Animal feed comprising phytase without mineral supplements

Abstract

Provided herein are animal feeds containing a phytase polypeptide or fragment thereof, wherein the feed is free or substantially free of exogenously added trace minerals.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 498,065, filed April 25, 2023, and U.S. Provisional Patent Application No. 63 / 520,797, filed August 21, 2023, the disclosure of each of which is incorporated herein by reference in its entirety. By incorporating references

[0002] The sequence list provided as a 64 KB file named 20240419_NB42195-WO-PCT_Seq List.xml, which will be created and submitted simultaneously on April 22, 2024, is incorporated into this paper by reference in its entirety. Technical Field

[0003] This field relates to animal diets containing no or substantially no exogenous trace minerals and engineered phytase peptides, and their use in enhancing animal performance. Background Technology

[0004] Trace minerals (TMs) are essential for healthy bone development, enzyme structure and function, and catalytic biological reactions in animal diets (Suttle, 2010). The amounts of key TMs in commonly used ingredients and components in commercial broiler diets are considered insufficient (due to low availability) or too variable to support optimal growth and development.

[0005] Of course, there is evidence that stopping TM supplementation (especially zinc) from day 1 leads to poor growth and leg problems (Bao et al., 2007, 2009, 2010; Singh et al., 2015). Therefore, it is common practice to supplement diets with zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and selenium (Se) from inorganic or organic sources.

[0006] Adding exogenous microbial phytase to broiler diets is an effective way to increase phosphorus (P) availability. The efficacy of phytase in broilers has been extensively studied and is widely accepted (Selle and Ravindran, 2007; Humer et al., 2015). The substrate of phytase, phytate (IP6, inositol hexaphosphate), reacts with mineral cations (Ca) in the neutral pH environment of the small intestine. 2+ Zn 2+ Fe 2+ Mn 2+ Fe 3+ Mg 2+ Cu 2+ Co 2+Phytase binds to potent antinutrients with strong affinity (Selle and Ravindran, 2007; Selle et al., 2009). These complexes are insoluble at neutral pH, thus reducing the availability of both phytate and bound minerals, or TM, to animals (Selle et al., 2009). If phytase is active in the early gastrointestinal tract (GIT) and can rapidly and completely degrade phytate, it should reduce the binding of phytate to TM and improve the bioavailability of TM. A novel concordant sequence bacterial 6-phytase variant has recently been developed and commercialized, which efficiently degrades phytate in broilers and releases phosphorus (P) in the early GIT, improving the digestibility and utilization of energy, P, crude protein, and amino acids (Dersjant-Li et al., 2020; Christensen et al., 2020; Dersjant-Li et al., 2021; Dersjant-Li et al., 2022).

[0007] Therefore, it is necessary to determine the extent to which novel phytases can "replace" exogenously added TM in commercial animal diets throughout the entire growth cycle, while still ensuring normal growth performance and tissue-level TM utilization. Summary of the Invention

[0008] This article specifically provides animal diets containing phytase that contain little or no exogenously added trace minerals. When fed to animals (e.g., but not limited to poultry), these diets ensure normal growth at all developmental stages compared to diets containing trace mineral supplements.

[0009] Therefore, in one aspect, this document provides an animal diet comprising (i) an engineered phytase polypeptide or a fragment thereof containing phytase activity; and (ii) a lack of one or more exogenously added trace minerals. In some embodiments, the one or more trace minerals are one or more trace minerals selected from the group consisting of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and selenium (Se). In some embodiments of any of the embodiments disclosed herein, the phytase polypeptide or a fragment thereof containing phytase activity has at least 82% sequence identity with the amino acid sequence shown in SEQ ID NO:1. In some embodiments of any of the examples disclosed herein, the phytase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:3 ... NO:35, SEQ ID NO:36, and SEQ ID NO:37. In some embodiments of any of the embodiments disclosed herein, the diet further comprises one or more of the following: (a) a direct-feeding microorganism comprising at least one bacterial strain, (b) at least one other enzyme, and / or (c) an essential oil. In some embodiments of any of the embodiments disclosed herein, an engineered phytase polypeptide or a fragment thereof is present in an amount of at least about 0.1 g / ton of feed. In some embodiments of any of the embodiments disclosed herein, the diet comprises about 0.62% to 0.72% calcium in a starter diet, about 0.54% to 0.64% calcium in a growing diet, and / or about 0.42% to 0.55% calcium in a finishing diet. In some embodiments of any of the embodiments disclosed herein, the finishing diet comprises about 0.46% to 0.55% or about 0.42% to 0.50% calcium.In some embodiments of any of the examples disclosed herein, the diet contains about 1.18% to 1.22% amino acids in the starter diet, about 1.06% to 1.10% in the growing diet, and / or about 0.88% to 1% in the finishing diet. In some embodiments of any of the examples disclosed herein, the finishing diet contains about 0.96% to 1.0% or about 0.88% to 0.92% amino acids. In some embodiments of any of the examples disclosed herein, the amino acids include digestible lysine. In some embodiments of any of the examples disclosed herein, the diet contains about 2824 to 2950 kcal / kg in the starter diet, about 2924 to 3050 kcal / kg in the growing diet, and / or about 2970 to 3120 kcal / kg in the finishing diet. In some embodiments, the finishing diet contains about 2970 to 3100 kcal / kg or about 2994 to 3120 kcal / kg of metabolizable energy. In some embodiments of any of the examples disclosed herein, the diet contains about 0.13% to 0.17% sodium. In some embodiments disclosed herein, phytase is used at a rate between about 500 FTU / kg and about 2000 FTU / kg (e.g., about 500 FTU / kg, 550 FTU / kg, 600 FTU / kg, 650 FTU / kg, 700 FTU / kg, 750 FTU / kg, 800 FTU / kg, 850 FTU / kg, 900 FTU / kg, 950 FTU / kg, 1000 FTU / kg, 1050 FTU / kg, 1100 FTU / kg, 1150 FTU / kg, 1200 FTU / kg, 1250 FTU / kg, 1300 FTU / kg, 1350 FTU / kg, 1400 FTU / kg, 1450 FTU / kg, 1500 FTU / kg, 1550 FTU / kg, 1600 FTU / kg, 1650 FTU / kg, 1700 FTU / kg, etc.). The diet is present at doses of 1750 FTU / kg, 1800 FTU / kg, 1850 FTU / kg, 1900 FTU / kg, 1950 FTU / kg, or 2000 FTU / kg or more. In some embodiments of any of the examples disclosed herein, the diet contains a phytate source comprising one or more of corn, wheat, soybean meal, rapeseed, rice, and / or wheat bran. In some embodiments of any of the examples disclosed herein, the diet further comprises oat bran. In some embodiments of any of the examples disclosed herein, the diet is deficient in meat and / or bone meal. In some embodiments of any of the examples disclosed herein, the diet further comprises one or more additional feed enzymes selected from the group consisting of xylanase, protease, amylase, and glucosylamylase.In some embodiments of any of the embodiments disclosed herein, the feed is an starter diet. In some embodiments of any of the embodiments disclosed herein, the feed is a growing diet. In some embodiments of any of the embodiments disclosed herein, the feed is a finishing diet. In some embodiments of any of the embodiments disclosed herein, the animal is poultry. In some embodiments of any of the embodiments disclosed herein, the animal is a pig. In some embodiments of any of the embodiments disclosed herein, the animal is a ruminant. In some embodiments, the ruminant is a calf.

[0010] In another aspect, this document provides a method for improving animal performance in terms of one or more indicators, the method comprising administering to the animal an effective amount of any animal diet disclosed herein. In some embodiments of any of the embodiments disclosed herein, one or more indicators are selected from the group consisting of: increased feed efficiency, increased weight gain, decreased feed conversion ratio, improved digestibility of nutrients or energy in the feed, improved nitrogen retention, improved ability to avoid the negative effects of necrotizing enterocolitis, and improved immune response. In some embodiments of any of the embodiments disclosed herein, the animal is poultry, pig, or ruminant. In some embodiments, poultry are selected from the group consisting of turkeys, ducks, chickens, geese, pheasants, quails, and emus. In some embodiments, chickens are selected from the group consisting of laying hens and broilers.

[0011] In another aspect, this document provides a method for reducing pathogenic bacterial communities in animals, the method comprising administering to the animal an effective amount of any animal diet disclosed herein. In some embodiments, a) one or more trace minerals are iron (Fe); and / or b) the diet is further free of or substantially free of exogenously added inorganic phosphates. In some embodiments of any of the examples disclosed herein, the pathogenic bacterial population is one or more bacteria selected from the group consisting of: *Actinobacillus*, *Bordetalla*, *Campylobacter* (e.g., *C. jejuni*), *Clostridium*, *Corynebacterium*, *Escherichia coli* (e.g., enterotoxigenic *Escherichia coli*, ETEC), *Globicatella*, *Listeria*, *Mycobacterium*, *Salmonella*, *Staphylococcus*, and *Streptococcus*. In some embodiments of any of the examples disclosed herein, the animal is poultry, pig, or ruminant. In some embodiments, the poultry is selected from the group consisting of turkeys, ducks, chickens, geese, pheasants, quails, and emus. In some embodiments, the chicken is selected from the group consisting of laying hens and broilers. In some embodiments, the ruminant is a calf. Detailed Implementation

[0012] Global awareness of sustainability regarding the appropriate use of finite resources such as trace minerals and the pollution resulting from their use is growing. While commercially raised animals such as poultry have the lowest environmental impact among all animal proteins, significant amounts of exogenously added trace minerals are still used in their diets in commercial practice. The inventors of this application have unexpectedly discovered that the use of next-generation biosynthetic bacteria 6-phytase in broiler diets can completely eliminate the need for dietary supplementation with one or more trace minerals and / or significantly reduce the need for supplementation with one or more trace mineral sources. All animals fed diets supplemented with phytase at low or no trace mineral content exhibited comparable or better growth and feed conversion compared to animals fed diets containing one or more exogenously added trace minerals. Furthermore, tests showed that these animals did not exhibit the symptoms of bone loss or demineralization characteristic of animals experiencing trace mineral deficiency. Conversely, compared to controls, animals fed diets supplemented with phytase but without exogenously added trace minerals did not show deterioration in bone fracture strength. This represents the first report of a 100% plant-based, completely trace mineral-free, and commercially relevant broiler diet that demonstrates normal growth characteristics at all growth stages. Therefore, using the phytase-supplemented, trace mineral-free diets disclosed herein offers both economic advantages in the form of reduced feed costs (particularly for large-scale industrial poultry flocks) and significant environmental benefits due to reduced pollution as a byproduct of large-scale animal production.

[0013] In addition, the inventors of this invention have unexpectedly discovered that the combined use of next-generation biosynthetic bacteria 6-phytase with animal diets low in trace minerals can reduce the burden of pathogenic bacteria commonly associated with specific animal-based sources (e.g., but not limited to, Campylobacter species, such as Campylobacter jejuni).

[0014] All patents, patent applications and publications cited are incorporated herein by reference in their entirety for all purposes.

[0015] Many terms and abbreviations are used in this disclosure. Unless otherwise specified, the following definitions apply.

[0016] As used herein, the singular forms “a / an” and “the” include a plural indicator unless the context clearly indicates otherwise. For example, the term “a compound” or “at least one compound” may include multiple compounds, including mixtures thereof. For example, the terms “a / an,” “the,” “one or more,” and “at least one” are used interchangeably herein.

[0017] The terms “and / or” and “or” are used interchangeably herein and refer to a specific disclosure of each of two specified features or components, having or not having the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0018] Words that use the singular form include the plural form, and vice versa.

[0019] The terms “comprises / comprising,” “includes / including,” “having,” and their variations are used interchangeably and mean “including, but not limited to.” It should be understood that wherever the term “comprising” is used to describe an aspect herein, similar aspects described with the terms “consisting of” and / or “substantially consisting of” are also provided.

[0020] The term "composed of" means "including and limited to".

