrin. As used herein, the term granule refers to a small, compact particle of a substance. The particle may be formed from a matrix of materials or may include a core with one or more optional coating layers. As used herein, the term multilayer granule refers to a composition comprising a core and at least one coating layer. As used herein, the term matrix granule refers to a granule having a homogeneous structure that includes matrix material and homogeneously dispersed enzyme. As used herein, the term kernel is used interchangeably with the term seed. As used herein, the expression coating layer and the term layer are used interchangeably. nnccon / nznz / a / Y The one or more coating layers generally encapsulate the core to form a substantially continuous layer, such that the core surface has few or no uncoated areas. The materials (e.g., the agents, components, and enzyme detailed herein) used in the granule and / or multilayer granule are suitable for use in food and / or animal feed. The materials may be food grade or feed grade. As used herein, the term outer coating layer refers to the multilayer granule coating layer that is farthest from the core (i.e., the last coating layer applied). As used herein, the term enzyme coating layer or enzyme layer refers to an enzyme layer comprising at least one enzyme. In some embodiments, the enzyme layer comprises at least two enzymes. In some embodiments, the enzyme layer comprises at least three enzymes. As used herein, the term functional proximity means that the phosphate and phytase are so close that the phosphate is able to stabilize the phytase. For example, the phosphate and phytase are both located in the same layer of a granule; for example, both the phosphate and phytase are located in the core and / or both the phosphate and phytase are located in the same coating layer. In another example, the phosphate and phytase are located in adjacent coating layers of a granule. In a further example, the phytase is located in the core and the phosphate is located in the adjacent coating layer. As used herein, the term heat treatment refers to pelleting with steam or exposure to dry heat at a temperature above 90°C. As used herein, the terms pellets and pelleting refer to solid, rounded, spherical, and cylindrical pellets or tablets and to the processes for forming such solid shapes, in particular, extruded solid feed pellets and solid animal feed. Known pellet manufacturing processes generally include mixing the feed or food ingredients together for approximately 1 to approximately 5 minutes at room temperature, transferring the resulting mixture to a hopper, conveying the mixture to a steam conditioner, optionally transferring the steam-conditioned mixture to an expander, transferring the mixture to the pellet mill or extruder, and finally, transferring the pellets to a pellet cooler. Fairfield, D. 1994. Chapter 10, Pelleting Cost Center. In Feed Manufacturing Technology IV. (McEllhiney, nnccon / nznz / a / Y ed.).), American Feed Industry Association, Arlington, Va., pp. 110-139. As used herein, the term heat-treated feed pellets or animal feed pellets refers to non-granulated mixtures that are subjected to heat treatment (such as steam conditioning), typically at a temperature of at least 90°C for at least 30 seconds. The mixture may then be extruded to form the animal feed pellets. As used herein, the term stability refers to any of several effects in which the enzymatic activity or other functional property of a phytase enzyme is beneficially maintained or enhanced. A phytase may exhibit stability by showing recovered activity, improved thermostability, and / or reversibility of inactivity. Stability may also refer to the activity maintained in the phytase composition either before or after its combination with feed or feed pellets. As used herein, the term recovered activity refers to the amount of phytase activity after heat treatment, compared to the amount of phytase activity before heat treatment. Recovered activity may be expressed as a percentage. The percentage of recovered activity is calculated as follows: (activity after \ . , ) treatment · % of recovered activity H ----v—;---r--- ix 100 % ; activity before; k of the treatment7 As used herein, the term "relative improvement in recovered activity" refers to the amount of activity recovered from the stabilized phytase compared to the amount of activity recovered from the unstabilized phytase. Recovered activity may be expressed as a percentage. The relative increase in recovery is calculated as follows: % of recovered activity from stabilized phytase (% of recovered activity from unstabilized phytase) % of recovered activity from unstabilized phytase As used herein, a URF, or phytase renewal unit, or phytase activity unit, is the amount of enzyme capable of generating 1 pmol of inorganic phosphate per minute from an excess of sodium phytate at pH 5.5 and 37°C. Phytase activity is analyzed according to the official method of the Association of Analytical Chemists (AOAC) 2000.12, as described in "Determination of phytase activity in feed by a colorimetric enzymatic method: collaborative interlaboratory study" (Engelen, AJ, van der Heeft, FC, Randsdorp PH, Somers, WA, Schaefer, J., van der Vat, BJ; J AOAC Int. May-June 2001; 84:629-33). nnccon / nznz / a / Y Briefly, the ground samples are extracted in 220 mM sodium acetate trihydrate, 68.4 mM dehydrated calcium chloride, 0.01% Tween 20, pH 5.5. The supernatant is then analyzed. The assay measures the release of inorganic phosphate from rice phytase at pH 5.5 for 60 min at 37 °C. The assay is stopped with molybdate / vanadate acid reagent, and the phosphate is quantified by the intensity of a yellow vanadomolybdophosphorus complex. As used herein, the melting temperature (Tm) of an enzyme is the temperature at which 50% of the enzyme protein is in its unfolded, non-natural state, i.e., the temperature at which the free energy of the folded and unfolded states is equal and can be measured using techniques known in the art. One suitable technique is differential scanning calorimetry (DSC). Other suitable techniques for determining melting temperatures include light scattering, circular dichroism, and enzymology experiments. For easier reference, the elements of these compositions and methods can be arranged under one or more headings. Note that the compositions and methods under each heading also apply to the compositions and methods under the other headings. As used herein, the singular articles *un*, *una*, and *el* encompass plural referents, unless the context clearly indicates otherwise. All references cited herein are incorporated by reference in their entirety. The following Abbreviations / acronyms have the following meanings: Unless otherwise specified: °C degrees Celsius g gram g / l grams per liter g / mol grams per liter mo1 / mo1 molar ratio high-performance liquid chromatography h hour kg kilogram M molar mg milligram mi milliliter min minute mM millimolar cm centimeters mm millimeters nm nanometer PCR polymerase chain reaction rpm revolutions per minute pg microgram μA microliter μM micromolar nnccon / nznz / a / Y p / v weight / volume URF / g phytase units / gram UFC ultrafiltered concentrate kW kilowatt atm atmosphere molar ratio mo1:mo1 II. Stabilization of phytase using phosphate The present compositions and methods are based on the surprising observation that the thermal stability of a phytase enzyme increases after pre-incubation with a molar excess of inorganic phosphate relative to phytase. Phosphate is an inexpensive formulation ingredient and can advantageously replace more expensive phytase replacement excipients such as phytate. The aspects and procedures of the compositions and methods are described below. Phosphate sources The phosphate used to stabilize phytase can be derived from an inorganic salt, such as a sodium, calcium, potassium, or aluminum salt, or from an inorganic compound. The phosphate source is not critical for the present compositions and methods. Suitable phosphate salts include, at a minimum, monophosphates, such as sodium phosphate (mono- or dibasic), polyphosphates, such as sodium hexametaphosphate and sodium trimetaphosphate, and pyrophosphates, such as di-, tri-, and tetrasodium