Means and methods for reducing phosphorus secretions in fish

Abstract

The present invention relates to a fish feed or a fish feed additive comprising one or more polypeptides having a phytase activity wherein the phytase activity comprises an EC: 3.1. 3.26 (i.e., 4-phytase) phytase activity and / or an EC 3.1. 3.8 (i.e., 3-phytase) phytase activity and / or an EC 3.1. 3.72 (i.e., 5-phytase) phytase activity wherein the phytase activity comprises one or more polypeptides having a phytase activity. Wherein the dose level of the phytase activity is at least 10% lower than an effective (e.g., optimal) dose of phytase activity configured to maximize fish growth response. The present invention relates to a method for reducing phosphorus secretions and / or phosphorus excreta in fish, said method comprising the administration of an animal feed or animal feed additive according to any one of the claims.

Description

Technical Field

[0001] The present invention relates to a fish feed or a fish feed additive, the fish feed or fish feed additive comprising one or more polypeptides having phytase activity, wherein the phytase activity comprises EC: 3.1.3.26 (i.e. 4-phytase) phytase activity and / or EC 3.1.3.8 (i.e. 3-phytase) phytase activity and / or EC 3.1.3.72 (i.e. 5-phytase) phytase activity, wherein the dosage level of the phytase activity is at least 10% lower than the effective dosage of the phytase activity configured to maximize the growth response / weight gain of the fish. The present invention relates to a method for reducing phosphorus secretion and / or phosphorus excretion of fish, the method comprising administering an animal feed or an animal feed additive according to any one of the above claims. Background Art

[0002] In animal feed, phosphorus is mainly present in the form of phytates, for example in cereals and legumes. However, monogastric animals (such as pigs, poultry and fish) cannot absorb phytates or phytic acid directly. This leads to excrement discharge of phosphorus-producing phytates into drains in areas of intensive livestock farming. In addition, phytic acid binds to metals (such as calcium, copper or zinc) and has a negative impact on the metabolism of monogastric animals. In order to compensate for the phosphate deficiency of these animals and ensure adequate growth and health, inorganic phosphates are added to animal feed.

[0003] However, the addition of inorganic phosphates is costly and has a further adverse effect on the environment. By using phytase in animal feed, phytate is hydrolyzed into a substrate that can be utilized by animals. This can improve animal production performance (e.g., increased FCR or BWG), reduce the burden of phosphate on the environment and reduce farmers' costs. Therefore, phytase is usually added to the diet of animals, and is particularly added to the diet of monogastric animals such as poultry and pigs, and it is well known in the art that this can improve the production performance of animals, such as improving weight gain and / or feed conversion rate (Selle & Ravindran, 2007, "Microbial phytase in poultry nutrition", Anim. Feed Sci. Tech., 135: 1-41 and Selle and Ravindran, 2008, "Phytate degrading enzymes in pig nutrition", Livestock Sci., 1 13: 99-122). However, even when phytase is added to animal feed to maximize growth response / weight gain, phosphorus excrement is still very important. Therefore, there remains a need to reduce the environmental burden of phosphorus excreted in animal feed.

[0004] Therefore, the technical problem to be solved by the present application is to meet this demand. The technical problem is solved by providing the technical solutions reflected in the claims, the technical solutions described in the specification, and the implementation schemes illustrated in the following examples and drawings. Summary of the invention

[0005] Thus, in a first aspect, the present invention relates to a fish feed or a fish feed additive, comprising one or more polypeptides having phytase activity, wherein the phytase activity comprises EC: 3.1.3.26 (i.e. 4-phytase) phytase activity and / or EC 3.1.3.8 (i.e. 3-phytase) phytase activity and / or EC 3.1.3.72 (i.e. 5-phytase) phytase activity, wherein the dosage level of the phytase activity is at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30% or at least 35%) lower than the effective dose of the phytase activity for maximizing fish growth response / weight gain.