[0021] The term “consistently made of” means a specified material or a specified step of a method of a composition, as well as other materials or steps that do not substantially affect the essential characteristics of the material or method.

[0022] Throughout this application, various embodiments may be presented in the form of ranges. It should be understood that the descriptions in the form of ranges are merely for convenience and brevity and should not be construed as an inflexible limitation on the range of the embodiments described herein. Therefore, the description of a range should be considered as precisely disclosing all possible subranges within that range, as well as individual numerical values. For example, a description of a range such as 1 to 6 should be considered as precisely disclosing subranges such as 1 to 2, 1 to 3, 1 to 4 and 1 to 5, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0023] As used herein, the term “about” may allow for a degree of variability in a value or range, such as within 10%, 5%, or 1% of the specified limits of the specified value or range.

[0024] The term "phytase" (inositol hexaphosphate phosphohydrolase) refers to a class of phosphatases that catalyze the hydrolysis of phytic acid (inositol hexaphosphate or IP6) (a poorly digestible organic form of phosphorus found in cereal and oilseed seeds) to release inorganic phosphorus in a usable form.

[0025] The terms “animal” and “subject” are used interchangeably herein and refer to any living organism belonging to the animal kingdom, including but not limited to mammals (excluding humans), non-human animals, livestock, farm animals, zoo animals, breeding stock, etc. For example, all non-ruminant and ruminant animals may be mentioned. In this embodiment, the animal is a non-ruminant, i.e., a monogastric animal. Examples of monogastric animals include, but are not limited to, pigs (pigs and swine), such as piglets, growing pigs, multiparous sows; poultry, such as turkeys, ducks, chickens, broilers, laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans, such as shrimp and prawns. In another embodiment, the animal is a ruminant, including but not limited to cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorn, and wildebeest.

[0026] As used in this article, "poultry" refers to birds such as turkeys, pheasants, geese, emus, ducks, chickens, broilers, and laying hens.

[0027] The reference book "Commercial Poultry Nutrition" (3rd edition, 2005, ISBN 0-9695600-5-2; incorporated herein by reference) is the standard textbook in the field of nutrition for chicken production. The following is an overview of the background considered relevant to this invention.

[0028] The main energy-delivering components in poultry diets are corn, soybean, soybean oil, and amino acids. Corn is the primary contributor to metabolizable energy. The energy value of corn is comprised of endosperm starch, primarily composed of amylopectin, and germ, mainly oil. Typical energy values ​​for corn alone at 85% dry matter, expressed in kcal / kg, range from 3014 to 3313. Energy levels in starter and growing diets typically range from 3000 to 3100 kcal / kg. Wheat is also commonly used in poultry diets in many countries. Such diets have similar energy levels to those described above. Soybeans have become the world standard for protein sources compared to other protein sources. The amino acid profile of soybeans is excellent for most types of poultry, and when combined with corn or sorghum, methionine is often the only factor contributing to deficiencies. Additionally, fats and oils provide concentrated energy sources in diets, and even relatively small variations in their levels can have a significant effect on dietary energy (ME). Finally, diets are supplemented with synthetic amino acids such as methionine and lysine. Other important sources used are barley, sorghum and other grains, as well as by-products that contribute energy.

[0029] Chickens used for optimized commercial broiler production are typically fed different diets depending on their age. For example, chickens used for broiler production may be fed three or four diets. These diets are typically referred to as “starter,” “growing,” and “finishing.” “Pre-starter” diets are also possible. Therefore, the engineered phytases disclosed herein may be included at different dosage levels in starter-only diets, growing-only diets, finishing-only diets, combinations of any two phases, or combinations of all phases, as long as they contain no or substantially no exogenously added trace minerals.

[0030] As used herein, “trace minerals” refers to essential minerals present in trace amounts in animal feed that are associated with organic molecules (e.g., polysaccharides, amino acids) and act as cofactors for enzyme function. Non-limiting examples of trace minerals include one or more (e.g., 1, 2, 3, 4, or 5) of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and selenium (Se). When the animal diets disclosed herein also include appropriate doses of the engineered phytase peptides disclosed herein, the diets are free of or substantially free of exogenously added trace minerals (e.g., ZnSO4 or ZnO or zinc methionine or other organic or other forms of trace mineral premixes).

[0031] "Inorganic phosphorus" and "inorganic phosphate" are used interchangeably herein to refer to dietary supplements typically added to poultry feed to ensure that animals receive sufficient phosphate to meet their nutritional requirements. Additionally, in some non-limiting embodiments, the poultry diets disclosed herein may be free of or substantially free of inorganic phosphates when they also contain appropriate doses of the engineered phytase peptides disclosed herein and contain sufficient phytate as a substrate.

[0032] The expression “substantially none or substantially no” used in this document to describe the amount of trace minerals and / or inorganic phosphates in the feed formulations disclosed herein means that any amount present is trace, unintentionally included, and / or less than about 0.1% in the feed.

[0033] "Starter," "growing," and "finishing" diets are typically distinguished by their crude protein content, which is usually provided by ingredients such as soybean meal (SBM). For example, starter diets for broilers may optionally contain about 22-25% by weight of crude protein, such as 22%, 23%, 24%, or 25%, with 23% or 25% being preferred. In another example, growing diets for broilers may optionally contain about 21-23% by weight of crude protein, such as 21%, 22%, or 23%, with 22% being preferred. In yet another example, finishing diets for broilers may optionally contain about 19-23% by weight of crude protein, such as 19%, 20%, 21%, 22%, or 23%, with 19%, 20%, or 21% being preferred.

[0034] Alternatively, “starter diet,” “growing diet,” and “finishing diet” can be distinguished by their metabolizable energy (ME) content, which is typically lowest for starter diets and highest for finishing diets, with growing diets having levels in between. For example, starter diets for broilers may have an ME of approximately 3000 or 3025 kcal / kg (± 50, 40, 30, 20, 10, 5, or less kcal / kg). In another example, growing diets for broilers may have an ME of approximately 3100 or 3150 kcal / kg (± 50, 40, 30, 20, 10, 5, or less kcal / kg). In yet another example, growing diets for broilers may have an ME of approximately 3200 kcal / kg (± 50, 40, 30, 20, 10, 5, or less kcal / kg).

[0035] Animal feeds described herein may be vegetarian or non-vegetarian products. Vegetarian products do not contain meat or fish products. Non-vegetarian feeds may contain one or both of the following: fish products (such as fishmeal) or meat products (such as meat derivatives or other meat products that do not contain inorganic phosphates and / or trace minerals).

[0036] The terms “mixer liquid application” and “MLA” are used interchangeably herein and refer to animal feed production in which heat-sensitive compounds (especially enzymes) are applied to animal feed in liquid form prior to conditioning and pelleting and remain functional in the feed after conditioning and pelleting.

[0037] The terms “feed,” “animal feed,” or “ration” are used interchangeably herein, referring respectively to any natural or artificial diet, meal, or component thereof intended or suitable for consumption, ingestion, or digestion by non-human animals. Preferably, the term “feed” is used to refer to a product fed to animals in the feeding of livestock.

[0038] As used herein, “feed additive” refers to one or more ingredients, substances (e.g., cells) that are used alone or together in nutrition, for example to improve the quality of food (e.g., animal feed), improve animal performance and / or health, and / or enhance the digestibility of food or substances in food.

[0039] As used in this article, the term "food" is used in a broad sense - and covers any form of food and food products used for humans as well as food (i.e. feed) used for animals.

[0040] Food or feed may be in solution or solid form, depending on the use and / or mode of application and / or mode of administration. In some embodiments, the enzymes mentioned herein may be used as food or feed substances or for the preparation or production of food or feed substances.

[0041] As used herein, the term "food or feed ingredient" includes formulations that are added to or may be added to food or feed, and includes formulations that may be used at low levels in various products. Food ingredients may be in solution or solid form, depending on their use and / or mode of application and / or mode of administration. The enzymes described herein may be used as food or feed ingredients or for preparation or production. These enzymes may or may not be added to food supplements. Feed compositions for monogastric animals typically include compositions comprising plant products containing phytates. Such compositions include, but are not limited to, cornmeal, soybean meal, rapeseed meal, cottonseed meal, corn, wheat, barley, and sorghum-based feeds.

[0042] As used herein, the term "pelletizing" refers to the production of pellets, which can be solid, round, spherical, or cylindrical tablets, particularly feed pellets and solid extruded animal feed. An example of a known feed pelleting manufacturing process typically involves mixing food or feed ingredients together at room temperature for at least one minute, transferring the mixture to a buffer hopper, conveying the mixture to a steam conditioner (i.e., conditioning), optionally transferring the steam-conditioned mixture to an expander, transferring the mixture to a 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. [Pelleting Cost Center, Feed Manufacturing Technology IV] (edited by McEllhiney), American Feed Industry Association, Arlington, VA, pp. 110-139).

[0043] The term "pellets" refers to a composition of animal feed (typically derived from grains) that has been heat-treated, such as by steam treatment (i.e., conditioning), and then pressed or extruded by machine. Pellets may incorporate enzymes in the form of liquid or dried formulations. Dried formulations may be coated or uncoated and may be in granular form. The term "granules" is used for granules composed of enzymes (such as phytases, for example any engineered phytase peptides disclosed herein) and other chemicals (such as salts and sugars), and can be formed using any of a variety of techniques, including fluidized bed granulation methods to form layered granules.

[0044] The term phytase activity, as used in relation to determination in solid or liquid preparations, refers to 1 FTU (phytase unit), which is defined as the amount of enzyme required to release 1 micromolar of inorganic orthophosphate from a 5.0 mM sodium phytate substrate (from rice) within one minute under reaction conditions of 37°C and pH 5.5. This is also defined in ISO 2009 Phytase Assay (a standard assay for determining phytase activity, see International Standard ISO / DIS 30024: 1-17, 2009).

[0045] Alternatively, as used herein, one unit of phytase (U) can be defined as the amount of enzyme that releases 1 micromolar of inorganic orthophosphate from a 0.2 mM sodium phytate substrate (from rice) within one minute under reaction conditions of 25°C and pH 5.5 or 3.5, respectively, in a malachite green assay.

[0046] As used herein, the term "specific activity" is the number of enzyme units / ml divided by the (total) protein concentration in mg / ml. Therefore, specific activity values ​​are typically expressed in units / mg. Alternatively, "specific activity" is the number of enzyme units / ml divided by the phytase concentration in mg / ml.

[0047] As used herein, the term "differential scanning calorimetry" or "DSC" is a thermal analysis technique in which the difference in heat required to increase the temperature of a sample and a reference is measured as a function of temperature. Throughout the experiment, the sample and reference are maintained at nearly the same temperature. Typically, the temperature program for DSC analysis is designed such that the sample holder temperature increases linearly as a function of time. The reference sample should have a well-defined heat capacity over the temperature range to be scanned.

[0048] The term "probiotics" refers to indigestible food components that benefit the host by selectively stimulating the growth and / or activity of one or a limited number of beneficial bacteria.

[0049] As used herein, the term "direct-feed microorganisms" ("DFM") refers to a source of live (vibrant) microorganisms that, when applied in sufficient quantities, can confer benefits upon their recipient, namely probiotics. DFM can contain one or more such microorganisms, such as bacterial strains. Categories of DFM include Bacillus, lactic acid bacteria, and yeast. Therefore, the term DFM encompasses one or more of the following: direct-feeding bacteria, direct-feeding yeast, direct-feeding yeast, and combinations thereof.

[0050] Bacilli are unique, spore-forming Gram-positive bacilli. These spores are very stable and can withstand environmental conditions such as heat, moisture, and a certain pH range. When ingested by animals, these spores germinate into viable vegetative cells and can be used in powdered and pelleted diets. Lactic acid bacteria are Gram-positive cocci that produce lactic acid, which has antagonistic effects against pathogens. Because lactic acid bacteria appear to be somewhat heat-sensitive, they are not used in pelleted feeds. Types of lactic acid bacteria include *Bifidobacterium*, *Lactobacillus*, and *Streptococcus*.