diphosphate. Other forms of phosphate, such as potassium, magnesium, calcium, iron, and ammonium salts of phosphoric acid, pyrophosphates, and others, are likely to work. The phosphate used to stabilize the phytases according to the present compositions and methods must be added at significantly higher levels than would normally or inherently be present in the ingredients of a food or animal feed than the phytase (such as in the form of a liquid, powder, matrix granule, or multilayer granule) with which the phytate is mixed prior to pelleting. In other words, the phosphate used to stabilize the phytase is exogenous phosphate; the phosphate present in the ingredients of a food or animal feed is endogenous phosphate. The calculated amount of phosphate in the present compositions and methods does not include residual amounts of endogenous phosphate. The phosphate that is the subject of the present compositions and methods is exogenous phosphate arranged in functional proximity to the phytase for the intended purpose of stabilizing the phytase. Furthermore, the phosphate used to stabilize the phytase according to the present compositions and methods does not originate from phytate or any phytate hydrolysis product, including, for example, phytate or other inositol phosphates used to stabilize the phytase. Additionally, phytate, IP5, IP4, IP3, IP2, or even IPO (inositol) may be added to a composition comprising phytase and phosphate, but is not the source of the phosphate. Alternatively, the present compositions may expressly omit phytate, IP5, IP4, IP3, IP2, and / or IPO, or may include only low levels of inositol or inositol phosphates, i.e., less than 10 mM or even less than 5 mM, or an inositol or inositol phosphate to phytase ratio of less than 10:1 or even less than 5:1. The amount of phosphate required to stabilize phytase is a 50:1 molar ratio compared to phytase at a concentration of 50 mM or higher. This distinguishes the present compositions and methods from those described by Gebert in WO2013 / 119468, which required 10 millimolar phytate. Due to the lower cost of phosphate compared to phytate (10–20 times less than phytate) and the fact that phosphate can be added at a lower mass percentage for a given amount of phytase, the present compositions and methods significantly reduce formulation costs. Phosphate is also available with much higher purity than the physical acid, resulting in improved consistency of formulated products. Phosphate is widely available as a bulk commercial product rather than as a specialty chemical and does not produce end products such as inositol or inositol phosphates. In some forms, phytase is stabilized with phosphate at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, at least 500 mM, at least 550 mM or even at least 600 mM relative to a liquid phytase composition (e.g., a CFU) prior to granulation. In some forms, the molar ratio of phosphate to total phytase protein in functional proximity is at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1 or even at least 300:1, with reference to solid or liquid phytase compositions. In some forms, the relative improvement in recovered activity of phosphate-stabilized phytase is at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more, compared to an otherwise identical phytase that is not phosphate-stabilized, calculated using the % recovered activity formula provided herein. Any means may be used to provide phosphate to phytase, i.e., to bring the phosphate and phytase into functional proximity. For example, a liquid comprising phosphate may be mixed with a liquid comprising phytase. In a further example, the phytase and phosphate are spray-dried onto a solid support. In a particular embodiment, the phytase and phosphate are in functional proximity within a granule, for example, a multilayer granule or a matrix granule. The phosphate may be added in the form of a salt, for example, as the mono- or dibasic salt of phosphoric acid, as phosphoric acid, or as any combination thereof. B. Suitable phytases A phytase is a protein or polypeptide that can catalyze the hydrolysis of phosphoric acid esters, including those in inositol phosphates such as phytate, thereby releasing inorganic phosphate. Some phytases can also hydrolyze at least some inositol phosphates with intermediate degrees of phosphorylation, including IP5, IP4, IP3, IP2, IP1, and IPO. Phytase enzymes are added to food and animal feed to increase phosphate availability, thereby enhancing the product's nutritional value. Processing of food or animal feed, for example, with heat and high pressure, can denature phytase and reduce its activity. The phytase used in the present compositions and methods may be any phytase suitable for use in food or animal feed. In some embodiments, the enzyme is a 6-phytase (also called 4-phytase or phytate 6-phosphatase). In some embodiments, the enzyme is a histidine acid phytase (HAP), a group comprising members found among prokaryotes (e.g., appA phytase from Escherichia coli) and eukaryotes (phyA and B from Aspergillus sp., HAP phytases from yeasts and plants). HAP phytases share a common active site motif at their amino-terminal end and a motif at their carboxy-terminal end. In one modality, the phytase is from Escherichia coli, Citrobacter braakii, Peniophora iycii, or Aspergillus niger. In some modality, the phytase is from a Buttiauxella sp., for example, the phytase from the Buttiauxella sp. strain P 1-29 deposited with reference no. NCIMB 41248 or variants thereof, such as BP-11, BP17, and BP-111. The amino acid sequence of the wild-type phytase from the Buttiauxella sp. strain P 1-29 deposited with reference no. NCIMB 41248 is shown below as SEC. ID NO.: 1: NDTPASGYQVEKWILSRHGVRAPTKMTQTMRDVTPNTWPEWPVKLGYITPRGEHL ISLMGGFYRQKFQQQGILSQGSCPTPNSIYVWADVDQRTLKTGEAFLAGLAPQCGLTIHHQ QNLEKADPLFHPVKAGTCSMDKTQVQQAVEKEAQTPIDNLNQHYIPFLALMNTTLNFSTSA WCQKHSADKSCDLGLSMPSKLSIKDNGNKVALDGAIGLSSTLAEIFLLEYAQGMPQAAWGN IHSEQEWASLLKLHNVQFDLMARTPYIARHNGTPLLQAISNALNPNATESKLPDISPDNKI LFIAGHDTNIANIAGMLNMRWTLPGQPDNTPPGGALVFERLADKSGKQYVSVSMVYQTLEQ LRSQTPLSLNQPAGSVQLKIPGCNDQTAEGYCPLSTFTRWSQSVEPGCQLQ The amino acid sequence of BP-11, a variant phytase from Buttiauxella sp. comprising 11 nnccon / nznz / a / Y amino acid substitutions compared to the natural enzyme (SEC. ID NO.: 1) is shown below as SEC. ID NO.: 2: NDTPASGYQVEKWILSRHGVRAPTKMTQTMRDVTPNTWPEWPVKLGYITPRGEHL ISLMGGFYRQKFQQQGILSQGSCPTPNSIYVWTDVDQRTLKTGEAFLAGLAPQCGLTIHHQ QNLEKADPLFHPVKAGICSMDKTQVQQAVEKEAQTPIDNLNQHYIPSLALMNTTLNFSKSP WCQKHSADKSCDLGLSMPSKLSIKDNGNEVSLDGAIGLSSTLAEIFLLEYAQGMPQAAWGN IHSEQEWALLLKLHNVYFDLMERTPYIARHKGTPLLQAISNALNPNATESKLPDISPDNKI LFIAGHDTNIANIAGMLNMRWTLPGQPDNTPPGGALVFERLADKSGKQYVSVSMVYQTLEQ LRSQTPLSLNQPAGSVQLKIPGCNDQTAEGYCPLSTFTRWSQSVEPGCQLQ The amino acid sequence of BP-17, a variant phytase from Buttiauxella sp. comprising 12 amino acid substitutions compared to the natural enzyme (SEC. ID NO.: 1) is shown below as SEC. ID NO.: 3: NDTPASGYQVEKVVILSRHGVRAPTKMTQTMRDVTPNTWPEWPVKLGYITPRGEHL ISLMGGFYRQKFQQQGILSQGSCPTPNSIYVWTDVAQRTLKTGEAFLAGLAPQCGLTIHHQ QNLEKADPLFHPVKAGICSMDKTQVQQAVEKEAQTPIDNLNQHYIPSLALMNTTLNFSKSP WCQKHSADKSCDLGLSMPSKLSIKDNGNEVSLDGAIGLSSTLAEIFLLEYAQGMPQAAWGN IHSEQEWALLLKLHNVYFDLMERTPYIARHKGTPLLQAISNALNPNATESKLPDISPDNKI LFIAGHDTNIANIAGMLNMRWTLPGQPDNTPPGGALVFERLADKSGKQYVSVSMVYQTLEQ LRSQTPLSLNQPAGSVQLKIPGCNDQTAEGYCPLSTFTRWSQSVEPGCQLQ The amino acid sequence of BP-111, a variant phytase from Buttiauxella sp. comprising 21 amino acid substitutions compared to the natural one (SEC. ID NO.: 1) is shown below as SEC. ID NO.: 4: nnccon / nznz / a / Y NDTPASGYQVEKWILSRHGVRAPTKMTQTMRDVTPYTWPEWPVKLGYITPRGEHL ISLMGGFYRQKFQQQGILPRGSCPTPNSIYVWTDVAQRTLKTGEAFFLAGLAPQCGLTIHHQ QNLEKADPLFHPVKAGICSMDKTQVQQAVEKEAQTPIDNLNQRYIPELALMNTILNFSKSP WCQKHSADKPCDLALSMPSKLSIKDNGNEVSLDGAIGLSSTLAEIFLLEYAQGMPQVAWGN IHSEQEWALLLKLHNVYFDLMERTPYIARHKGTPLLQAISNALNPNATESKLPDISPDNKI LFIAGHDTNIANIAGMLNMRWTLPGQPDNTPPGGALVFERLADKSGKQYVSVSMVYQTLEQ LRSQTPLSLNQPPGSVQLKIPGCNDQTAEGYCPLSTFTRWSQSVEPGCQLQ In some embodiments, the phytase is one or more of QUANTUM®, QUANTUM® BLUE, PHYZYMEXPTM, AXTRA® PHY, RONOZYMETM HYPHOS or NATUPHOS. Phytases are described, for example, in documents WO2006038128, US2017143004, US2006141562, US2016362666, US2016289655, US9365840, US8663963 and US2015159149. The phytases for