[0006] In another aspect, the present invention relates to a method for reducing phosphorus secretion and / or phosphorus excretion of fish, preferably wherein the phosphorus is in the form of phosphate (e.g., phosphate anion [PO4] 3- ) in the form of an animal feed or an animal feed additive of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 : Daily soluble phosphorus excretion in fish fed different experimental diets. Bar graphs are mean ± SD (n = 4). Different superscripts indicate statistically significant differences (P < 0.05). DETAILED DESCRIPTION

[0008] As used herein, the term "animal" may refer to any animal other than humans. Examples of animals are non-ruminants and ruminants. Ruminants include animals such as sheep, goats, cattle (such as beef cattle, dairy cows, calves), deer, yaks (yanks), camels, llamas (llama) and kangaroos. Non-ruminants include monogastric animals, including but not limited to pigs (pig or swine) (including but not limited to piglets, growing pigs, sows); poultry, such as turkeys, ducks, quails, guinea fowls, geese, pigeons (including squabs) and chickens (including but not limited to meat chickens (hereinafter referred to as broilers), chickens, laying hens (hereinafter referred to as laying hens)); horses (including but not limited to hot-blooded horses, cold-blooded horses and warm-blooded horses); crustaceans (including but not limited to shrimp and prawns) and fish (including but not limited to amberjack, arapaima, thornfish, bass, bluefish, scorpionfish (boca chico), bream, bullhead, cachama, carp, catfish, catla, milkfish, chanos, salmon, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut alibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, perch, sea bream The following are some of the most common marine mammals: whitefish, trout, tuna, turbot, vendace, walleye, and whitefish).

[0009] As used herein, the term "animal feed" (e.g., fish feed) may refer to any compound, preparation, or mixture suitable for or intended to be ingested by an animal (e.g., fish). Animal feed for monogastric animals typically comprises concentrates and vitamins, minerals, enzymes, microorganisms for direct feeding, amino acids, and / or other feed ingredients (e.g., in premixes), while animal feed for ruminants typically comprises forage (including roughage and silage) and may also comprise concentrates and vitamins, minerals, enzymes, microorganisms for direct feeding, amino acids, and / or other feed ingredients (e.g., in premixes). Animal feed additives (e.g., fish feed additives) are formulated as enzyme products, which may further comprise, for example, vitamins, minerals, enzymes, amino acids, preservatives, and / or antibiotics; i.e., premixes. Animal feed additives / premixes are typically mixed with concentrates and / or forage (e.g., vegetable proteins, beans, or other plant materials) in a feed mill. The animal feed is typically fed to monogastric animals as pelleted feed.

[0010] As used herein, the term "phytase" may refer to an enzyme that hydrolyzes phytate (inositol hexakisphosphate) to inositol and inorganic phosphate and is known to be a valuable feed additive (also known as myo-inositol hexakis phosphate phosphohydrolases, e.g., EC: 3.1.3.26 (i.e., 4-phytase) or EC 3.1.3.8 (i.e., 3-phytase) or EC 3.1.3.72 (i.e., 5-phytase)).

[0011] Phytases with different pH optimum conditions, substrate specificity and hydrolysis specificity have been identified in plants and fungi. Acid phytases from wheat bran and Aspergillus have been extensively studied, and the stereospecificity of hydrolysis has been well established. Based on the specificity of initial hydrolysis, the International Union of Pure and Applied Chemistry and the International Union of Biochemistry (IUPAC-IUB, 1975) recognize two types of phytases: 6-phytase (for example, found in plants) and 3-phytase (found in fungi). 6-phytase hydrolyzes phosphate esters at the L-6 (or D-4) position of phytic acid, while 3-phytase hydrolyzes phosphate esters at the D-3 position.

[0012] The ENZYME website on the Internet (http: / / www.expasy.ch / enzyme / ) is an information database related to the nomenclature of enzymes. The website is mainly based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), describing each characteristic enzyme with an EC (Enzyme Commission) number (Bairoch A. the enzyme database, 2000, Nucleic Acids Res 28: 304-305). See also the manual Enzyme Nomenclature from NC-IUBMB, 1992.

[0013] According to the ENZYME website, three different types of phytases are known: one called 3-phytase (phytate 3-phosphohydrolase, EC 3.1.3.8), one called 6-phytase (phytate 6-phosphohydrolase, EC 3.1.3.26) and one called 5-phytase (phytate 5-phosphohydrolase, EC 3.1.3.72). For the purposes of the present invention, all three types are included in the definition of the present phytase.