[0051] The terms “probiotics,” “probiotic culture,” and “DFM” are used interchangeably herein and define live microorganisms (including, for example, bacteria or yeast) that, when ingested in adequate quantities or applied topically, beneficially influence a host organism (i.e., by conferring one or more proven health benefits such as health, digestion, and / or performance benefits) on the host organism. Probiotics can improve the microbial balance of one or more mucosal surfaces. For example, mucosal surfaces can be the intestines, urinary tract, respiratory tract, or skin. As used herein, the term “probiotics” also encompasses live microorganisms that can stimulate beneficial branches of the immune system while simultaneously reducing inflammatory responses in mucosal surfaces (e.g., the digestive tract). While there is no lower or upper limit to probiotic intake, it has been shown that at least 10 6 -10 12 Preferably at least 10 6 -10 10 Preferably 10 8 -10 9 CFU, when administered at a daily dose, will effectively achieve beneficial health effects in the subjects.

[0052] As used in this article, the term “CFU” means “colony-forming unit” and is a measure of viable cells, where a colony represents a collection of cells derived from a single progenitor cell.

[0053] The term “isolated” means a substance in a form or environment not found in nature and does not reflect the degree to which the isolated substance has been purified, but rather indicates separation or isolation from its natural form or natural environment. Non-limiting examples of isolated substances include (1) any substance not naturally occurring, (2) any substance removed at least partially from one or more of the naturally occurring components associated with it, including, but not limited to, any host cell, enzyme, engineered enzyme, nucleic acid, protein, peptide, or cofactor; (3) any substance artificially modified relative to a substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components associated with it naturally. The terms “isolated nucleic acid molecule,” “isolated polynucleotide,” and “isolated nucleic acid fragment” are used interchangeably and refer to a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or modified nucleotide bases. An isolated nucleic acid molecule in the form of a DNA polymer may consist of one or more fragments of cDNA, genomic DNA, or synthetic DNA.

[0054] The terms “purify,” “purified,” and “purification” mean the removal of unwanted components, material contaminants, mixtures, or defects to make the substance substantially pure or clean. For example, when applied to nucleic acids or peptides, purification typically means that the nucleic acid or peptide is substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified peptides or polynucleotides form discrete bands in electrophoresis gels, chromatographic eluents, and / or media subjected to density gradient centrifugation). For example, a nucleic acid or peptide that produces substantially one band in an electrophoresis gel is “purified.” Purified nucleic acids or peptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., based on molar weight percentages). In a relevant sense, a composition is enriched for a molecule when there is a significant increase in the concentration of that molecule after the application of purification or enrichment techniques. The term "enrichment" refers to the presence of a compound, peptide, cell, nucleic acid, amino acid, or other specified material or component in the composition at a relative or absolute concentration higher than that of the starting composition.

[0055] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acids linked together by peptide bonds. A “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues. Throughout this disclosure, single-letter and three-letter codes for amino acids are used in accordance with the definitions of the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC-IUB. The single-letter X refers to any one of the twenty amino acids. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. Mutations may be named by a single-letter code of the parental amino acid, followed by a position number, and then a single-letter code of the variant amino acid. For example, mutating glycine (G) at position 87 to serine (S) is represented as “G087S” or “G87S.” When describing modifications, a position is followed by an amino acid listed in parentheses, indicating that at that position, any of the listed amino acids can be substituted. For example, 6(L,I) means that position 6 can be substituted with either leucine or isoleucine. Sometimes, in a sequence, a forward slash ( / ) is used to define substitutions; for example, F / V indicates that the position may contain either phenylalanine or valine.

[0056] As used herein, the terms "corresponding to" or "corresponds to" or "correspond to" regarding amino acid residue positions refer to an amino acid residue at the listed position in a protein or peptide, or an amino acid residue that is similar to, homologous to, or equivalent to the listed residue in the protein or peptide. As used herein, "corresponding region" typically refers to a similar position in a related or reference protein.

[0057] The terms "derived from" and "obtained from" refer not only to proteins produced or potentially produced by strains of the organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences. Additionally, the term refers to proteins encoded by DNA sequences derived from synthetic and / or cDNA sources and possessing the identifying characteristics of the protein in question.

[0058] The term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide. The abbreviations used in this article to identify specific amino acids are shown in Table A.

[0059] Table A. Amino acid abbreviations (single letter and three letter)

[0060]

[0061] Those skilled in the art will recognize that the amino acid sequences disclosed herein can be modified while retaining the function associated with the disclosed amino acid sequences. For example, gene alterations that result in chemically equivalent amino acids at a given site without affecting the functional properties of the encoded protein are common, as is well known in the art.

[0062] The term "codon-optimized," when referring to the coding regions of genes or nucleic acid molecules used to transform various hosts, means altering the codons in the coding regions of genes or nucleic acid molecules to reflect the typical codon usage of the host organism, without changing the polypeptide encoded by the DNA.

[0063] As used herein, the term "transformation" refers to the transfer or introduction of nucleic acid molecules into a host organism. Nucleic acid molecules can be introduced as linear or circular DNA. The nucleic acid molecule can be a self-replicating plasmid or it can integrate into the genome of the producing host. A producing host containing transformed nucleic acid is referred to as a "transformed," "recombinant," or "transgenic" organism or "transformer."

[0064] The terms “recombination” and “engineered” refer to the artificial combination of two originally separate nucleic acid sequence fragments, for example, through chemical synthesis or through manipulation of isolated nucleic acid fragments using genetic engineering techniques. For example, this involves the natural or laboratory insertion of one or more fragments of DNA from different molecules, from another part of the same molecule, or an artificial sequence, resulting in the introduction of a new sequence into the gene and subsequently into the organism. The terms “recombination,” “transgenic,” “transformed,” “engineered,” “genetically engineered,” and “modified for the expression of exogenous genes” are used interchangeably herein.

[0065] The terms “recombinant construct,” “expression construct,” “recombinant expression construct,” and “expression cassette” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, such as regulatory and coding sequences that are not naturally occurring together. For example, a construct may contain regulatory and coding sequences derived from different sources or regulatory and coding sequences derived from the same source but arranged in a manner different from their natural counterparts. Such constructs may be used alone or in combination with a vector. If a vector is used, the choice of vector depends on the method to be used for transforming host cells, as is well known to those skilled in the art. For example, a plasmid vector may be used. Those skilled in the art will readily understand the genetic elements that must be present on the vector for successful transformation, selection, and propagation of host cells. Skilled technicians will also recognize that different independent transformation events can lead to different expression levels and patterns (Jones et al., (1985) EMBO J [Journal of the European Society for Molecular Biology] 4:2411-2418; De Almeida et al., (1989) Mol Gen Genetics [Molecular and General Genetics] 218:78-86), and therefore typically screen for multiple events to obtain strains exhibiting the desired expression levels and patterns. Such screening can be performed using standard molecular biology, biochemistry, and other assays, including Southern blotting of DNA, Northern blotting of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting of protein expression, enzyme or activity assays, and / or phenotypic analysis.

[0066] The terms “production host,” “host,” and “host cell” are used interchangeably herein and refer to any plant, organism, or cell of any plant or organism, whether human or non-human, into which a recombinant construct can be stably or transiently introduced to express genes. The term encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during reproduction.

[0067] The term "identity percentage" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing sequences. In the art, "identity" also means the degree of sequence correlation between polypeptide or polynucleotide sequences, which, as appropriate, is determined by the number of matching nucleotides or amino acids between such sequence strings. "Identity" and "similarity" can be readily calculated using known methods, including but not limited to those described in the following literature: Computational Molecular Biology (edited by Lesk, AM), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (edited by Smith, DW), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (edited by von Heinje, G.), Academic Press (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Devereux). (J. Editor) Stockton Press, New York (1991). Methods for determining identity and similarity have been incorporated into publicly available computer programs.

[0068] As used herein, “identity %” or “identity percentage” or “PID” refers to protein sequence identity. Percentage identity can be determined using standard techniques known in the art. Useful algorithms include the BLAST algorithm (see Altschul et al., J Mol Biol [Journal of Molecular Biology], 215:403-410, 1990; and Karlin and Altschul, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5873-5787, 1993). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most biologically relevant sequences, but is not recommended for query sequences with fewer than 20 residues (Altschul et al., NucleicAcids Res, 25:3389-3402, 1997; and Schaffer et al., NucleicAcids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searching include: adjacent word threshold = 11; E-value cutoff = 10; scoring matrix = NUC.3.1 (match = 1, mismatch = -3); gap open = 5; and gap extension penalty = 2. Exemplary default BLAST parameters for amino acid sequence searching include: word length = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap open = 11; and gap extension penalty = 1. The percentage (%) of amino acid sequence identity is determined by dividing the number of matching identical residues by the total number of residues in the "reference" sequence. The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0069] As used in this article, “homological protein” or “homological phytase” refers to proteins that have significant similarity in primary, secondary, and / or tertiary structures. When aligning proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology searches of protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST with a threshold (E-value cutoff) of 0.001. (Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI BLAST a new generation of protein databasesearch programs. Nucleic Acids Res [Nucleic Acids Research] 1997, Group 1;25(17):3389-402). Using this information, protein sequences can be grouped.

[0070] Sequence alignment and identity percentage calculations can be performed using the Megalign program of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, Wisconsin), the AlignX program of Vector NTI v. 7.0 (Informax Inc., Bethesda, Maryland), or the EMBOSS open software package (EMBL-EBI; Rice et al., Trends in Genetics 16, (6):276-277 (2000)). Multiple sequence alignments can be performed using the CLUSTAL alignment method (such as CLUSTALW; for example, version 1.83) (Higgins and Sharp, CABIOS [Bioinformatics], 5:151-153 (1989); Higgins et al., Nucleic Acids Res. [Nucleic Acids Research] 22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res. [Nucleic Acids Research] 31 (13):3497-500 (2003), obtained from the European Molecular Biology Laboratory via the European Institute for Bioinformatics) with default parameters. Suitable parameters for CLUSTALW protein alignment include vacancy presence penalty = 15, vacancy extension = 0.2, matrix = Gonnet (e.g., Gonnet250), protein ENDGAP = -1, protein GAPDIST = 4, and KTUPLE = 1. In one embodiment, fast or slow alignment uses the default settings for slow alignment. Alternatively, the parameters for using the CLUSTALW method (e.g., version 1.83) can be modified to use KTUPLE = 1, gap penalty = 10, gap extension = 1, matrix = BLOSUM (e.g., BLOSUM64), window = 5, and top storage diagonal = 5. Alternatively, multiple sequence alignments can be derived using MAFFT alignments from Geneious® version 10.2.4 with default settings, a score matrix of BLOSUM62, a gap opening penalty of 1.53, and an offset of 0.123.

[0071] The MUSCLE program (Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput [MUSCLE: High-precision and high-throughput multiple sequence alignment] Nucl. Acids Res. [Nucleic Acids Research] (2004) 32 (5): 1792-1797) is another example of a multiple sequence alignment algorithm.

[0072] The term "engineered phytase polypeptide" means that the polypeptide is not naturally occurring and has phytase activity.

[0073] Note that a fragment of an engineered phytase polypeptide is a part or subsequence of the engineered phytase polypeptide that can function like the engineered phytase polypeptide; that is, it retains phytase activity.

[0074] The term "vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, bacteriophage particles, expression cassettes, etc.

[0075] As used herein, an "expression vector" means a DNA construct containing a DNA sequence operably linked to appropriate control sequences capable of effecting DNA expression in a suitable host. Such control sequences can include a promoter that effects transcription, optional operator sequences that control transcription, sequences encoding appropriate ribosome binding sites on the mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0076] As used herein, the term "expression" refers to the production of a functional end product, either in precursor or mature form (e.g., mRNA or protein). Expression can also refer to the translation of mRNA into a polypeptide.