use as described herein may be precursor, immature, or full-length, in which case they include a signal sequence, or mature, in which case they lack a signal sequence. The mature forms of the polypeptides are generally the most useful. Unless otherwise stated, the numbering of amino acid residues used herein refers to the mature forms of the respective phytase polypeptides. The amylase polypeptides herein may also be truncated to remove the amino-terminal or carboxy-terminal ends, such that the resulting polypeptides retain phytase activity. Phytases are generally produced in microbial cells, such as fungal or bacterial cells. In some embodiments, phytase is produced in a Trichoderma host cell. This phytase is produced in a Trichoderma host cell that involves a deletion of the endo-N-acetylglucosamidase gene (sometimes referred to as endo-T), which encodes an enzyme that removes N-linked glycosylation from proteins, including phytases. An illustrative strain with endo-T deletion is described in WO 09 / 114380. The amount of phytase units added to animal feed depends on the feed's composition. Feeds containing lower amounts of available phosphorus typically require higher levels of phytase activity. The required amount of exogenous phytase can be determined by a specialist. In some formulations, the amount of phytase incorporated into animal feed ranges from 0.1 to 20 units of phytase activity per gram of feed. Typically, the amount of phytase is approximately 50 grams (12,000 U / g of phytase granules) per metric ton (1,000 kg) of feed; this equates to 0.6 units of phytase per gram of feed. In some embodiments, phytase is in solution. In others, it is in solid form. In further embodiments, phytase is spray-dried onto a solid carrier or incorporated into a powder. In still others, phytase is incorporated into a granule, such as a matrix granule or a multilayer granule. In most embodiments, phytase is ultimately incorporated into a food or animal feed. In some forms, phytase is present in a liquid or solid composition containing phosphate in an amount of at least 0.5% w / w, less 1.0% w / w, less 1.5% w / w, less 2.0% w / w, less 2.5% w / w, less 3.0% w / w, less 3.5% w / w, less 4.0% w / w, less 4.5% w / w, or even at least 5.0% w / w or higher. III. Compositions comprising phytase and phosphate Compositions comprising phytase and phosphate include liquid compositions as well as solid compositions, such as powders (including spray-dried powders) and granules (including solid granules and multilayer granules). Examples of the compositions will be described. Liquid compositions of animal feed and food In some forms, animal feed is a liquid, such as liquid feed. In other forms, animal feed is a solid. As used herein, the term "animal" includes all animals, examples of which include non-ruminants (i.e., monogastric animals, such as pigs and poultry, fish, dogs, cats, and humans) and ruminants (such as cattle, sheep, goats, and horses). Animal feed may include vegetable proteins. Suitable sources of vegetable protein are soybeans, soybean meal, cereals (such as corn, wheat, oats, barley, rye, and sorghum), cereal flours (such as corn flour, wheat flour, oat flour, barley flour, rye flour, sorghum flour, and rapeseed flour), brans (such as wheat bran and oat bran), oilseeds (such as rapeseed or sunflower seeds), oilseed meals (such as rapeseed meal), cottonseed meal, cabbage, beetroot, and sugar beetroot. Animal feed may comprise animal proteins. Suitable animal proteins may include fishmeal and whey. Animal feed may comprise additives. Suitable additives include enzyme inhibitors, vitamins, trace elements, macroelements, coloring agents, flavoring compounds, antimicrobial peptides (such as Leucocin A, Thanatin, and Trypticin), and enzymes. The liquid phytase composition can be applied to a solid animal feed composition either by direct mixing into the feed mass before pelleting, or by liquid application after pelleting (PPLA) of liquid phytase into the finished pellets. B. Solid compositions of animal feed and food Types and methods of production Solid carriers and granules can be produced using various manufacturing techniques and from a variety of materials. Solid carriers include inert solid materials in or onto which phytase and phytic acid are incorporated, for example, by pulverizing, mixing, adsorbing, or otherwise forming the particles, such as granules or powders. Examples of solid carriers include, but are not limited to, sodium sulfate, magnesium sulfate, granulated sucrose, starch-sucrose pellets (ASNP), and maltodextrin. The materials used in the core must be suitable for use in food and / or animal feed. Granules can be prepared, for example, by rotary atomization, wet granulation, dry granulation, spray drying, disc granulation, extrusion, tray coating, spheronization, drum granulation, fluidized bed agglomeration, high-shear granulation, fluidized bed spray coating, crystallization, precipitation, emulsion gelation, rotary disc atomization, and other molding strategies and bead-forming processes. See, for example, US4689297, US5324649, US454332, US6248706, US6534466, EP656058B1, and EP804532B1 and US Patent No. [Number missing in original text]. The granule core can be the granule itself or the inner core of a layered granule. The materials used in the core must be suitable for use in food and / or animal feed. Particle nuclei The core may comprise one or more water-soluble or water-dispersible agents, including, but not limited to, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate; citric acid; sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose); plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate); fibrous material (e.g., cellulose and cellulose derivatives such as hydroxypropylcellulose, carboxymethylcellulose, and hydroxyethylcellulose); phosphate; and combinations thereof. Suitable dispersible agents include, but are not limited to, clays; pellets (sugar and starch combinations; e.g., starch-sucrose pellets - ASNP); talc; silicates; carboxymethylcellulose; starch; and combinations thereof. In some embodiments, the core comprises mainly sodium sulfate. In some embodiments, the core consists essentially of sodium sulfate. In one particular embodiment, the core consists solely of sodium sulfate. In some embodiments, the core comprises both phytase and phosphate in functional proximity. In other embodiments, the core comprises one or more enzymes in addition to phytase. In other embodiments, the core comprises one or more enzymes other than phytase. In other embodiments, the core is inert and does not comprise any enzymes. In some embodiments, the core is an enzyme powder, including CFU containing an enzyme. The enzyme powder may be spray-dried and may optionally be mixed with any of the water-soluble or water-dispersible agents listed herein. The enzyme may be, or may include, the phytase to be stabilized, in which case the enzyme powder must also include phosphate. Coating layers In some embodiments, the core is coated with at least one coating layer. In one particular embodiment, the core is coated with at least two coating layers. In another particular embodiment, the core is coated with at least three coating layers. The materials used in one or more coating layers may be suitable for use in food and / or animal feed (see, for example, nnccon / nznz / a / Y documents US20100124586, WO9932595 and US5324649). In some embodiments, a coating layer comprises one or more of the following materials: an inorganic salt (e.g., sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, a sugar (e.g., sucrose, lactose, glucose, and fructose), a plasticizer (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxypropyl methylcellulose, carmellose, and hydroxyethylcellulose), clay, pellets (a combination of sugar and starch), silicate, carmellose, phosphate, starch (e.g., corn starch), fats, oils (e.g., rapeseed oil and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (EVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water), anti-caking agents (e.g., talc, clay, amorphous silica, and titanium dioxide),Antifoaming agents (such as FOAMBLAST 882® and EROL 6000K®) and talc. Documents US20100124586, WO9932595, and US5324649 detail suitable components for the coating