[0014] Examples of phytases used in the present invention include, but are not limited to: phytases derived from Escherichia coli (Ecoli) strains disclosed in EP 684313, phytases derived from Buttiauxella strains, Ascomycete phytases (e.g., derived from Aspergillus fumigatus strains, Aspergillus terreus strains, and Myceliophthora thermophila strains); phytases disclosed in JP 11000164 (a phytase derived from a Penicillium strain); phytases disclosed in US Pat. No. 6,139,902 (a phytase derived from an Aspergillus strain); phytases disclosed in WO 98 / 13480 (Monascusanka phytase); and phytases disclosed in WO 2008 / 116878 and WO 2009 / 116987. Phytase disclosed in 2010 / 034835 (Hafnia phytase). Preferred phytases used in the present invention are derived from the family Enterobacteriaceae, and more preferably from Escherichia species, Citrobacter species, Buttiauxella species or Hafnia species.

[0015] For the purposes of the present invention, preferred phytases are those contained in the following commercial products: HiPhos, NP and P (DSM Nutritional Products AG); Natuphos TM (BASF); and Blue (AB Enzymes); (Huvepharma); XP (Verenium / DuPont) and PHY (DuPont).

[0016] For the purposes of the present invention, phytase activity is determined by the release of inorganic phosphate from a sodium phytate solution, wherein one phytase activity unit is the amount of enzyme that releases 1 μmol of inorganic phosphate per minute from a 0.0051 M sodium phytate solution in 0.25 M sodium acetate at a pH of 5.5 and 37° C. (Engelen, AJ, et al., 1994, "Simple and rapid determination of phytase activity", J. AOAC Int. 77: 760-764). Examples of the names of the activity units are FYT, FTU, and U. Phytase activity can be determined by the method described in Example 1 of US2017119017 ("Determination of phytase activity").

[0017] Specific activity is measured on highly purified samples (SDS polyacrylamide gels should show the presence of only one component). The enzyme protein concentration can be determined by amino acid analysis and the phytase activity is expressed in FYT units. The specific activity is characteristic of the specific phytase variant in question and is calculated as the phytase activity (measured in FYT units) per mg of phytase enzyme protein.

[0018] In order to determine the amount of phytase protein in milligrams per kilogram of feed or feed additive, the enzyme from the feed composition or feed additive is purified and the specific activity of the purified enzyme is determined using the relevant test. The phytase activity of the feed composition or feed additive is also determined using the same test and based on these two determinations the dosage of phytase protein in milligrams per kilogram of feed is calculated.

[0019] In one embodiment, the present invention provides a fish feed or a fish feed additive, wherein the fish feed or the fish feed additive comprises one or more polypeptides having phytase activity, wherein the phytase activity comprises EC: 3.1.3.26 (i.e., 4-phytase) phytase activity and / or EC 3.1.3.8 (i.e., 3-phytase) phytase activity and / or EC 3.1.3.72 (i.e., 5-phytase) phytase activity, wherein the dosage level of the phytase activity is at least 10% (e.g., at least 15%, at least 20%, 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%) lower than the maximum (or optimal) dose of phytase activity calculated to maximize the fish growth response (e.g., the maximum phytase activity calculated to achieve the maximum fish growth response / weight gain under given conditions) (e.g., a standard commercial dose of the phytase), preferably wherein the dosage level of the phytase activity is at least 10% (e.g., at least 15%, at least 20%, 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%) lower than the maximum (or optimal) dose of the phytase activity calculated to maximize the fish growth response (e.g., the maximum phytase activity calculated to achieve the maximum fish growth response / weight gain under given conditions) (e.g., a standard commercial dose of the phytase). Preferably, the dosage level of phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet as described in Example 1 herein) to about or less than 2000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY2000" diet as described in Example 1 herein), further preferably, wherein the dosage level of phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet as described in Example 1 herein) to about or less than 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet as described in Example 1 herein).

[0020] In another embodiment, the present invention provides a method for reducing phosphorus secretion and / or phosphorus excretion of fish, preferably, the phosphorus is in the form of phosphate (e.g., phosphate anion [PO4] 3- ) in the form of an animal feed or an animal feed additive of the present invention.

[0021] In yet another embodiment, the phytase is applied / present in the fish feed / method at a concentration that is at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30% or at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%) lower than a standard commercial dosage of the phytase.

[0022] In yet another embodiment, the dosage level of phytase activity is about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet set forth in Example 1 herein), about or less than 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet set forth in Example 1 herein), or about or less than 2000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY2000" diet set forth in Example 1 herein).