[0077] The expression of a gene involves the transcription of the gene and the translation of the mRNA into a precursor or mature protein. A "mature" protein is a polypeptide that has been post-translationally processed; that is, a polypeptide in which any signal sequence, prepeptide, or propeptide present in the primary translation product has been removed. A "precursor" protein is the primary product of mRNA translation; that is, the propeptide and prepeptide are still present. The propeptide and prepeptide can be, but are not limited to, intracellular localization signals. "Stable transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, including both the nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus of a host organism or into an organelle containing DNA, resulting in gene expression without integration or stable inheritance.

[0078] Therefore, in one embodiment, a recombinant construct is described that includes a regulatory sequence that functions in a production host, the regulatory sequence being operatively linked to a nucleotide sequence encoding an engineered phytase polypeptide and fragments thereof as described herein.

[0079] This recombinant construct may contain a regulatory sequence that functions in the production host, operatively linked to a nucleotide sequence encoding any engineered phytase polypeptide or fragment thereof described herein. Furthermore, the production host is selected from the group consisting of bacteria, fungi, yeast, plants, or algae. A preferred production host is the filamentous fungus *Trichoderma reesei*.

[0080] Alternatively, cell-free protein synthesis as described in Chong, Curr Protoc Mol Biol. [Latest Molecular Biology Experimental Methods] 2014; 108: 16.30.1–16.30.11 is possible.

[0081] This article also describes a method for producing engineered phytase peptides or fragments thereof, the method comprising:

[0082] (a) Transforming a production host using the recombinant constructs described herein; and

[0083] (b) Cultivate the production host of step (a) under conditions that produce engineered phytase peptides or fragments thereof.

[0084] Optionally, engineered phytase peptides or fragments thereof can be recovered from the production host.

[0085] On the other hand, a culture supernatant containing phytase can be obtained by any of the methods disclosed herein.

[0086] In another embodiment, a polynucleotide sequence encoding any of the engineered phytase polypeptides or fragments thereof as described herein is described.

[0087] Numerous and well-known in those skilled in the art, numerous start control regions or promoters may be included in the expression vector. A “constitutive promoter” is a promoter that is active under most environmental and developmental conditions. An “inducible” or “repressed” promoter is a promoter that is active under environmental or developmental regulation. In some embodiments, the promoter is inducible or repressed due to variations in environmental factors, including but not limited to availability of carbon, nitrogen, or other nutrients, temperature, pH, osmotic pressure, presence of heavy metals, concentration of inhibitors, pressure, or combinations thereof, as known in the art. In some embodiments, inducible or repressed promoters may be induced or repressed by metabolic factors, such as the levels of certain carbon sources, certain energy sources, certain catabolites, or combinations thereof, as known in the art.

[0088] In one embodiment, the promoter is a natural promoter of the host cell. For example, in some cases, when Trichoderma reesei is the host, the promoter can be a natural Trichoderma reesei promoter, such as the cbh1 promoter deposited in GenBank with accession number D86235. Other suitable non-restrictive examples of promoters that can be used for fungal expression include cbh2, egl1, egl2, egl3, egl4, egl5, xyn1 and xyn2, the repressed acid phosphatase gene (phoA) promoter of Penicillium chrysogenus (see, for example, Graessle et al., (1997) Appl. Environ. Microbiol. [Applied and Environmental Microbiology], 63:753-756), the glucose-repressed PCK1 promoter (see, for example, Leuker et al., (1997), Gene [Gene], 192:235-240), the maltose-inducible glucose-repressed MET3 promoter (see Liu et al., (2006), Eukary. Cell [Eukaryotic Cell], 5:638-649), the pKi promoter, and the cpc1 promoter. Other examples of useful promoters include promoters from the glucosylamylase genes of *Aspergillus awamori* and *Aspergillus niger* (see, for example, Nunberg et al., (1984) Mol. Cell Biol. [Molecular and Cell Biology] 154:2306-2315 and Boel et al., (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3:1581-1585). Furthermore, the promoter of the *Trichoderma reesei* xln1 gene may be useful (see, for example, EPA 137280A1).

[0089] The DNA fragment controlling transcription termination can also be derived from various genes native to the preferred production host cell. In some embodiments, including a termination control region is optional. In some embodiments, the expression vector includes a termination control region derived from the preferred host cell.

[0090] The terms “production host,” “production host cell,” “host cell,” and “host strain” are used interchangeably herein and refer to a suitable host containing a polynucleotide expression vector or DNA construct encoding a phytase polypeptide or a fragment thereof. The production host can be selected from a group consisting of bacteria, fungi, yeast, plants, and algae. Typically, the selection will depend on the gene encoding the engineered phytase polypeptide or a fragment thereof and its origin.

[0091] In particular, the host strain is preferably a filamentous fungal cell. In a preferred embodiment of the invention, "host cell" means a cell and protoplast produced by a filamentous fungal strain, and in particular by a Trichoderma or Aspergillus species.

[0092] The term "filamentous fungi" refers to all filamentous forms of the subphylum Eumycotina (see Alexopoulos, CJ (1962), INTRODUCTORY MYCOLOGY, Wiley, New York). These fungi are characterized by vegetative mycelia with cell walls composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of this invention differ from yeast in morphology, physiology, and genetics. Vegetative growth of filamentous fungi occurs through hyphal elongation, and carbon catabolism is obligate aerobic. In this invention, the filamentous fungal parent cells can be, but are not limited to, cells of the following species: *Trichoderma* (e.g., *Trichoderma longibrachiatum*, and now also known as *Hypocrea jecorina*), *Trichoderma viride*, *Trichoderma koningii*, *Trichoderma harzianum*); species of *Penicillium*, species of *Humicola* (e.g., *Humicola insolens* and *Humicola grisea*), species of *Chrysosporium* (e.g., *C. lucknowense*), species of *Gliocladium*, and species of *Aspergillus* (e.g., *Aspergillus oryzae*). oryzae), Aspergillus niger and Aspergillus avocado), species of Fusarium, Neurospora, Hypocrea and Emericella (see also Innis et al., (1985) Sci. [Science] 228:21–26).

[0093] As used herein, the term “Trichoderma” or “Trichoderma species” refers to any genus of fungi that was previously or currently classified as Trichoderma.

[0094] Expression cassettes can be incorporated into a production host, particularly the cells of a microbial production host. The production host cell can be a microbial host found in the fungal family and can grow under a wide range of temperature, pH, and solvent tolerance conditions. For example, any of bacteria, yeast, plants, algae, or fungi (such as filamentous fungi) is expected to be a suitable host for the expression vector.

[0095] Including expression cassettes in the host cell allows for the expression of target proteins, enabling them to be located intracellularly, extracellularly, or a combination of both. Extracellular expression makes it easier to recover the desired protein from fermentation products compared to methods used for recovering proteins produced by intracellular expression.

[0096] Methods for converting nucleic acids into filamentous fungi (such as Aspergillus species, for example Aspergillus oryzae or Aspergillus niger, Aspergillus oryzae, Aspergillus terreus, and Trichoderma reesei) are well known in the art. Suitable procedures for converting Aspergillus host cells are described, for example, in EP238023.

[0097] The appropriate procedure for transforming Trichoderma host cells is described, for example, in Steiger et al. 2011, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 77:114-121. DNA uptake into the host Trichoderma species depends on the calcium ion concentration. Typically, about 10 mM CaCl2 to 50 mM CaCl2 is used in the uptake solution. In addition to the need for calcium ions in the uptake solution, other compounds typically included are buffer systems such as TE buffer (10 M mTris, pH 7.4; 1 mM EDTA) or 10 mM MOPS (pH 6.0) buffer (morpholinopropanesulfonic acid) and polyethylene glycol (PEG). PEG is believed to act as a fusion cell membrane, allowing the contents of the culture medium to be delivered into the cytoplasm of the Trichoderma species and to transfer plasmid DNA into the nucleus. This fusion often leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[0098] Typically, conversions using the term "10" are employed. 5 Up to 10 7 / mL, preferably 2 × 10 6 A suspension of Trichoderma species protoplasts or cells subjected to osmotic treatment at a density of / mL. 100 μL of these protoplasts or cells are mixed with the desired DNA in a suitable solution (e.g., 1.2 M sorbitol; 50 mM CaCl2). Typically, a high concentration of PEG is added to the uptake solution. 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. However, adding approximately 0.25 volume is preferred. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc., can also be added to the uptake solution to aid transformation. Similar procedures can be used for other fungal host cells. (See, for example, U.S. Patent Nos. 6,022,725 and 6,268,328, both of which are incorporated herein by reference).

[0099] Preferably, a genetically stable transformant is constructed using a vector system, thereby stably integrating the nucleic acid encoding a phytase polypeptide or a fragment thereof into the host strain chromosome. The transformant is then purified using known techniques.

[0100] After the expression vector is introduced into the cell, the transfected or transformed cells are cultured under conditions that are conducive to gene expression under the control of the promoter sequence.

[0101] Typically, cells are cultured in standard media containing physiological salts and nutrients (see, for example, Pourquie, J. et al., BIOCHEMISTRY AND GENETICS OF CELLULOSE DEGRADATION, edited by Aubert, JP et al., Academic Press, pp. 71–86, 1988; and Ilmen, M. et al., (1997) Appl. Environ. Microbiol. 63:1298–1306). Commonly prepared commercial media (e.g., yeast malt extract (YM) broth, Luria Bertani (LB) broth, and Sabouraud Dextrose (SD) broth) can also be used in this invention.

[0102] The culture conditions are also standard (e.g., incubating the culture at approximately 28°C in a suitable medium in a shaking culture or fermenter until the desired phytase expression level is reached). Preferred culture conditions for a given filamentous fungus are known in the art and can be found in scientific literature and / or from fungal sources such as the American Type Culture Collection and the Fungal Genetics Stock Center.

[0103] After fungal growth has been established, cells are exposed to conditions that effectively induce or allow phytase expression, particularly as defined herein. In cases where the phytase coding sequence is under the control of an inducible promoter, an inducer (e.g., sugar, metal salt, or antimicrobial agent) is added to the culture medium at a concentration that effectively induces phytase expression. Engineered phytase peptides or fragments thereof secreted from host cells can be used as whole-broth formulations requiring minimal post-production processing.

[0104] The preparation of used whole fermentation broth from recombinant microorganisms can be achieved using any culture method known in the art, thereby resulting in the expression of engineered phytase peptides or fragments thereof.

[0105] The term "used whole fermentation broth" is defined herein as the ungraded contents of fermentation material comprising culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It should be understood that the term "used whole fermentation broth" also encompasses cellular biomass that has been lysed or permeated using methods well known in the art.

[0106] After fermentation, the fermentation broth is obtained. Microbial cells and various suspended solids (including residual crude fermentation material) are removed using conventional separation techniques to obtain a phytase solution. Commonly used methods include filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, post-centrifugation ultrafiltration, extraction, or chromatography.

[0107] It is possible to optionally recover the desired protein from the production host. Alternatively, a culture supernatant containing an engineered phytase polypeptide or a fragment thereof can be obtained using any method known to those skilled in the art.

[0108] Examples of these techniques include, but are not limited to, affinity chromatography (Tilbeurgh et al., (1984) FEBS Lett. [European Federation of Biochemical Societies Letters] 16:215), ion exchange chromatography (Goyal et al., (1991) Biores. Technol. [Bioresource Technology] 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol. [European Journal of Applied Microbiology and Biotechnology] 17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. [Journal of Applied Biochemistry] 6:336; and Ellouz et al., (1987) Chromatography [Chromatography] 396:307), including ion exchange using materials with high-resolution capabilities (Medve et al., (1998) J. Chromatography A [Chromatography Journal A]). Chromatography can be performed using various methods, including: (808:153), hydrophobic interaction chromatography (see Tomaz and Queiroz, (1999) J. Chromatography A [Chromatography Journal A] 865:123); two-phase partitioning (see Brumbauer et al., (1999) Bioseparation [Bioseparation] 7:287); ethanol precipitation; reversed-phase HPLC, silica gel or anion exchange resin chromatography such as DEAE, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration (e.g., Sephadex G-75). The desired degree of purification will vary depending on the intended use of the engineered phytase peptide or fragment thereof. In some embodiments, purification will not be necessary.