layers. In some embodiments, the coating layer comprises sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose). In some embodiments, the coating layer comprises a polymer, such as polyvinyl alcohol (PVA). The PVA suitable for incorporation into the one or more coating layers of the multilayer granule includes partially hydrolyzed, fully hydrolyzed, and intermediately hydrolyzed PVAs having low to high viscosity grades. In some embodiments, the coating layer comprises an inorganic salt, such as sodium sulfate. In some embodiments, the coating layer comprises phosphate. Enzymatic coating layers In some embodiments, at least one coating layer is an enzymatic coating layer. In some embodiments, the core is coated with at least two enzymatic coating layers. In another embodiment, the core is coated with at least three or more enzymatic coating layers. In some embodiments, the enzymes are a phytase in combination with one or more additional enzymes selected from the group consisting of phytases, xylanases, phosphatases, amylases, esterases, redox enzymes, lipases, transerases, cellulases, hemicellulases, beta-glucanases, oxidases (e.g., hexose oxidases and maltose oxidoreductases), proteases, and mixtures thereof. In general, at least one enzyme coating layer comprises at least one phytase and phosphate. The lists of enzymes above are merely examples and are not intended to be exhaustive. Any enzyme may be used in the granules described herein, including natural, recombinant, and variant enzymes from bacterial, fungal, yeast, plant, insect, and animal sources, and acidic, neutral, or alkaline enzymes. In some embodiments, the enzymatic coating layer may further comprise one or more additional materials selected from the group consisting of: sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin and fructose), starch (e.g., corn starch), fats, oils (e.g., rapeseed oil and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (PVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate and water), anti-caking agents (e.g., talc, clays, amorphous silica and titanium dioxide), anti-foaming agents (such as FOAMBLAST 882® and EROL 6000K® available from Ouvrie PMC, Lesquin, France) and talc. Documents US20100124586, WO9932595, and US5324649 detail suitable components for granules. FOAMBLAST 882® is a defoamer made with food-grade ingredients and is available from Emerald Foam Control, LLC. Location and distribution of phosphate in the granules When the granule is coated, in some embodiments, the core includes both phytase and phosphate. In some embodiments, the coating includes both phytase and phosphate. In some embodiments, the core includes phytase and the coating includes phosphate. In some embodiments, the core includes phosphate and the coating includes phytase. In some embodiments, a single enzyme coating layer comprises both phosphate and phytase. In some embodiments, multiple enzyme coating layers comprise both phosphate and phytase. In some embodiments, the phosphate and phytase are located in adjacent layers and are therefore in functional proximity. C. Animal feed and food pellets The pellets may comprise granules and / or multilayer granules as described herein, according to any of several known pelleting methods, examples of which are described in more detail below. In some embodiments, the pellet comprises phytase, phosphate, and at least one food or feed ingredient. In some embodiments, the pellet comprises at least one food or feed ingredient and a granule comprising phosphate and phytase. The pellets of the present compositions and methods can be produced by a method in which the temperature of a feed mixture is raised to a high level to eliminate bacteria. The temperature is typically raised by steam treatment prior to pelleting, a process known as conditioning. Subsequently, the conditioned feed mixture can be passed through a die to produce pellets of a particular size. The feed mixture can be prepared by mixing the pellets and / or multilayer pellets described herein with animal feed or food, as described herein. In general, the steam conditioner treats the mixture for approximately 20 to 90 seconds, and sometimes for several minutes, at approximately 85°C to 95°C or higher. The amount of steam can vary depending on the moisture content and initial temperature of the feed mixture. The addition of approximately 4% to 6% steam has been reported in pelleting processes, and this amount is selected to produce a moisture content of approximately 18% in the mass before pelleting, or up to 28% moisture in the mass intended for extrusion. An optional expansion process may occur for approximately 4 to approximately 10 seconds at a temperature range of approximately 100°C to approximately 140°C. The pellet mill portion of the manufacturing process normally operates for approximately 3 to approximately 5 seconds at a temperature of approximately 85°C to approximately 95°C. Before pelleting, non-pellet mixtures (called premixes or precursors, base mixes, dough, and pellet diluents) typically contain vitamins and trace elements. Base mixes usually contain feed and animal feed ingredients, such as dicalcium phosphate, limestone, salt, and a vitamin and mineral premix, but not grains or protein ingredients. Diluents include, but are not limited to, grains (e.g., wheat flour and rice fiber) and clays, such as phyllosilicates (the magnesium silicates sepiolite, bentonite, kaolin, montmorillonite, hectorite, saponite, beidelite, attapulgite, and stevensite). Clays also function as carriers and fluidizing agents or diluents for feed and animal feed premixes. Dough typically comprises a complete animal diet.For example, the dough comprises or consists of corn, soy flour, soybean oil, salt, DL-methionine, limestone, dicalcium phosphate, and vitamins and minerals. In one example, the dough consists of 61.10% corn, 31.43% soy flour, 4% soybean oil, 0.40% salt, 0.20% DL-methionine, 1.16% limestone, 1.46% dicalcium phosphate, and 0.25% vitamins and minerals. In one embodiment, a feed or animal feed is produced by mixing at least one feed or food ingredient (such as a mass) with a phytase and phosphate in solution, steam conditioning the resulting mixture, followed by pelleting the mixture. A liquid phytase composition can be applied to a solid animal feed composition either by direct mixing into the feed mass before pelleting, or by post-pelleting (PPLA) liquid application of liquid phytase to the finished pellets. In one embodiment, a food or animal feed is produced by mixing at least one food or animal feed ingredient (such as a mass) with a phytase and phosphate in a solid state (such as in a granule, such as a multilayer granule), steam conditioning the resulting mixture followed by pelleting the mixture. For example, a non-granulated mixture of corn flour and soybean meal (such as 60% corn flour and 40% soybean meal) can be mixed with multilayer granules comprising phytase and phosphate and then steam-conditioned at 90°C for 30 seconds. Subsequently, the mixture is extruded to form animal feed pellets. In some embodiments, the compositions of the present compositions and methods may reside in heat-treated food or animal feed pellets. The heat-treated food or animal feed pellets may be subjected to heat treatment (such as steam conditioning) at a temperature of at least 90 °C for at least 30 seconds (e.g., at least 30-60 seconds at 90-100 °C). Subsequently, the mixture may be extruded to form the animal feed pellets. IV. Methods and processes using phosphate to stabilize phytase The methods of use for these compositions relate primarily to the addition of phytase to food and animal feed products that are to be heat-treated, such as by steam pelleting. The stability of phytase can be significantly improved in the liquid and / or solid states compared to phytase that has not been stabilized by the use of phosphate. These methods represent an improvement over those described by Gebert in WO2013 / 119468, which required a phytate concentration of at least 10 millimolar. Although the amount of phosphate required to stabilize the phytase is greater than the amount of phytate required, the lower cost of phosphate compared to phytate significantly reduces formulation costs. In some forms, phytase is stabilized