[0023] In another embodiment, the phytase is administered in an amount such that the specific activity in the final fish feed is between about 500 FYT / kg feed to about 2000 FYT / kg feed (e.g., 500 FYT / kg feed, 1000 FYT / kg feed, or 2000 FYT / kg feed), preferably between about 500 FYT / kg feed to about or less than 1000 FYT / kg feed.

[0024] Technical solution of the present invention

[0025] The present invention can also be summarized as the following technical solutions:

[0026] 1. A fish feed or a fish feed additive, comprising one or more polypeptides having phytase activity, wherein the phytase activity comprises EC: 3.1.3.26 (i.e., 4-phytase) phytase activity and / or EC 3.1.3.8 (i.e., 3-phytase) phytase activity and / or EC 3.1.3.72 (i.e., 5-phytase) phytase activity, wherein the dosage level of the phytase activity is at least 10% (e.g., at least 15%, at least 20%, 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%) lower than the maximum (or optimal) dose of phytase activity calculated to maximize the fish growth response (e.g., the maximum phytase activity calculated to achieve the maximum fish growth response / weight gain under given conditions) (e.g., a standard commercial dose of the phytase), preferably wherein the dosage level of the phytase activity is at least 10% (e.g., at least 15%, at least 20%, 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%) lower than the maximum (or optimal) dose of the phytase activity calculated to maximize the fish growth response (e.g., the maximum phytase activity calculated to achieve the maximum fish growth response / weight gain under given conditions) (e.g., a standard commercial dose of the phytase). Preferably, the dosage level of phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet as described in Example 1 herein) to about or less than 2000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY2000" diet as described in Example 1 herein), further preferably, wherein the dosage level of phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet as described in Example 1 herein) to about or less than 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet as described in Example 1 herein).

[0027] 2. The fish feed or fish feed additive according to any one of the preceding technical solutions, wherein the fish feed or fish feed additive comprises phytate-bound phosphorus.

[0028] 3. The fish feed or fish feed additive according to any one of the preceding technical solutions, wherein the fish feed or fish feed additive does not contain inorganic P (Pi) additives, preferably, the fish feed or fish feed additive does not contain inorganic P (Pi) additives such as DCP (dicalcium phosphate) and / or MCP (monocalcium phosphate) and / or PP (potassium phosphate) and / or MSP (monosodium phosphate), most preferably, the fish feed or fish feed additive does not contain inorganic P (Pi) additives such as DCP (dicalcium phosphate) and MCP (monocalcium phosphate).

[0029] 4. The fish feed or fish feed additive according to any one of the preceding technical solutions, wherein the dosage level of the phytase activity is about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet described in Example 1 herein), about or less than 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet described in Example 1 herein), or about or less than 2000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY2000" diet described in Example 1 herein).

[0030] 5. The fish feed or fish feed additive according to any one of the preceding technical solutions, wherein one unit of phytase activity is the amount of enzyme that releases 1 μmol of inorganic phosphate per minute from a 0.0051 M sodium phytate solution in 0.25 M sodium acetate at a pH of 5.5 and 37°C.

[0031] 6. The fish feed or fish feed additive according to any one of the preceding technical solutions, wherein the one or more polypeptides having phytase activity are derived from a bacterial source, preferably, the bacterial source is Citrobacter braakii bacteria, and further preferably, the one or more polypeptides having phytase activity are "RONOZYME HiPhos" phytase (for example, and / or other suitable phytases disclosed in WO2017001701) or have UniProtKB Accession Number: Q2VY22-1.

[0032] 7. A method for reducing phosphorus secretion and / or phosphorus excretion of fish, preferably, the phosphorus is in the form of phosphate (e.g., phosphate anion [PO4] 3- ) in the form of an animal feed or an animal feed additive according to any one of the above technical solutions.

[0033] 8. The method according to any one of the preceding technical solutions, wherein the amount of phytase applied is such that the specific activity in the final fish feed is between about 500 FYT / kg feed and about 2000 FYT / kg feed (e.g., 500 FYT / kg feed, 1000 FYT / kg feed or 2000 FYT / kg feed), preferably between about 500 FYT / kg feed and about or less than 1000 FYT / kg feed.

[0034] 9. A method according to any one of the aforementioned technical solutions, wherein the phosphorus secretion and / or phosphorus excretion is reduced by at least 5% (for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or at least 35%).