[0109] On the other hand, it may be necessary to concentrate the solution containing engineered phytase peptides or fragments thereof to optimize recovery. Using an unconcentrated solution requires increased incubation time to collect the enriched or purified enzyme precipitate. The enzyme-containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme-containing solution can be achieved using any of the techniques discussed herein. Exemplary methods for enrichment and purification include, but are not limited to, rotary vacuum filtration and / or ultrafiltration.

[0110] Alternatively, precipitants such as metal halide precipitants can be used to concentrate the desired protein product. Sodium chloride, a metal halide precipitant, can also be used as a preservative. Metal halide precipitants are used in amounts that effectively precipitate engineered phytase peptides or fragments thereof. After routine testing, selecting at least an effective and optimal amount of metal halide to effectively induce enzyme precipitation, and the precipitation conditions used to maximize recovery, including incubation time, pH, temperature, and enzyme concentration, will be apparent to those skilled in the art. Typically, at least about 5% w / v (weight / volume) to about 25% w / v of metal halide is added to the concentrated enzyme solution, and typically at least 8% w / v.

[0111] Another alternative to precipitating enzymes is the use of organic compounds. Exemplary organic compound precipitants include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of the organic compound precipitant can be performed before, simultaneously with, or after the addition of the metal halide precipitant, and the addition of the two precipitants, the organic compound, and the metal halide can be performed sequentially or simultaneously. Typically, the organic precipitant is selected from the group consisting of: alkali metal salts of 4-hydroxybenzoic acid (such as sodium or potassium salts) and straight-chain or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 12 carbon atoms, and blends of two or more of these organic compounds. Other organic compounds include, but are not limited to, methyl 4-hydroxybenzoate (referred to as methyl p-hydroxybenzoate) and propyl 4-hydroxybenzoate (referred to as propyl p-hydroxybenzoate). For further description, see, for example, U.S. Patent No. 5,281,526. The addition of organic precipitants offers the advantage of high flexibility in precipitation conditions regarding pH, temperature, concentration, precipitant type, protein concentration, and incubation time. Typically, at least about 0.01% w / v and no more than about 0.3% w / v of an organic precipitant is added to concentrated enzyme solutions.

[0112] After the incubation period, the enriched or purified enzyme is then separated from the dissociated pigments and other impurities and collected using conventional separation techniques such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, pressure filtration, cross-flow membrane microfiltration, and cross-current membrane microfiltration. Further enrichment or purification of the enzyme precipitate can be achieved by washing the precipitate with water. For example, the enriched or purified enzyme precipitate can be washed with water containing a metal halide precipitant or with water containing both metal halides and organic compounds as precipitants.

[0113] Sometimes it is advantageous to delete a gene from the expression host, where the gene defect can be cured by the expression vector. In cases where it is necessary to obtain fungal host cells with one or more inactivated genes, known methods can be used (e.g., methods disclosed in U.S. Patent Nos. 5,246,853, 5,475,101, and WO 92 / 06209). Gene inactivation can be accomplished by complete or partial deletion, inactivation by insertion, or by any other means that renders the gene ineffective for its intended purpose (such as preventing the expression of a functional protein).

[0114] Any cloned gene from Trichoderma species or other filamentous fungal hosts can be deleted, such as the cbh1, cbh2, egl1, and egl2 genes. In some embodiments, gene deletion can be accomplished by inserting the desired gene to be inactivated into a plasmid using methods known in the art. The deletion plasmid is then cut at appropriate restriction enzyme sites within the desired gene coding region, and the gene coding sequence or a portion thereof is replaced with a selective marker. Flanking DNA sequences (preferably between about 0.5 and 2.0 kb) from the locus of the gene to be deleted are retained on either side of the marker gene. A suitable deletion plasmid will typically have unique restriction enzyme sites present therein, allowing the fragment containing the deleted gene, flanking DNA sequences, and selective marker genes to be removed as a single linear slice.

[0115] Depending on the host cell used, post-transcriptional and / or post-translational modifications can occur. A non-limiting example of post-transcriptional and / or post-translational modifications is the "splicing" or "truncation" of a polypeptide. In another case, such splicing can result in the production of a mature phytase polypeptide and further removal of the N- or C-terminal amino acid to produce a truncated form of phytase that retains its enzymatic activity.

[0116] Other examples of post-transcriptional or post-translational modifications include, but are not limited to, myristylation, glycosylation, truncation, lipidation, and phosphorylation of tyrosine, serine, or threonine. Those skilled in the art will recognize that the type of post-transcriptional or post-translational modifications a protein may undergo can vary depending on the host organism expressing the protein.

[0117] Peptides may undergo further sequence modifications after expression. These include, but are not limited to, oxidation, deglycosylation, and glycosylation. Glycosylation is known to affect phytase activity when incubated with glucose or other reducing sugars (especially at temperatures above 30°C and under neutral or alkaline pH conditions). Protein engineering that removes lysine residues can be used to prevent such modifications. Examples of this can be found in US 8,507,240. For example, yeast expression can produce highly glycosylated peptides, resulting in a significant increase in molecular weight. Additionally, WO 2013 / 119470 (incorporated herein by reference), internationally published on August 15, 2013, relates to phytases with increased stability (believed to be due to increased glycosylation).

[0118] As used herein, the term "glycosylation" refers to the attachment of a glycan to a molecule such as a protein. Glycosylation can be an enzymatic reaction. Attachment can be formed via covalent bonds. The phrase "highly glycosylated" means that a molecule (such as an enzyme) is glycosylated at many sites and at all or almost all available glycosylation sites (e.g., N-linked glycosylation sites). Alternatively or in addition, the phrase "highly glycosylated" can refer to extensive glycolytic branching at all or substantially all N-linked glycosylation sites (e.g., the size and number of glycolytic moieties associated with a particular N-linked glycosylation site). In some embodiments, engineered phytase peptides are glycosylated at all or substantially all consensual sequence N-linked glycosylation sites (i.e., NXS / T consensual sequence N-linked glycosylation sites).

[0119] As used herein, the term "glycan" refers to the carbohydrate portion of a polysaccharide, oligosaccharide, or glycoconjugate (such as a glycoprotein). Glycans can be homopolymers or heteropolymers of monosaccharide residues. They can be linear or branched molecules.

[0120] Phytases can undergo glycosylation to varying degrees. Such glycosylation is known to improve stability during storage and application. (Extensive...)

[0121] The activity of any of the engineered phytase peptides or fragments disclosed herein can be determined as discussed above.

[0122] It is believed that applying robust, engineered phytase peptides or fragments thereof to feed in liquid form is advantageous compared to applying such phytases in coated pellet form. Coated pellets are currently the commercial method for manufacturing phytase products suitable for high-temperature conditioning and granulation. The benefits of liquid application of robust enzymes include: 1) the enzyme will begin to function immediately after animal ingestion, as it does not need to be released from the coated pellet before interacting with the feed; 2) improved distribution of the enzyme throughout the feed, thus ensuring more consistent delivery of the enzyme to the animal, which is particularly important for young animals consuming small amounts of feed; 3) uniform distribution in the feed makes it easier to measure the enzyme in the feed; and 4) in the case of robust phytases (such as the engineered phytase peptides and fragments disclosed herein), it can begin to degrade phytates already present in the feed.

[0123] In other words, the novel engineered phytase peptides and their fragments are so robust that no special coatings or formulations are required before conditioning and pelleting for their application in feed, as they have been engineered to withstand the stresses of conditioning and pelleting used in industrial feed production. Therefore, the robustness of the novel engineered phytase peptides and their fragments described herein allows them to be applied in uncoated granule or particle form, or in an uncoated and unprotected form when placed in a liquid.

[0124] It should be noted that engineered phytase peptides and their fragments can be inexpensively formulated on solid carriers without particularly requiring a protective coating, and remain active throughout conditioning and granulation. When certain areas where harsh conditions are used require, or if conditions permit (e.g., in the case of over-conditioned feeds above 90°C), a protective coating that provides additional thermal stability when applied in solid form may be beneficial for achieving granulation stability.

[0125] The disclosed engineered phytase peptides or fragments thereof are derived using a combination of methods and techniques known in the field of protein engineering, including phylogenetic analysis, site evaluation libraries, combinatorial libraries, high-throughput screening, and statistical analysis.

[0126] In one aspect, this disclosure relates to an engineered phytase polypeptide or a fragment thereof that also has at least 82% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0127] Those skilled in the art will understand that such at least 82% sequence identity also includes 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0128] Those skilled in the art will understand that such at least 79% sequence identity also includes 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0129] The following may also be mentioned, as they are provided in some embodiments:

[0130] a) An engineered phytase polypeptide or fragment thereof having at least 81% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO: 2, 3, 8, 10, 12, 18, 19, 24, 26, 27, 28, 30, 31, 32, 33, and / or 36.

[0131] b) An engineered phytase polypeptide or fragment thereof having at least 82% (e.g., 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequences of SEQ ID NO: 1, 4, 5, 7, 9, 11, 14, 15, 17, 21, 25, 34, and / or 35;

[0132] c) An engineered phytase polypeptide or fragment thereof that also has at least 83% (e.g., 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequence of SEQ ID NO:13.

[0133] d) An engineered phytase polypeptide or fragment thereof having at least 79% (e.g., 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequences of SEQ ID NO:6 and / or 22; and / or

[0134] e) An engineered phytase polypeptide or fragment thereof having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with the amino acid sequences of SEQ ID NO: 16, 20, 23, 29, and / or 37.

[0135] In another aspect, the polypeptide comprises either a core domain of an engineered phytase polypeptide or a fragment of an engineered phytase polypeptide core domain. A “core domain fragment” is defined herein as a polypeptide lacking one or more amino acids from its amino and / or carboxyl termini. As used herein, the phrase “core domain” refers to a polypeptide region encompassing the amino acids essential for maintaining the structure and function of the polypeptide, such as phytate hydrolysis. The amino acids in the core domain may be further modified to improve thermal stability or catalytic activity under various conditions, such as, but not limited to, pH. In some non-limiting embodiments, the core domain of the engineered phytase polypeptide or fragment thereof disclosed herein corresponds to amino acid positions 14-325 of SEQ ID NO:1. In other non-limiting embodiments, the core domain corresponds to amino acid positions 13-326, 12-327, 11-328, 10-329, 9-330, 8-331, 7-332, 6-333, 5-334, 4-335, 3-336, 2-337, or 1-338 of SEQ ID NO:1. In other embodiments, the N-terminus of the core domain corresponds to amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of SEQ ID NO:1, and the C-terminus of the core domain corresponds to SEQ ID NO:1. The amino acid positions of NO:1 are 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 3 63, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, or 413.

[0136] Therefore, this article also provides:

[0137] f) An engineered phytase polypeptide or a fragment of its core domain having at least 78% (e.g., 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 14-325 of SEQ ID NO:6, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1;

[0138] g) An engineered phytase polypeptide or a fragment of its core domain having at least 79% (e.g., 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 14-325 of SEQ ID NO:2, 8, 27, and / or 37, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1;

[0139] h) An engineered phytase polypeptide or a fragment of its core domain having at least 81% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 14-325 of SEQ ID NO:3, 10, 12, 18, 25, 26, 28, 30, 32, and / or 35, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1;

[0140] i) An engineered phytase polypeptide or a fragment of its core domain having at least 82% (e.g., 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 14-325 of SEQ ID NO:1, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1;

[0141] j) An engineered phytase polypeptide or a fragment of its core domain having at least 83% (e.g., 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 14-325 of SEQ ID NO:19, 20, 23, and / or SEQ ID NO:24, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1; and / or

[0142] k) An engineered phytase polypeptide or a fragment of its core domain having at least 84% (e.g., 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with amino acids 14-325 of SEQ ID NO:29, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1.