with phosphate at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, at least 500 mM, at least 550 mM or even at least 600 mM with respect to a liquid phytase composition (e.g., a CFU) prior to granulation. In some forms, the molar ratio (i.e., mol:mol) of phosphate to total phytase protein in functional proximity is at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, or even at least 300:1, with reference to solid or liquid phytase compositions. In some forms, phytase is present in a liquid composition containing phosphate in an amount of at least 0.5% w / w, less 1.0% w / w, less 1.5% w / w, less 2.0% w / w, less 2.5% w / w, less 3.0% w / w, less 3.5% w / w, less 4.0% w / w, less 4.5% w / w, or even at least 5.0% w / w. In some embodiments, the recovered activity of phosphate-stabilized phytase is at least 5%, at least 10%, at least 15%, at least 20%, at least 30% or more after heat treatment, compared to an otherwise identical phytase that is not phosphate-stabilized, calculated using the % recovered activity formula provided herein. In some embodiments, the relative improvement in recovered activity of phosphate-stabilized phytase is at least 10%, at least 20%, at least 30%, at least 40%, nnccon / nznz / a / Y at least 50% or more, compared to an otherwise identical phytase that is not phosphate-stabilized, calculated using the % recovered activity formula provided herein. VI. Combinations of several modalities The forms of compositions and methods described herein, including those described in the various section headings and other forms that will be obvious to those skilled in the art, may be combined unless such combination impairs the stated purposes and advantages of the present compositions and methods. EXAMPLES The following examples are intended to illustrate modalities of the present compositions and methods and should not be interpreted in any way as limiting. Example 1. Determination of the melting point of phytase in the presence of excipients An experiment was conducted to determine whether the addition of different excipients during thermal exposure increases the melting point of the enzyme phytase in the liquid state. Excipient solutions were prepared at their maximum solubility, and the concentrations were subsequently titrated from highest to lowest before their addition to the phytase CFU. The enzyme / excipient combinations were then exposed to a temperature profile of 21–95 °C, and the melting points were measured by differential scanning fluorimetry. Materials The following equipment and reagents were used: UFC of phytase BP-17 (70,000 URF / g 123.4 g / 1 of phytase) Sodium phosphate, dibasic, anhydrous (JT Baker, Lot V3114 9, FW 141.96) Concentrated phosphoric acid for pH adjustment Hydrate of the sodium salt of phytic acid (Sigma Aldrich, P8810-500G) Sodium citrate dihydrate (JT Baker, FW 294.1) Concentrated citric acid for pH adjustment Sodium sulfate, anhydrous powder (FW 142.04) Sulfuric acid for pH adjustment Sodium polyphosphate, crystalline (Spectrum, SO169, P2O5 al 60-71%) 10% NaOH for pH adjustment pH meter Whatman Autovial syringe-free filters, 0.45 pm NanoDSF Prometheus NT.48 with standard capillaries (NanoTemper Technologies) Corning 3641 96-well flat-bottom non-stick polystyrene microtiter plates Corning 15 ml polypropylene centrifuge tubes (Sigma Aldrich) nnccon / nznz / a / Y UFC preparation The phytase concentration in the CFU was measured at 123.9 g / L by HPLC. The pH of the CFU was adjusted from 5.03 to 5.49 by adding 22 pK of 10% NaOH to 5 mL of the CFU, diluting the phytase concentration to 123.36 g / L. Preparation of the stabilizers A 20% phytic acid solution was prepared by dissolving 1 gram of phytic acid in 5 mL of 0.1 M acetate buffer at pH 5.5. The pH was adjusted from 4.2 to 5.47 using 20% ​​NaOH, and the solution was then filtered using a 0.45 µm non-syringe filter. A 25% anhydrous sodium sulfate solution was prepared by dissolving 5 grams in 20 mL of water. The pH was adjusted to 5.6 using sulfuric acid, and the solution was then filtered using a 0.45 µm non-syringe filter. A 20% sodium polyphosphate solution was prepared by dissolving 5 grams in 25 mL of water. The pH was adjusted to 5.6 using concentrated phosphoric acid, and the solution was then filtered using a 0.45 µm non-syringe filter. A 25% sodium citrate solution was prepared by dissolving 5 grams in 20 ml of water. The pH was adjusted to 5.6 using 80% citric acid, and then the solution was filtered using a 0.45 µm syringeless filter.A 20% solution of dibasic sodium phosphate was prepared by dissolving 2 grams in 10 ml of water. The pH was adjusted to 5.6 using concentrated phosphoric acid, and then the solution was filtered using a 0.45 µm syringe-free filter. nnccon / nznz / a / Y Preparation of plates for excipient dilutions A 100 µA volume of excipient stock solution was added to well A in column 1 of a 96-well plate. A 50 µA volume of water was added to each well BH in column 1 of the same 96-well plate. The excipient stock solution was titrated twice along the column of the plate by transferring 50 µA to each subsequent well. An 80 µA volume of pH-adjusted CFU was added to each well in column 1 of a new 96-well plate. A 20 µA volume from each well of the titrated excipient was transferred directly to the corresponding well containing the CFU. Differential scanning fluorimetry setup The 96-well plate containing the enzyme and excipient combinations was mixed in a plate shaker for 5 seconds. Standard capillaries for nanoDSF were filled by immersing one capillary in each well of the 96-well plate and then loading it into the Nanotemper instrument. The Nanotemper instrument was programmed to operate at 10% excitation power and 21–95 °C with a ramp rate of 1 °C / minute. The Nanotemper instrument monitors the intrinsic tryptophan fluorescence change of the proteins after unfolding by measuring wavelengths at 330 and 350 nm. The protein melting point (Tm) is determined by analyzing the change in the ratio of fluorescence intensities (F350 / F330) and is the maximum of the derivative of the fluorescence vs. temperature curve. As shown in Figure 1, the melting point (Tm) of phytase increased by approximately 2.7 °C in the presence of a 14:1 molar ratio of sodium phytate to phytase. As shown in Figures 2 and 3, the melting point of phytase increased by approximately 5.5 °C in the presence of a 130:1 molar ratio of sodium phosphate to phytase. Phosphate does not appear to stabilize the background proteins present in the phytase CFU, which are represented by the first peak of the chromatogram; however, it specifically affects the phytase enzyme, which is represented by the second peak of the chromatogram. As shown in Figure 4, the stabilizing effect of sodium phosphate is greater than that of sodium phytate, sodium polyphosphate, and sodium sulfate at equivalent molar ratios with respect to phytase, when normalized to anion equivalents.Furthermore, the stabilization mechanism does not appear to be a Hofmeister effect, i.e., related to the degree to which the salts act as lyotropes in salt precipitation or the physical stabilization of the protein conformation by interacting with the solvation water around the protein, since the increase in Tm is much greater in the presence of phosphate than of sulfate, whereas sulfate (see, for example, Hofmeister F. (1888) Arch. Exp. Pathol. Pharmacol. 24:247-260; reviewed at nnccon / nznz / a / Y Zhang Y. and Cremer PS (2006) Current Opinion in Chemical Biology. 