[0035] 10. The method according to any one of the aforementioned technical solutions, wherein the method is also a method for reducing the weight gain of fish to reduce the phosphorus secretion and / or phosphorus excretion of the fish.

[0036] 11. A method according to any one of the aforementioned technical solutions, wherein the method is an in vitro method.

[0037] 12. The method according to any one of the aforementioned technical solutions, wherein the phosphorus secretion and / or phosphorus excretion is postprandial soluble phosphorus secretion and / or phosphorus excretion.

[0038] 13. A method according to any one of the above technical solutions, wherein the phosphorus is soluble phosphorus; preferably, the soluble phosphorus is cumulative soluble phosphorus secretion and / or phosphorus excretion (for example, cumulative soluble phosphorus excretion 24 hours after a meal, for example, as described in Example 1 and / or as described in Example 2). Figure 1 shown).

[0039] 14. Use of the fish feed or fish feed additive described in any one of the above technical solutions for reducing phosphorus secretion and / or phosphorus excretion of fish.

[0040] 15. The fish feed or fish feed additive, method or use according to any one of the preceding technical solutions, wherein the fish is selected from the group consisting of Tilapia (e.g., Tilapiasparrmanii or banded tilapia), Oreochromis (e.g., Oreochromis niloticus or Nile tilapia), Dicentrarchus (e.g., Dicentrarchus labrax or European seabass) and Sparus (e.g., Sparus aurata or gilthead seabream).

[0041] 16. The fish feed or fish feed additive, method or use according to any one of the above technical solutions, wherein:

[0042] a) when the dosage level of the phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet as set forth in Example 1 herein) to about 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet as set forth in Example 1 herein), the fish is selected from the genus Carassius auratus or the genus European seabass (e.g., Nile mouthbrooder or European seabass); or

[0043] b) when the dosage level of the phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g. corresponding to the "PHY500" diet as set forth in Example 1 herein) to about 2000 phytase activity units (FYT) / kg fish feed (e.g. corresponding to the "PHY1000" diet as set forth in Example 1 herein), the fish is selected from the genus Carassius auratus, Sparus and European Seabass (e.g. Nile mouthbrooder, European seabass or Gilthead seabream).

[0044] 17. The fish feed or fish feed additive, method or use according to any one of the above technical solutions, wherein the fish feed or fish feed additive is aquatic feed or aquatic feed additive.

[0045] 18. The fish feed or fish feed additive, method or use according to any one of the preceding technical solutions, wherein the phytase is applied / present in an amount such that the specific activity in the final fish feed is between about 500 FYT / kg feed and about 2000 FYT / kg feed (e.g., 500 FYT / kg feed, 1000 FYT / kg feed or 2000 FYT / kg feed), preferably between about 500 FYT / kg feed and about 1000 FYT / kg feed.

[0046] ***

[0047] Unless otherwise stated, the following terms used herein, including the specification and claims, have the definitions given below.

[0048] Those skilled in the art will recognize or be able to determine many equivalent technical solutions of the specific technical solutions of the present invention described herein by using no more than routine experiments. These equivalent technical solutions are intended to be included in the present invention.

[0049] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more different agents of that type, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or substitute the methods described herein.

[0050] Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to ascertain many equivalent technical solutions to the specific technical solutions of the invention described herein using no more than routine experimentation. These equivalent technical solutions are intended to be included in the present invention.

[0051] The term "and / or" used anywhere as used herein includes the meanings of "and", "or" and "all or any other combinations of the elements connected by the term".

[0052] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, specific values ​​are also included, for example, about 20 includes 20.

[0053] In this specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprising" may be replaced with the term "containing" or "including", or with the term "having" as is sometimes used herein.

[0054] When "consisting of..." is used herein, any element, step or component not specified in the claim element is excluded. When "consisting essentially of..." is used herein, materials or steps that have no substantial impact on the basic and novel features of the claim are not excluded.

[0055] In each instance herein, any of the terms "includes / comprising / containing", "consisting essentially of" and "consisting of" may be replaced with any of the other two terms.

[0056] It should be understood that the present invention is not limited to the specific methods, protocols, materials, reagents and substances, etc. described herein, and these methods, protocols, materials, reagents and substances, etc. may vary. The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the claims.