[0143] On the other hand, any of the engineered peptides or fragments disclosed herein have a specific activity of at least about 100 U / mg at pH 3.5. Specific activity ranges (U / mg, at pH 3.5) include, but are not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.

[0144] On the other hand, some engineered peptides or fragments thereof disclosed herein exhibit a specific activity of at least about 100 U / mg at pH 5.5. Specific activity ranges (U / mg, at pH 5.5) include, but are not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.

[0145] On the other hand, any of the engineered phytase peptides or fragments thereof disclosed herein are stable in liquid form at a pH of about 3.0 or lower. This is particularly relevant when the engineered phytase peptides or fragments thereof described herein pass through the animal digestive tract, as discussed below.

[0146] In another embodiment, a trace mineral-free diet comprising any of the engineered phytase peptides or fragments thereof described herein is described.

[0147] Importantly, feed additive enzymes (such as phytase) undergo very harsh conditions as they pass through the animal digestive tract, namely low pH and the presence of digestive enzymes. Pepsin is one of the most important proteolytic digestive enzymes present in the gastrointestinal tract of monogastric animals. Pepsin exhibits low specificity and high pH tolerance in acidic regions (stable from pH 1.5–6.0 up to pH 8.0). The engineered phytase peptides or fragments thereof described herein are largely resistant to pepsin, and this tolerance is essential for good in vivo performance.

[0148] A trace mineral-free diet containing any of the engineered phytase peptides or fragments thereof described herein may be used (i) alone or (ii) in combination with a direct-feeding microorganism containing at least one bacterial strain or (iii) in combination with at least one other enzyme or (iv) in combination with a direct-feeding microorganism containing at least one bacterial strain and at least one other enzyme, or (v) any of (i), (ii), (iii) or (iv) further comprising at least one other feed additive component, and optionally, the engineered phytase peptides or fragments thereof are used at a concentration of at least 0.1 g / tonne of feed (such as at least about 0.1 g / tonne, 0.2 g / tonne, 0.3 g / tonne, 0.4 g / tonne, 0.5 g / tonne, 0.6 g / tonne, 0.7 g / tonne, 0.8 g / tonne, 0.9 g / tonne, 1 g / tonne, 1.1 g / tonne, 1.2 g / tonne, 1.3 g / tonne, 1.4 g / tonne, 1.5 g / tonne, 1.6 g / tonne, etc.). The amounts present are 1.7 g / ton, 1.8 g / ton, 1.9 g / ton, 2 g / ton, 2.1 g / ton, 2.2 g / ton, 2.3 g / ton, 2.4 g / ton, 2.5 g / ton, 2.6 g / ton, 2.7 g / ton, 2.8 g / ton, 2.9 g / ton, 3 g / ton or more.

[0149] In some non-limiting embodiments, phytase is present in the diet in the range of about 200 FTU / kg to about 1000 FTU / kg feed, more preferably about 300 FTU / kg feed to about 750 FTU / kg feed, and even more preferably about 400 FTU / kg feed to about 500 FTU / kg feed. In one embodiment, phytase is present in the feed in an amount exceeding about 200 FTU / kg feed, suitably exceeding about 300 FTU / kg feed, and suitably exceeding about 400 FTU / kg feed. In one embodiment, phytase is present in the feed in an amount less than about 1000 FTU / kg feed, and suitably less than about 750 FTU / kg feed. In some embodiments, phytase is present in the feed additive composition in an amount ranging from about 40 FTU / g to about 40,000 FTU / g of the composition, more preferably from about 80 FTU / g of the composition to about 20,000 FTU / g of the composition, and even more preferably from about 100 FTU / g of the composition to about 10,000 FTU / g of the composition, and even more preferably from about 200 FTU / g of the composition to about 10,000 FTU / g of the composition. In one embodiment, phytase is present in the feed additive composition in an amount exceeding about 40 FTU / g of the composition, suitably exceeding about 60 FTU / g of the composition, suitably exceeding about 100 FTU / g of the composition, suitably exceeding about 150 FTU / g of the composition, and suitably exceeding about 200 FTU / g of the composition. In one embodiment, phytase is present in the feed additive composition in an amount of less than about 40,000 FTU / g of the composition, suitably less than about 20,000 FTU / g of the composition, suitably less than about 15,000 FTU / g of the composition, and suitably less than about 10,000 FTU / g of the composition.

[0150] In some non-limiting embodiments, “1 FTU” (phytase unit) is defined as the amount of enzyme required to release 1 μmol of inorganic orthophosphate from the substrate within one minute under the reaction conditions defined in ISO 2009 Phytase Assay (a standard assay for determining phytase activity), and 1 FTU is found in International Standard ISO / DIS 30024:1-17, 2009. In one embodiment, the enzyme is classified using the EC classification method described above, and the EC classification method specifies the enzyme that has the activity when tested in the assay for determining 1 FTU taught herein.

[0151] The terms “feed additives,” “feed additive components,” and / or “feed additive ingredients” are used interchangeably in this document.

[0152] Feed additives can be described as products used for animal nutrition with the aim of improving feed quality and the quality of animal-derived food or improving animal performance and health, such as by providing enhanced digestibility of feed materials.

[0153] Feed additives are categorized into several types, such as sensory additives, which stimulate animals' appetite and naturally encourage them to eat more; nutritional additives, which provide specific nutrients that may be lacking in animal diets; and animal husbandry additives, which enhance the overall nutritional value of animal diets through additives in the feed.

[0154] As used in this article, "diet free of trace minerals" means a diet that does not contain or is substantially free of trace minerals, such as those added exogenously as feed additives.

[0155] Examples of such feed additives include, but are not limited to, probiotics, essential oils (such as, but not limited to, thymol and / or cinnamaldehyde), fatty acids, short-chain fatty acids (such as propionic acid and butyric acid), vitamins, minerals, amino acids, etc.

[0156] Feed additive compositions or formulations may also contain at least one component selected from the group consisting of: protein, peptide, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben, and propylparaben.

[0157] At least one other enzyme (i.e., any of the engineered phytase polypeptides or fragments thereof disclosed herein) may be included in the feed additive compositions or formulations disclosed herein, which may include, but are not limited to, xylanase, amylase, another phytase, β-glucanase and / or protease.

[0158] Xylanases are a class of enzymes that degrade the linear polysaccharide β-1,4-xylan into xylose, thereby breaking down hemicellulose (a major component of plant cell walls). Xylanases, such as endo-β-xylanase (EC 3.2.1.8), hydrolyze the xylan backbone.

[0159] In one embodiment, the xylanase can be any commercially available xylanase. Suitably, the xylanase can be an endo-1,4-Pd-xylanase (classified as EG3.2.1.8) or a 1,4β-xylosidase (classified as EG3.2.1.37). In one embodiment, this disclosure relates to a composition comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein, combined with a xylanase (e.g., endo-1,4-Pd-xylanase) and another enzyme. All EC enzyme classifications mentioned herein refer to the classifications provided in the Enzyme Nomenclature-Recommendations (1992) - ISBN 0-12-226164-3 of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, which are incorporated herein.

[0160] In another embodiment, the xylanase may be a xylanase derived from the genera *Bacillus*, *Trichoderma*, *Thermomyces*, *Aspergillus*, *Pythium*, and *Penicillium*. In yet another embodiment, the xylanase may be a xylanase from Axtra XAP® or Avizyme 1502®, both commercially available from Danisco A / S. In one embodiment, the xylanase may be a mixture of two or more xylanases. In yet another embodiment, the xylanase is an endo-1,4-β-xylanase or a 1,4-β-xylosidase.

[0161] In one embodiment, this disclosure relates to a trace mineral-free diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and xylanase. In one embodiment, the trace mineral-free diet comprises a composition of 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 xylanase units / g.

[0162] In one embodiment, the trace mineral-free diet comprises a composition of 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, and greater than 8000 xylanase units / g.

[0163] It should be understood that one xylanase unit (XU) is the amount of enzyme that releases 0.5 µmol of reducing sugar equivalent (xylose, by the dinitrosalicylic acid (DNS) assay – reducing sugar method) per minute from oat-spelt-xylan substrate at pH 5.3 and 50°C (Bailey et al., Journal of Biotechnology, Vol. 23, (3), May 1992, 257-270).

[0164] Amylases are a class of enzymes that hydrolyze starch into shorter-chain oligosaccharides, such as maltose. The glucose moiety is then more readily transferred from maltose to monoglycerides or glycosyl monoglycerides compared to the original starch molecule. The term amylase includes α-amylase (EG3.2.1.1), G4-forming amylase (EG3.2.1.60), β-amylase (EG3.2.1.2), and γ-amylase (EC3.2.1.3). Amylases can be of bacterial or fungal origin, chemically modified, or protein-engineered mutants.

[0165] In one embodiment, the amylase may be a mixture of two or more amylases. In another embodiment, the amylase may be an amylase (e.g., α-amylase) from Bacillus licheniformis and an amylase (e.g., α-amylase) from Bacillus amyloliquefaciens. In one embodiment, the α-amylase may be an α-amylase from Axtra XAP® or Avizyme 1502®, both of which are commercially available from Danisco. In yet another embodiment, the amylase may be a pepsin-resistant α-amylase, such as a pepsin-resistant Trichoderma (e.g., Trichoderma reesei) α-amylase. Suitable pepsin-resistant α-amylases are taught in UK Application No. 101 1513.7 (which is incorporated herein by reference) and PCT / IB2011 / 053018 (which is incorporated herein by reference).

[0166] It should be understood that one amylase unit (AU) is the amount of enzyme that releases 1 mmol of glycosidic bonds per minute from a water-insoluble cross-linked starch polymer substrate at pH 6.5 and 37°C (this may be referred to herein as the assay for determining 1 AU).

[0167] In one embodiment, this disclosure relates to a trace mineral-free diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein and amylase. In one embodiment, this disclosure relates to a trace mineral-free diet comprising any of the engineered phytase polypeptides or fragments thereof disclosed herein, xylanase, and amylase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 amylase units / g of composition.

[0168] In one embodiment, the trace mineral-free diet contains 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500- Compositions with amylase units / g of 7000, 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000.

[0169] As used herein, the term protease is synonymous with peptidase or proteinase. A protease may be a subtilisin (EG3.4.21.62), a bacillolysin (EG3.4.24.28), an alkaline serine protease (EG3.4.21.x), or a keratinase (EG3.4.XX). In one embodiment, the protease is a subtilisin. Suitable proteases include those of animal, plant, or microbial origin.

[0170] Chemically modified or protein-engineered mutants are also suitable. The protease can be a serine protease or a metalloproteinase. For example, an alkaline microbial protease or a trypsin-like protease. In one embodiment, this document provides a composition comprising any one or more of the engineered phytase peptides or fragments thereof disclosed herein, and one or more proteases.

[0171] Examples of alkaline proteases are subtilisinases, especially those derived from Bacillus species, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, for example, U.S. Patent No. 6,287,841), subtilisin 147, and subtilisin 168 (see, for example, WO 89 / 06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and Fusarium proteases (see, for example, WO 89 / 06270 and WO 94 / 25583). Examples of useful proteases also include, but are not limited to, the variants described in WO 92 / 19729 and WO 98 / 20115.

[0172] In one embodiment, the protease is selected from the group consisting of: subtilisin, subtilis metalloproteinase, basic serine protease, keratinase, and Nocardiopsis protease.

[0173] It should be understood that one protease unit (PU) is the amount of enzyme that releases one microgram of a phenolic compound (expressed as a tyrosine equivalent) from a substrate (0.6% casein solution) within one minute at pH 7.5 (40 mM Na2PO4 / lactate buffer) and 40°C. This can be referred to as the assay used to determine 1 PU.