10:658-63). As shown in Figure 5, the stabilization mechanism also does not appear to be an effect of the charge, since the increase in melting temperature is much greater in the presence of phosphate than in citrate when normalized with respect to charge equivalents. Example 2: Thermal inactivation of phytase BP-17 in the presence of stabilizing salts An experiment was conducted to determine whether the addition of excipients under heat stress conditions allows the phytase enzyme to retain its activity. A temperature was selected at which the phytase enzyme is completely inactivated in the absence of excipient after 30 minutes of incubation. Excipient solutions were prepared at their maximum solubility, and the concentrations were titrated from highest to lowest before their addition to the phytase CFU. Subsequently, the enzyme / excipient combinations were exposed to a temperature of 70 °C, and the residual activity was measured after 2, 5, 10, 20, 30, and 60 minutes of incubation using a colorimetric activity assay. Materials The following equipment and reagents were used: Phytase CFU BP-17 (70,000 URF / g, 123.4 g / L of phytase) • Phytase Standard BP-17 (22,000 URF / g) nnccon / nznz / a / Y • Phytase Control BP-17 (36,500 URF / g) • 0.2 mL PCR Tube Strips with 12 Wells (VWR 53509-306) • Autoclaved Water • Thermocycler with Temperature Gradient Capability • 96-Well PCR Prep Racks • Large Benchtop Centrifuge • Anhydrous Dibasic Sodium Phosphate (Sigma Aldrich), 141.96 g / mol • Sodium Citrate Dihydrate (JT Baker), 294.1 g / mol • Phosphoric Acid and Citric Acid for pH Adjustment • pH Meter • Buffer Test: 0.1 M acetate buffer at pH 5.5 with 0.05% (w / v) Tween 20 • Substrate buffer: acetate buffer 0.1 M at pH 5.5 • Hydrate of the sodium salt of phytic acid (Sigma Aldrich) • Pi Blue Stop Solution: POPB-500 (BioAssay Systems US) • Corning® 3641 96-well flat-bottom polystyrene non-stick microtiter plate nnccon / nznz / a / Y • VWR® aluminum seals for 96-well plates • iEMS incubator • SpectraMax 340 microplate reader (Molecular Devices) Preparation of stabilizing saline solutions A 20% (or 1.41 M) sodium phosphate solution was prepared by dissolving 5 grams of anhydrous dibasic sodium phosphate in 25 mL of water. The pH was adjusted to 5.5 using concentrated phosphoric acid. A 25% (or 0.85 M) sodium citrate solution was prepared by dissolving 5 grams of sodium citrate dihydrate in 20 mL of water. The pH was adjusted to 5.5 using 80% citric acid. Preparation of PCR strips An 80 µA volume of 20% sodium phosphate stock solution was added to the first well of eight 12-well PCR strips. A 40 µA volume of Q-water was added to wells 2, 3, 4, 5, and 6 of each PCR strip. The sodium phosphate stock solution was serially diluted 2-fold sequentially from well 1 to well 6. An 80 µA volume of 25% sodium citrate stock solution was added to well 7 of each PCR strip. A 40 µA volume of water was added to wells 8, 9, 10, 11, and 12 of each PCR strip. The sodium citrate stock solution was serially diluted 2-fold sequentially from well 7 to well 11, leaving well 12 containing only water. The nnccon / nznz / a / Y phytase BP-17 CFU was diluted 50-fold in autoclaved water. A volume of 40 µA of the diluted CFU was added to each well of the eight 12-well PCR strips containing the diluted excipients. Thermal inactivation The thermocycler was set to 70 °C. One PCR strip was sealed and immediately placed on ice for time zero measurement. The remaining PCR strips were sealed, placed inside the PCR instrument, incubated for various time increments (0.5, 2, 5, 10, 20, 30, and 60 minutes), then removed from the PCR instrument and immediately placed on ice. After collecting each PCR strip on ice, they were placed in a 96-well PCR tube rack and centrifuged for 2 minutes at 3500 rpm. Phytase activity assay Because some precipitation occurred during thermal inactivation, the PCR strips were centrifuged, and the supernatant was removed and diluted with assay buffer (0.1 M acetate buffer at pH 5.5 + 0.05% w / v Tween 20) to a final activity of 0.012 URF / ml in a 96-well non-adherent plate (Corning 3641). A 40 µL volume of the enzyme dilution plate was transferred to a separate reaction plate (Costar 9017, intermediate bonding plate). A 40 µL volume of substrate nnccon / nznz / a / Y (150 µM sodium phytate) was then added to each well of the reaction plate. The reaction plate was sealed and incubated for 10 minutes at 25 °C and 900 rpm. Then, a volume of 170 μA of Pi Blue stop / color reagent (POPB-500 from BioAssay Systems US) was added to each well of the reaction plate to stop the reaction.The plate was sealed and incubated for 30 minutes at 25 °C and 400 rpm for color display and then read in a spectrophotometer at 620 nm. As shown in Figure 6, approximately 90% of the residual phytase activity (calculated using the % activity recovered as described herein) is retained after 60 minutes of incubation in the presence of 352 mM sodium phosphate. At half this concentration, approximately 60% of the phytase activity is retained after 60 minutes of incubation. Below this concentration, the phytase enzyme appears to be practically inactivated. As shown in Figure 7, approximately 90% of the phytase activity is retained after 60 minutes of incubation in the presence of 425 mM sodium citrate. At half this concentration, approximately 50% or less of the phytase activity is retained after 60 minutes of incubation. Below this concentration, the phytase enzyme appears to be inactivated.To achieve similar activity profiles under heat stress conditions, a higher concentration of sodium citrate is required compared to sodium phosphate. In this respect, sodium phosphate appears to be a better stabilizer for phytase than sodium citrate. Example 3. Inhibition Mechanism A kinetic analysis was carried out to determine the stabilization mechanism of both sodium phosphate and sodium citrate with respect to the phytase enzyme by measuring phytase activity in the presence of concentrations of both titrated substrate and titrated excipient. Materials The following equipment and reagents were used: UFC of phytase BP-17 (70,000 URF / g 123.4 g / 1 of phytase) Anhydrous dibasic sodium phosphate (Sigma Aldrich), 141.96 g / mol Sodium citrate dihydrate (JT Baker), 294.1 g / mol Phosphoric acid and citric acid for pH adjustment pH meter Dilution buffer: 0.1 M acetate buffer at pH 5.5 with 0.05% (w / v) Tween 20 4-Nitrophenyl phosphate di(tris) salt (Sigma Aldrich) Substrate buffer: 0.25 M acetate buffer at pH 5.5 Stop buffer: 200 mM borate at pH 10.2 Corning 3641 96-well microtiter plate, flat bottom, non-stick polystyrene. VWR aluminum seals for 96-well plates. Eppendorf Thermomixer™ R with microplate block (Fisher Scientific) SpectraMax 340 Microplate Reader (Molecular Devices) Enzyme and substrate preparation The BP-17 phytase CFU was diluted 7,000 times in 0.1 M acetate buffer at pH 5.5. The substrate stock solution (400 mM) was prepared by dissolving 0.923 grams of 4-nitrophenyl di(tris)phosphate salt in 5 mL of 0.25 M acetate buffer at pH 5.5. A volume of 200 µA of the substrate stock solution was added to columns 1 and 7 of a 96-well plate. A volume of 100 µA of 0.25 M acetate buffer at pH 5.5 was added to columns 2-6 and 8-12 of the same 96-well plate. Serial dilutions were performed by transferring 100 μM from column 1 to column 2 of the 96-well plate, and then this dilution pattern was repeated along the plate to column 6, so that the following substrate concentrations were prepared: 400, 200, 100, 50, 25, and 12.5 mM. The same serial dilutions were performed along the second half of the plate between columns 7 to 12. Preparation of sodium phosphate A 20% (or 1.41 M) sodium phosphate solution was prepared by dissolving 5 grams of anhydrous dibasic sodium phosphate (nnccon / nznz / a / Y) in 25 mL of 0.1 M acetate buffer at pH 5.5. The 20% sodium phosphate solution was then diluted 7-fold by combining 1 mL of the stock solution with 6 mL of 0.1 M acetate buffer at pH 5.5, yielding a 200 mM concentration. A 200 µL volume of the sodium phosphate solution (200 mM) was added to each well in row A of a 96-well plate. A 100 µL volume of the 0.1 M acetate buffer at pH 5.5 was added to each well in rows BH of the same 96-well plate. Serial dilutions (factor 2) were performed by transferring 100 μA from row A to row B of the 96-well plate, and then this dilution pattern was repeated along the plate to row G, creating the following sodium phosphate concentrations: 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 mM. Preparation of sodium citrate A 25% (or 0.85 M) sodium citrate solution was prepared by dissolving 5 grams of sodium citrate dihydrate in 20 mL of 0.1 M acetate buffer at pH 5.5. A volume of 200 µL of the sodium citrate (0.85 M) solution was added to each well in row A of a new 96-well plate. A volume of 100 µL of the 0.1 M acetate buffer at pH 5.5 was added to each well in rows BH of the same 96-well plate. Serial dilutions (factor 2) were performed by transferring 100 μA from row A to row B of the nnccon / nznz / a / Y 