[0057] All publications cited in the body of this specification (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to such disclosures earlier than those disclosed by reason of prior inventions. In the event that the material incorporated by reference into the text contradicts or is inconsistent with this specification, the description of this specification shall prevail.

[0058] Example

[0059] The following examples illustrate the present invention. These examples should not be construed as limiting the scope of the present invention. These examples are included for illustrative purposes, and the present invention is limited only by the claims.

[0060] Example 1: Postprandial soluble phosphorus excretion

[0061] Postprandial total soluble phosphorus was measured by standardized collection of water from the rearing tanks on 3 non-consecutive days after the end of the growth performance trial and at all subsequent relevant samplings. In order to reduce the basal phosphorus level in the rearing system, the rearing water was completely replaced before the measurements. In addition, to avoid any potential daily carryover effect, phosphorus excretion was measured under aerated static water conditions. On the measurement days, fish were fed a fixed diet corresponding to the lowest feed intake recorded during the growth study (1.6% biomass for Nile tilapia and 1.4% biomass for European seabass and gilthead seabream). One tank without fish was used as a control. Within 23 h after the meal, the fish were fed a fixed diet using a multichannel peristaltic pump ( Water samples from each pool were collected continuously into 2 L glass flasks kept in ice at the Food Testing Laboratory (Eurofins, Lisboa, Portugal) in Gilson, Middleton, WI, USA. After each daily cycle, frozen water samples were immediately sent to an external laboratory (Eurofins Food Testing Lisboa, Portugal) for total phosphate (PO4) analysis. Based on the fixed water flow rate (4.5 L / min for tilapia and 5.6 L / min for European seabass and gilthead seabream) and fish biomass for each pool, the 24-hour cumulative soluble phosphorus excretion ( Figure 1, Tables 1-6, wherein the phytase activities from the "DSM Phytases Matrix Calculator" available at https: / / phytases.dsm.com were used (the dosage levels of RONOZYME HiPhos were 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to a "PHY500" diet), 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to a "PHY1000" diet), and 2000 phytase activity units (FYT) / kg fish feed (e.g., a "PHY2000" diet)).

[0062] Table 1: P-excretion (mg / kg / day) in Nile tilapia feeding trials:

[0063] mg / kg / day diet P excrement STD CTRL 53.21 6.88 PHY500 38.69 8.68 PHY1000 23.37 7.41 PHY2000 99.24 1.69 DCP 118.82 3.87

[0064] Table 2: P-excretion (% intake) in Nile tilapia feeding trials:

[0065]

[0066]

[0067] Table 3: P-excretion (mg / kg / day) in European seabass feeding trials:

[0068] mg / kg / day diet P excrement STD CTRL 36.34 1.85 PHY500 28.46 4.07 PHY1000 29.74 2.89 PHY2000 61.27 0.47 MCP 127.81 2.59

[0069] Table 4: P-excretion (% intake) in the European seabass feeding trial:

[0070] % Intake diet P excrement STD CTRL 34.42 1.75 PHY500 27.29 3.90 PHY1000 28.70 2.79 PHY2000 58.35 0.44 MCP 92.73 1.88

[0071] Table 5: P-excretion (mg / kg / day) in the feeding trial of black sea bream:

[0072]

[0073]

[0074] Table 6: P-excretion (% intake) in the feeding trial of gilthead sea bream:

[0075] % Intake diet P excrement STD CTRL 44.28 1.14 PHY500 24.68 0.29 PHY1000 15.60 0.87 PHY2000 36.38 1.23 MCP 68.06 4.74

[0076] Conclusions: Tilapia and European seabass fed the PHY500 diet, PHY1000 diet, and seabream fed the PHY2000 diet showed significantly reduced soluble phosphorus excretion (P<0.05) compared to fish fed the CTRL and DCP / MCP supplemented diets. In addition, the DCP-supplemented diet (tilapia) and the MCP-supplemented diet (seabass and seabream) showed significantly increased soluble phosphorus excretion (P<0.05) compared to fish fed the other diets.