[0174] In one embodiment, this disclosure relates to a trace mineral-free diet comprising any of the engineered phytase peptides or fragments thereof disclosed herein, and a protease. In another embodiment, this disclosure relates to a trace mineral-free diet comprising any of the engineered phytase peptides or fragments thereof disclosed herein, as well as xylanase and a protease. In yet another embodiment, this disclosure relates to a trace mineral-free diet comprising any of the engineered phytase peptides or fragments thereof disclosed herein, as well as amylase and a protease. In yet another embodiment, this disclosure relates to a trace mineral-free diet comprising any of the engineered phytase peptides or fragments thereof disclosed herein, as well as xylanase, amylase, and a protease.

[0175] In one embodiment, the trace mineral-free diet contains about 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 protease units / g composition.

[0176] In one embodiment, the trace mineral-free diet contains approximately 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500- Compositions with 7000, 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000 protease units / g.

[0177] In other embodiments, the diet may have reduced (e.g., significantly reduced) trace mineral levels compared to those recommended by the National Research Council (NRC) or broiler farmers. In some embodiments, the diet contains trace mineral levels between 0.05% and 75% of the levels recommended by the National Research Council (NRC) or broiler farmers, for example, approximately 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, and 17% of the levels recommended by the National Research Council (NRC) or broiler farmers (e.g., the recommended levels discussed in the Examples section below). The trace mineral levels are any one of the following: 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%. In some embodiments, the trace minerals are one or more of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and / or selenium (Se).

[0178] At least one direct-feed microorganism (DFM) may contain at least one viable microorganism, such as a viable bacterial strain, viable yeast, or viable fungus. Preferably, the DFM contains at least one viable bacterium.

[0179] It is possible that DFM can be a spore-forming bacterial strain, therefore the term DFM can consist of or contain spores, such as bacterial spores. Thus, as used herein, the term "viable microorganism" can include microbial spores, such as endospores or conidia. Alternatively, the DFM in the feed additive compositions described herein may not consist of or contain microbial spores, such as endospores or conidia.

[0180] Microorganisms can be naturally occurring microorganisms or transformed microorganisms.

[0181] As described herein, a DFM may comprise microorganisms from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera, and combinations thereof.

[0182] Preferably, the DFM comprises one or more bacterial strains selected from the following Bacillus species: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilis, and Bacillus amyloliquefaciens.

[0183] As used herein, “Bacillus” includes all species within the genus Bacillus as known to those skilled in the art, including but not limited to Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus gibsonii, Bacillus thuringiensis, and Bacillus thuringiensis. It should be recognized that the genus Bacillus continues to undergo taxonomic recombination. Therefore, this genus aims to include species that have been reclassified, including but not limited to organisms such as Bacillus stearothermophilus (now named Geobacillus stearothermophilus) or Bacillus polymyxa (now named Paenibacillus polymyxa). The production of resistant endospores under stress conditions is considered a defining characteristic of the genus *Bacillus*, although this characteristic also applies to recently named genera such as *Alicyclobacillus*, *Amphibacillus*, *Aneurinibacillus*, *Anoxybacillus*, *Brevibacillus*, *Filobacillus*, *Gracilibacillus*, *Halobacillus*, *Paenibacillus*, *Salibacillus*, *Thermobacillus*, *Ureibacillus*, and *Virgibacillus*.

[0184] On the other hand, DFM can be further combined with the following Lactococcus species: Lactococcus cremoris and Lactococcus lactis, and combinations thereof.

[0185] DFM can be further combined with the following Lactobacillus species: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillus reuteri, and Lactobacillus fermentum. Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus sausage, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, and Lactobacillus jensenii, and any combination thereof.

[0186] On the other hand, DFM can be further combined with the following Bifidobacteria species: Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudo-small chain, Bifidobacterium adolescentis, and Bifidobacterium angulatum, and any combination thereof.

[0187] The following bacterial species may be mentioned: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pumilus, Enterococcus, species of Enterococcus and Pediococcus, species of Lactobacillus, species of Bifidobacterium, Lactobacillus acidophilus, Pediococs acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Bacillus subtilis, Propionibacterium thoenii, Lactobacillus sausageii, Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius ssp. Salivarius, species of Propionibacteria sp., and combinations thereof.

[0188] The direct-feeding microorganisms described herein, which contain one or more bacterial strains, may be of the same type (genus, species, and strain), or may contain a mixture of genera, species, and / or strains.

[0189] Alternatively, DFM may be combined with one or more products disclosed in WO 2012110778 or the microorganisms contained in these products, and is summarized as follows: Bacillus spp. strain 2084 Registry No. NRRLB-50013, Bacillus spp. strain LSSAO1 Registry No. NRRLB-50104, and Bacillus spp. strain 15A-P4 ATCC Registry No. PTA-6507 (from EnvivaPro® (formerly known as Avicorr®)); Bacillus subtilis strain C3102 (from Calsporin®); Bacillus subtilis strain PB6 (from Clostat®); Bacillus pumilus (8G-134); Enterococcus spp. NCIMB 10415 (SF68) (from Cylactin®); Bacillus subtilis strain C3102 (from Gallipro® and GalliproMax®); Bacillus licheniformis (from Gallipro® Tect®); Enterococcus and Pediococcus (from Poultry star®); Lactobacillus, Bifidobacterium and / or Enterococcus (from Protexin®); Bacillus subtilis strain QST 713 (from Proflora®); Bacillus amyloliquefaciens CECT-5940 (from Ecobiol® and Ecobiol® Plus); Enterococcus faecalis faecium) SF68 (from Fortiflora®); Bacillus subtilis and Bacillus licheniformis (from BioPlus2B®); Lactobacillus faecium (from Lactiferm®); Bacillus spp. strains (from CSI®); Saccharomyces cerevisiae (from Yea-Sacc®); Enterococcus spp. (from Biomin IMB52®); Pediococcus lactis, Enterococcus spp., Bifidobacterium animalis subsp. animalis, Lactobacillus reuteri, Lactobacillus salivarius subsp. salivary (from Biomin C5®); Lactobacillus sausageii (from Biacton®); Enterococcus spp. (from Oralin E1707®); Enterococcus spp. (2 strains), Lactococcus lactis DSM1103 (from Probios-pioneer) PDFM®); Lactobacillus rhamnosus and Lactobacillus sausage (from Sorbiflore®); Bacillus subtilis (from Animavit®); Enterococcus (from Bonvital®); Saccharomyces cerevisiae (from Levucell SB 20®); Saccharomyces cerevisiae (from Levucell SC 0 & SC10® ME); Pediococcus lactis (from Bactocell); Saccharomyces cerevisiae (from Actisaf® (formerly BioSaf®)); Saccharomyces cerevisiae NCYC Sc47 (from Actisaf® SC47);Clostridium butyricum (from Miya-Gold®); Enterococcus spp. (from Fecinor and Fecinor Plus®); Saccharomyces cerevisiae NCYC R-625 (from InteSwine®); Saccharomyces cerevisiae (from BioSprint®); Enterococcus spp. and Lactobacillus rhamnosus (from Provita®); Bacillus subtilis and Aspergillus oryzae (from PepSoyGen-C®); Bacillus cereus (from Toyocerin®); Bacillus cereus var. toyoi NCIMB 40112 / CNCM I-1012 (from TOYOCERIN®), or other DFMs such as Bacillus licheniformis and Bacillus subtilis (from BioPlus® YC) and Bacillus subtilis (from GalliPro®).

[0190] DFM can be combined with Enviva® PRO, which is commercially available from Danisco. Enviva Pro® is a combination of Bacillus spp. strain 2084 Registry No. NRRL B-50013, Bacillus spp. strain LSSAO1 Registry No. NRRL B-50104, and Bacillus spp. strain 15A-P4 ATCC Registry No. PTA-6507 (as taught in US 7,754,469 B – incorporated herein by reference).

[0191] The DFM described in this article can also be combined with yeast species from the following genera: Saccharomycesspp.

[0192] Preferably, the DFM described herein contains microorganisms that are generally considered safe (GRAS) and preferably GRAS approved.

[0193] Those skilled in the art will readily recognize the specific microbial species and / or strains from the genera described herein, which are used in the food and / or agricultural industries and are generally considered suitable for animal consumption.

[0194] In some embodiments, it is important that the DFM is heat-resistant, i.e., heat-resistant. This is especially true when the feed is pelleted. Therefore, in another embodiment, the DFM can be a heat-resistant microorganism, such as heat-resistant bacteria, including, for example, species of the genus Bacillus.

[0195] In other respects, it may be desirable for DFM to include spore-producing bacteria, such as Bacillus species, for example, species of the genus Bacillus. Bacillus species are able to form stable endospores when growth conditions are unfavorable and are highly resistant to heat, pH, moisture, and disinfectants.

[0196] The DFM described herein can reduce or prevent the establishment of the gut by pathogenic microorganisms such as Clostridium perfringens and / or Escherichia coli and / or Salmonella species and / or Campylobacter species. In other words, DFM can be antipathogenic. As used herein, the term "antipathogenic" refers to the effect of DFM against pathogens (negative effect).

[0197] As stated above, a DFM can be any suitable DFM. For example, the following assay, “DFM assay,” can be used to determine the suitability of a microorganism as a DFM. The DFM assay used herein is explained in more detail in US 2009 / 0280090. For the avoidance of doubt, the DFM selected as an inhibitory strain (or antipathogenic DFM) according to the “DFM assay” taught herein is a suitable DFM used according to this disclosure, i.e., the DFM used in the feed additive compositions according to this disclosure.

[0198] Each tube is inoculated with a representative pathogen (e.g., bacteria) from a representative cluster.

[0199] Supernatant from potential DFMs growing aerobicly or anaerobicly was added to the inoculation tubes (except for the control tubes without supernatant) and incubated. After incubation, the optical density (OD) of each pathogen was measured in the control tubes and the supernatant-treated tubes.

[0200] Colonies of strains that produce lower OD compared to controls (containing no supernatant) (potential DFM) can then be classified as inhibitory strains (or resistant to pathogenic DFM). Therefore, the DFM assay used herein is explained in more detail in US 2009 / 0280090.

[0201] Preferably, the representative pathogen used in this DFM assay can be one (or more) of the following: Clostridium species, such as Clostridium perfringens and / or Clostridium difficile, and / or Escherichia coli and / or Salmonella species and / or Campylobacter species. In a preferred embodiment, the assay is performed with one or more of the following: Clostridium perfringens and / or Clostridium difficile and / or Escherichia coli, preferably Clostridium perfringens and / or Clostridium difficile, more preferably Clostridium perfringens.

[0202] Antipathogenic DFM includes one or more of the following bacteria, which are described in WO 2013029013:

[0203] Bacillus subtilis strain 3BP5, accession number NRRL B-50510,

[0204] Bacillus strain 918, ATCC accession number NRRL B-50508, and

[0205] Bacillus strain 1013 (ATCC accession number NRRL B-50509).

[0206] DFM can be prepared as one or more cultures and one or more carriers (if used) and can be added to a belt or paddle mixer and mixed for about 15 minutes, although the time can be increased or decreased. These components are blended to obtain a homogeneous mixture of culture and carrier. The final product is preferably a dry, flowable powder. DFM containing one or more bacterial strains can then be added to animal feed or feed premix, added to the animal's water, or administered in other ways known in the art (preferably simultaneously with the enzymes described herein).

[0207] The proportion of individual strains included in the DFM mixture can vary from 1% to 99%, and preferably from 25% to 75%.

[0208] The appropriate dosage of DFM in animal feed is approximately 1 × 10⁻⁶. 3 CFU / g feed to approximately 1 × 10⁻⁶ 10 CFU / g feed, appropriately at about 1 × 10 4 CFU / g feed to approximately 1 × 10⁻⁶ 8 Between CFU / g feed, appropriately around 7.5 × 10 4 CFU / g feed to approximately 1 × 10⁻⁶ 7 The range of CFU / g feed.