96-well plate and then repeating this dilution pattern along the plate to row G, creating the following sodium citrate concentrations: 850, 425, 212.5, 106.25, 53.13, 26.56, 13.28 and 0 mM. Preparation of the substrate / excipient plate Two identical 96-well substrate plates were prepared by adding 25 μA of the standardized substrate concentrations to columns 1-6 and columns 7-12 of both plates. A volume of 75 μA of the standardized sodium phosphate concentrations was transferred to each corresponding well of the first substrate plate to prepare the following substrate and excipient concentrations: 100, 50, 25, 12.5, 6.25 and 3.125 mM of substrate 150, 75, 37.5, 18.75, 9.38, 4.69 and 2.34 mM of sodium phosphate A 75 μA volume of the standardized sodium citrate concentrations was transferred to each corresponding well of the second substrate plate to prepare the following substrate and excipient concentrations: 100, 50, 25, 12.5, 6.25 and 3.125 mM of substrate 637.5, 425, 212.5, 106.25, 53.13, 26.56 and 13.28 mM of sodium citrate PNPP Trial A 5 µA volume of 7,000-fold diluted BP-17 phytase CFU was added to columns 1–6 of an empty 96-well plate. A 5 µA volume of 0.1 M acetate buffer at pH 5.5 was added to columns 7–12 of the same 96-well plate, which served as blanks. An identical plate was prepared in the same manner. A 55 µA volume of the substrate / phosphate combinations was added to each corresponding well of the first 96-well plate containing the diluted CFU. A 55 µA volume of the substrate / citrate combinations was added to each corresponding well of the second 96-well plate containing the diluted CFU. The two 96-well reaction plates were sealed and placed in thermomixers at 25 °C at 650 rpm for 15 minutes. After the reaction was complete, a volume of 70 μA of borate stop solution (200 mM) at pH 10.2 was added to each well.The plates were sealed and placed in the plate shaker for 1 minute at 25 °C and 650 rpm and then read in a spectrophotometer at 405 nm. The PNPP assay measures the phosphorylation of p-nitrophenyl phosphate to p-nitrophenol, which is converted into a soluble yellow product that can be measured in a spectrophotometer at a wavelength of 405 nm. As shown in Figure 8, increasing concentrations of sodium phosphate reduced phytase enzyme activity in the presence of increasing concentrations of phytic acid substrate under ambient test conditions. At a sodium phosphate concentration of 150 mM and a phytic acid substrate concentration of 100 mM, phytase enzyme activity showed very little. Sodium phosphate inhibits enzyme activity in the presence of phytic acid substrate; therefore, it must compete for the enzyme's active site. As shown in Figure 9, low concentrations of sodium citrate begin to reduce phytase enzyme activity in the presence of increasing concentrations of phytic acid substrate under ambient test conditions, but the loss of activity appears to equilibrate at concentrations of 26 mM or higher.Therefore, the stabilization mechanism for sodium citrate must be different from that of sodium phosphate on the phytase enzyme. Example 4. Granulation and pelleting performance Multilayer enzyme granules were produced using a fluidized bed spraying process as described in U.S. Patent No. 5,324,649 (see also U.S. Patent Publication No. 2015037491), which are incorporated by reference. The granules were prepared (a) without a receptor or (b) with phytate or (c) phosphate, as summarized in Table 1. nnccon / nznz / a / Y nnccon / nznz / a / Y Table 1. Composition of the granules Stabilizing Granule Percentage of stabilizer in the granule (% w / w) Percentage of stabilizer in the phytase layer (% w / w) Molarity of stabilizer in the phytase layer (mM) Effective molar ratio of phosphate:phytase A None 0.0 0.0 0 0 B Sodium phytate 0.5 3.6 9 26 C Sodium phosphate 1.9 12.2 250 122 Prior to pelleting, the activity recovered from the pellets was compared using a steam process simulator in a steam simulation device, which the applicants have found predicts pelleting stability. The steam process simulator consists of a saturated steam source connected to a flexible hose with an internal diameter of approximately 0.635 cm, connected to a 3-way valve that feeds a receiving chamber with a diameter of approximately 6 inches (15.24 cm), over which a matching-diameter sieve mesh can be placed. The 3-way valve is also connected to a vacuum line, such that the steam feed can be instantly interrupted while the vacuum line is activated. To perform the test in the steam process simulator, approximately 1–2 grams of phytase enzyme granules were placed on the sieve mesh. The steam feed line was opened so that the steam flowed slowly over the mesh and came into contact with the enzyme granules for a defined number of seconds. After this time, the three-way valve was turned to stop the steam feed and induce a reverse flow of ambient air through the granules, thus instantly stopping contact with the steam and inducing rapid cooling and drying. The sieve mesh and the steam-treated granules were then removed from the steam process simulator and left to air dry overnight.The phytase activity retained in the granules was subsequently evaluated, and the recovery of phytase activity was calculated as a percentage of the activity of the initial untreated granules before steam treatment. Figure 10 shows the percentage of phytase activity recovered in granules subjected to 48 seconds of simulated steam treatment, compared to granules that had not undergone simulated steam treatment. nnccon / nznz / a / Y Stabilization with phosphate provided an 11% better recovery than stabilization with phytate. The granules were combined with a mixture of 60% corn flour and 40% soy flour (i.e., the dough) at a ratio of 60 grams of granules to 120 kilograms of corn-soy flour, such that the final phytase activity in the mixture before granule formation was approximately 5 units / gram. The mixture was then pelleted using an animal feed pellet press. The pellets and corn-soybean meal were mixed in a horizontal helical ribbon mixer for approximately 8 minutes. The pellet mill was a Simon Heesen single-roller type, equipped with a die with an internal diameter of 17.3 cm and a pellet hole with a diameter of 3 mm. The die speed was 500 rpm, driven by a 7.5 kW motor. The typical feed rate was 300 kg per hour. The temperature in the conditioner was maintained at + / - 0.1°C, measured at the conditioner feed outlet. The conditioner had a cascade-type mixing system. The conditioning temperature was 100°C. The steam inlet pressure was 2 atm, and the temperature in the conditioner was controlled by manually adjusting three valves that regulate the steam supply.The residence time in the nnccon / nznz / a / Y conditioner was 30 or 60 seconds. Once the target temperature was reached, the system was left running for approximately 5 to 10 minutes before sampling. Samples were taken over 11.5-minute intervals, corresponding to 5–7.5 kg of pelleted feed, and immediately placed in a cooling box with a perforated bottom and an airflow of 1500 cubic meters per hour. After cooling for 15 minutes, the sample size was reduced fivefold using a sample divider, and 250 g were taken for laboratory testing. After pelleting and cooling, the pellets were ground, and their phytase activity was evaluated. Figure 11 shows the percentage of phytase activity recovered in pellets converted to granules at 100°C for 30 or 60 seconds, compared to the phytase activity of unpelletized granules, using the % activity recovery formula provided herein. Stabilization with phosphate resulted in a 4% better recovery than stabilization with phytate after 30 seconds and an 11% better recovery after 60 seconds. Although the above compositions and methods have been described in detail for illustrative purposes and to ensure clarity and understanding, it will be evident that certain changes and modifications may be made without departing from the spirit and scope of the present compositions and methods. Accordingly, the description should not be construed as limiting the scope of the present compositions and methods set forth in the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. It is hereby stated that, as of this date, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.