Claims

1. A fish feed or a fish feed additive, comprising one or more polypeptides having phytase activity, wherein: The phytase activity comprises EC: 3.1.3.26 (i.e. 4-phytase) phytase activity and / or EC 3.1.3.8 (i.e. 3-phytase) phytase activity and / or EC 3.1.3.72 (i.e. 5-phytase) phytase activity, wherein the dosage level of the phytase activity is at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30% or at least 35%) lower than the effective dosage of the phytase activity for maximizing the growth response and / or weight gain of the fish, preferably, wherein the dosage level of the phytase activity is about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet set forth in Example 1 herein). ) to about 2000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY2000" diet as described in Example 1 herein), further preferably, wherein the dosage level of phytase activity is about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet as described in Example 1 herein) to about 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet as described in Example 1 herein).

2. Fish feed or fish feed additive according to any one of the preceding claims, wherein the fish feed or fish feed additive comprises phytate-bound phosphorus.

3. The fish feed or fish feed additive according to any one of the preceding claims, wherein The fish feed or fish feed additive does not contain inorganic P(Pi) additives, preferably, the fish feed or fish feed additive does not contain inorganic P(Pi) additives such as DCP (dicalcium phosphate) and / or MCP (monocalcium phosphate) and / or PP (potassium phosphate) and / or MSP (monosodium phosphate), most preferably, the fish feed or fish feed additive does not contain inorganic P(Pi) additives such as DCP (dicalcium phosphate) and MCP (monocalcium phosphate).

4. The fish feed or fish feed additive according to any one of the preceding claims, wherein The dosage level of phytase activity is about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet set forth in Example 1 herein), about 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet set forth in Example 1 herein), or about 2000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY2000" diet set forth in Example 1 herein).

5. The fish feed or fish feed additive according to any one of the preceding claims, wherein One unit of phytase activity is the amount of enzyme which releases 1 μmol of inorganic phosphate per minute from a 0.0051 M sodium phytate solution in 0.25 M sodium acetate at pH 5.5 and 37°C.

6. The fish feed or fish feed additive according to any one of the preceding claims, wherein The one or more polypeptides having phytase activity are derived from a bacterial source, preferably the bacterial source is the bacterium Citrobacter braakii.

7. A method for reducing phosphorus secretion and / or phosphorus excretion of fish, preferably, the phosphorus is in the form of phosphate (e.g., phosphate anion [PO4] 3- ), the method comprising: Administration of an animal feed or an animal feed additive according to any one of the preceding claims.

8. A method according to any one of the preceding claims, wherein: The phosphorus secretion and / or phosphorus excretion is reduced by at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35%).

9. A method according to any one of the preceding claims, wherein: The phosphorus secretion and / or phosphorus excretion is postprandial soluble phosphorus secretion and / or phosphorus excretion.

10. A method according to any one of the preceding claims, wherein: The phosphorus is soluble phosphorus; preferably, the soluble phosphorus is cumulative soluble phosphorus secretion and / or phosphorus excretion (eg, cumulative soluble phosphorus excretion 24 hours after a meal, eg, as described in Example 1 herein and / or as shown in FIG. 1 ).

11. Use of a fish feed or a fish feed additive as claimed in any one of the preceding claims for reducing phosphorus secretion and / or phosphorus excretion of fish.

12. A fish feed or fish feed additive, method or use according to any one of the preceding claims, wherein: The fish is selected from the group consisting of Tilapia (e.g. Tilapiasparrmanii or banded tilapia), Oreochromis (e.g. Oreochromis niloticus or Nile tilapia), Dicentrarchus (e.g. Dicentrarchus labrax or European seabass) and Sparus (e.g. Sparus aurata or gilthead Seabream).

13. A fish feed or fish feed additive, method or use according to any one of the preceding claims, wherein: a) when the dosage level of the phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY500" diet set forth in Example 1 herein) to about 1000 phytase activity units (FYT) / kg fish feed (e.g., corresponding to the "PHY1000" diet set forth in Example 1 herein), the fish is selected from the genus Carassius auratus or the genus European seabass (e.g., Nile mouthbrooder or European seabass); or b) when the dosage level of the phytase activity is from about 500 phytase activity units (FYT) / kg fish feed (e.g. corresponding to the "PHY500" diet as set forth in Example 1 herein) to about 2000 phytase activity units (FYT) / kg fish feed (e.g. corresponding to the "PHY1000" diet as set forth in Example 1 herein), the fish is selected from the genus Carassius auratus, Sparus and European Seabass (e.g. Nile mouthbrooder, European seabass or Gilthead seabream).

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