Claims

Having described the invention as above, the following claims are claimed as property:

1. A method for increasing the stability of a phytase in a solid composition or for recovering the phytase in a pelleting process, characterized in that the solid composition comprises the method comprising introducing phosphate into the solid composition, wherein the phosphate and the phytase are in functional proximity and at a molar ratio of at least 50:1, and wherein the solid composition comprises a molar ratio of less than 10:1 of inositol or inositol phosphates to phytase.

2. The method according to claim 1, characterized in that the phytase and phosphate form, or are incorporated in or on, a solid support.

3. The method according to claim 1 or 2, characterized in that the phytase and phosphate form, or are incorporated into, a granule.

4. The method according to claim 3, characterized in that the granule is a matrix granule.

5. The method according to claim 3, characterized in that the granule is a multilayer granule.

6. The method according to claim 5, characterized in that the phytase and phosphate are incorporated into a single layer of the multilayer granule.

7. The method according to any of claims 3-6, characterized in that the phytase and phosphate are incorporated into the core of the granule.

8. The method according to any of claims 3-7, characterized in that the granule is included in a pellet of food or animal feed, wherein the percentage of activity recovered after heat treatment is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% or greater, after pelleting with steam.

9. The method according to any of claims 3-8, characterized in that the granule is incorporated into a pellet of food or animal feed, wherein the relative improvement in recovered activity is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% or more, after pelleting of the food or animal feed, compared to the activity recovered using, in the same pellet of food or animal feed, a granule having no phosphate and phytase in functional proximity and a molar ratio of at least 50:

1.

10. A method for increasing the stability of a phytase in a liquid composition or for the recovery of the phytase in a pelleting process characterized in that the liquid composition comprises the method comprising introducing phosphate into the composition, wherein the phosphate and the phytase are in functional proximity and the phosphate is present at a concentration of 50 mM or more, and wherein the liquid composition comprises inositol or inositol phosphates at less than 10 mM.

11. The method according to claim 10, characterized in that the phytase and phosphate are in solution or suspension.

12. The method according to claim 10 or 11, characterized in that the phytase and phosphate are included in a pellet of food or animal feed, wherein the percentage of activity recovered is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% after pelleting of the food or animal feed.

13. The method according to any of claims 10-12, characterized in that the phytase and phosphate are applied to the surface of a pellet of food or animal feed, wherein the percentage of activity recovered is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% after pelleting of the food or animal feed.

14. The method according to any of claims 10-13, characterized in that the relative improvement in recovered activity is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% after pelleting of the food or animal feed, compared to the activity recovered using, in the same food or animal feed pellet, a pellet having no phosphate and phytase in functional proximity, the phosphate being present at a concentration of 50 mM or more.

15. The method in accordance with any of the preceding claims, characterized in that the phosphate is not a product of the hydrolysis of phytate present in the solid or liquid composition.

16. The method according to any of the preceding claims, characterized in that the solid or liquid composition is essentially free of inositol or inositol phosphates.

17. The method in accordance with any of the preceding claims, characterized in that the phosphate is a monophosphate.

18. The method according to any of the preceding claims, characterized in that the DSC Tm of the phytase is increased by at least 2.7 °C compared to a control phytase not stabilized with phosphate at a phosphate to phytase molar concentration ratio of at least 50:1 or a phosphate concentration of at least 50 mM.

19. A granulated composition characterized in that it comprises phosphate and phytase, wherein the phosphate and phytase are in functional proximity and in a molar ratio of at least 50:1 or the phosphate is present in an amount of phosphate of at least 50 mM.

20. The granule according to claim 19, characterized in that it is a matrix granule.

21. The granule according to claim 19, characterized in that it is a multilayer granule.

22. The granule according to claim 20, characterized in that the phytase and phosphate are incorporated into a single layer of the multilayer granule.

23. The granule according to any of claims 19-22, characterized in that the phytase and phosphate are incorporated into the core of the granule.

24. The granule according to any of claims 19-22, characterized in that the phosphate is not a hydrolysis product of the phytate present in the solid or liquid composition.

25. The granule according to any of claims 19-22, characterized in that the solid or liquid composition does not contain phytate.

26. The granule according to any of claims 19-22, characterized in that the phosphate is a monophosphate.

27. A pellet composition characterized in that it comprises the granule in accordance with any of claims 19-26.

28. A pellet composition characterized in that it comprises a phytase and phosphate in functional proximity and in a molar ratio of at least 50:1 or wherein the phosphate is present at a concentration of at least 50 mM, wherein the phosphate is not a hydrolysis product of the phytate present in the pellet composition.