[0209] On the other hand, the dosage of DFM in feed can exceed approximately 1 x 10⁻⁶. 3 CFU / g feed, appropriately exceeding approximately 1 x 10 4 CFU / g feed, appropriately exceeding approximately 5 x 10 4 CFU / g feed, or appropriately exceeding about 1 x 10 5 CFU / g feed.

[0210] The dosage of DFM in diets that do not contain trace minerals is approximately 1 × 10⁻⁶. 3 CFU / g composition to about 1 × 10 13 CFU / g composition, preferably 1 × 10 5 CFU / g composition to about 1 × 10 13 CFU / g composition, more preferably at about 1 × 10⁻⁶. 6 CFU / g composition to about 1 × 10 12The CFU / g composition is between, and most preferably between, about 3.75 × 10⁻⁶. 7 CFU / g composition to about 1 × 10 11 Between CFU / g of the composition. On the other hand, the dosage of DFM in the feed additive composition is greater than about 1 × 10⁻⁶. 5 CFU / g composition, preferably greater than about 1 × 10⁻⁶. 6 CFU / g composition, and most preferably greater than about 3.75 × 10⁻⁶. 7 CFU / g composition. In one embodiment, the dose of DFM in the feed additive composition is greater than about 2 × 10⁻⁶. 5 CFU / g composition, suitably greater than about 2 × 10⁻⁶ 6 CFU / g composition, suitably greater than about 3.75 × 10⁻⁶ 7 CFU / g composition.

[0211] In some respects, at least one polypeptide with phytase activity, as described herein (or in other embodiments, in combination with one or more DFMs, such as any of those disclosed herein) can reduce the level of pathogenic bacteria in animals. Fresh food animal products, including poultry, are susceptible to contamination by microorganisms that come into contact with the surface of the meat immediately after slaughter and evisceration, including gastrointestinal organisms that can be transferred during processing. Contaminating microorganisms include bacteria such as species of Salmonella and Campylobacter, Listeria monocytogenes, Escherichia coli and other coliforms, and other intestinal organisms. Once pathogenic bacteria come into contact with tissue surfaces, they attach rapidly and are difficult to remove even with chlorine-based disinfectants permitted for use in poultry sprays and cooling cans.

[0212] The problems caused by Salmonella bacteria in poultry products are of particular concern. Currently, Americans spend approximately $20 billion annually on poultry products, averaging about 80 pounds per person. About 35% to 45% of poultry reaching American consumers is contaminated with Salmonella species. Improper cooking and the physical transfer of bacteria to food handling surfaces and subsequently to other foods lead to the spread of the microorganism, causing gastrointestinal illness and, in some cases, even death.

[0213] Furthermore, Campylobacter is a leading cause of foodborne gastroenteritis in humans worldwide, particularly in Europe, and poultry stockpiles are associated with over 80% of human cases. Campylobacter can cause a disease called Campylobacter disease. There are over 246,000 human cases annually, making it the most frequently reported foodborne illness in the EU. EFSA estimates that Campylobacter disease costs the EU public health system approximately €2.4 billion annually, resulting in lost productivity (worldwideweb.efsa.europa.eu / en / topics / topic / campylobacter).

[0214] Despite the importance of Campylobacter spp. to human health, there is currently no universally accepted solution for controlling it. Animal producers, the food industry, and regulatory agencies generally agree that a farm-to-table approach is needed to help reduce prevalence, contamination levels, and the risk of human infection. The poultry gut is a reservoir of Campylobacter spp., with the cecum containing approximately 10 [units unspecified]. 9 CFU / g. This storage facility caused cross-contamination of negative poultry and equipment during processing. Apart from the EU and markets exporting to European countries, nationwide Campylobacter screening programs requiring routine testing for Campylobacter are rare in other markets. For Campylobacter, a sustained 10-day monitoring period is expected. 8 CFU / gram cecal content (10) 6 The level of CFU / g cecal content (extended target) was used to reduce the level of the neck skin after cooling (sample type of regulatory agency).

[0215] Therefore, in some aspects, this document provides a method for reducing pathogenic bacterial populations in animals, the method comprising administering to the animal an effective amount of an animal diet containing engineered phytase peptides or fragments thereof containing phytase activity (such as any of those disclosed herein); and lacking one or more exogenously added trace minerals (e.g., but not limited to, iron and / or inorganic phosphates).

[0216] In some embodiments, compared with feed additive compositions containing phytase and low trace mineral content or animal feed diets described herein, pathogenic bacteria are reduced by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 105%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more (including percentages falling between these values). The pathogenic bacteria can be, but is not limited to, one or more of the following: *Streptococcus pneumoniae*, *Campylobacter* spp. (e.g., *Campylobacter jejuni*), *Neisseria gonorrhoeae*, *Staphylococcus aureus*, *Shigella* spp., *Enterococcus* spp., *Staphylococcus aureus*, *Streptococcus* spp., *Enterobacteriaceae* spp., *Acinetobacter* spp., *Clostridium* spp. (e.g., *C. difficile* or *C. perfingens*), *Escherichia coli* (e.g., *ETEC*), *Pseudomonas aeruginosa*, *Helicobacter pylori*, *Streptococcus anginosus*, and urinary tract pathogenic *Escherichia coli* (UPEC). In some embodiments, the animal is a pig. In other embodiments, the animal is poultry.

[0217] On another aspect, a trace mineral-free diet for use in animal feed is disclosed, the trace mineral-free diet comprising at least one polypeptide having phytase activity as described herein, used alone or in combination with at least one direct-feeding microorganism or with at least one other enzyme or in combination with at least one direct-feeding microorganism and at least one other enzyme, wherein the feed additive composition may be in any form such as granules. Such granules may be produced by methods selected from the group consisting of: high-shear granulation, drum granulation, extrusion, spheroidization, fluidized bed agglomeration, fluidized bed spray coating, spray drying, freeze drying, granulation, spray cooling, vortex disc atomization, coagulation, tableting, or any combination of the above processes.

[0218] In addition, the particles of the granular feed additive composition may have an average diameter greater than 50 micrometers and less than 2000 micrometers.

[0219] Those skilled in the art will understand that animal feed may include plant materials for poultry, pigs, ruminants, aquaculture, and pets, such as corn, wheat, sorghum, soybean, low-erucic acid rapeseed, sunflower, or mixtures of any of these plant materials or plant protein sources. Improvements are expected in animal performance parameters, such as growth, feed intake, and feed efficiency, as well as improved uniformity, reduced ammonia concentrations in animal sheds, and consequently, improved animal welfare and health.

[0220] Therefore, a method for improving the nutritional value of animal feed is disclosed, wherein any of the engineered phytases or fragments thereof, as described herein, can be added to the animal feed.

[0221] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g., phytase, such as any of the engineered phytase peptides disclosed herein) required, alone or in combination with one or more other active agents (e.g., but not limited to one or more additional enzymes, one or more DFMs, one or more essential oils, etc.) to impart improved performance to an animal on one or more indicators (e.g., but not limited to, increased feed efficiency, increased weight gain, decreased feed conversion ratio, improved digestibility of nutrients or energy in the feed, improved nitrogen retention, improved ability to avoid the negative effects of necrotizing enterocolitis, and improved immune response).

[0222] As used herein, the term “animal performance” can be determined by any indicator such as, but not limited to, feed efficiency and / or animal weight gain, and / or by feed conversion ratio and / or by the digestibility of nutrients in the feed (e.g., amino acid digestibility or phosphorus digestibility) and / or digestible or metabolizable energy in the feed, and / or by nitrogen retention and / or by the animal’s ability to avoid the adverse effects of disease or by the immune response of the subject.

[0223] Animal performance characteristics may include, but are not limited to: body weight; weight gain; mass; body fat percentage; height; body fat distribution; growth; growth rate; egg size; egg weight; egg quality; egg production rate; mineral absorption; mineral excretion and retention; bone density; bone strength; feed conversion ratio (FCR); average daily feed intake (ADFI); average daily weight gain (ADG); retention and / or excretion of any one or more of copper, sodium, phosphorus, nitrogen, and calcium; amino acid retention or absorption; mineralization, bone mineralization, carcass yield, and carcass quality.

[0224] "Improved animal performance on one or more metrics" means, compared to a diet containing the feed additive composition described herein or an animal feed diet, that, due to the use of such a diet, subjects experienced increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio and / or improved digestibility of nutrients or energy in the feed and / or improved nitrogen retention and / or improved ability to avoid the adverse effects of necrotizing enterocolitis and / or improved immune response. In some embodiments, compared with feed additive compositions containing phytase and low trace mineral content or animal feed diets described herein, one or more indicators are improved by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 105%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more (including percentages falling between these values).

[0225] Preferably, "improved animal performance" means increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio. As used herein, the term "feed efficiency" refers to the amount of weight gain that occurs when an animal is fed either randomly or in a prescribed amount of food over a period of time.

[0226] The term "increased feed efficiency" means that the use of the feed additive composition in feed results in increased weight gain per unit of feed intake compared to animals fed in the absence of the feed additive composition according to the invention.

[0227] As used in this article, the term "feed conversion ratio" refers to the amount of feed given to an animal to increase its body weight by a specified amount.

[0228] Improved feed conversion ratio means a lower feed conversion ratio.

[0229] "Lower feed conversion ratio" or "improved feed conversion ratio" means that the use of a feed additive composition in feed results in a lower amount of feed required to make an animal gain a specified amount of weight compared to the amount of feed required to make an animal gain the same amount of weight when the feed does not contain the feed additive composition.

[0230] The performance parameters can be improved relative to the control group in which the feed used does not contain phytase.

[0231] The term tibial ash refers to a quantitative method for bone mineralization. This parameter indicates whether phosphorus is deficient (e.g., low in a phosphorus-deficient negative control diet) or adequate (e.g., equivalent in a phytase-treated diet to a positive control diet that meets the phosphorus requirements of broilers).

[0232] The term "phosphorus-deficient diet" refers to a diet in which phosphorus levels are insufficient to meet the nutritional needs of an animal. For example, the phosphorus level in the formulated feed is far below the levels recommended by the National Research Council (NRC) or broiler farmers. The mineral levels in the animal feed are also below those required for optimal growth. If the diet lacks phosphorus, calcium will not be absorbed by the animal. Excessive calcium (Ca) can lead to poor phosphorus (P) digestibility and the formation of insoluble mineral-phytate complexes. Deficiencies in both P and Ca can result in reduced bone integrity, subnormal growth, and ultimately, weight loss.

[0233] The term "mineralization" encompasses the deposition or release of minerals. Minerals can be deposited or released from an animal's body. Minerals can be released from feed. Minerals can include any minerals required in an animal's diet and can include calcium, copper, sodium, phosphorus, iron, and nitrogen.

[0234] As used herein, nutrient digestibility refers to the fraction of nutrients that are lost from the gastrointestinal tract or a specific segment of the gastrointestinal tract (e.g., the small intestine). Nutrient digestibility can be measured as the difference between the nutrients administered to a subject and the nutrients excreted in the subject's feces, or the difference between the nutrients administered to a subject and the nutrients retained in the digestate in a specific segment of the gastrointestinal tract (e.g., the ileum).

[0235] As used herein, nutrient digestibility can be measured as follows: the difference between the amount of nutrients ingested over a period of time and the amount of nutrients excreted, obtained through complete collection in excrement; or using an inert label that is not absorbed by the animal, allowing researchers to calculate the amount of nutrients lost throughout the gastrointestinal tract or a segment thereof. Such an inert label can be titanium dioxide, chromium oxide, or acid-insoluble ash. Digestibility can be expressed as a percentage of nutrients in the feed or as a mass unit of digestible nutrients per mass unit of nutrients in the feed.

[0236] As used in this article, nutrient digestibility encompasses phosphorus digestibility, starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility. Digestible phosphorus (P) can be defined as ileal digestible P, which is the proportion of total P intake absorbed by the animal in the terminal ileum, or fecal digestible P, which is the proportion of total P intake not excreted in feces.

[0237] As used in this article, the term "survival rate" refers to the number of subjects who survive. The term "improved survival rate" is another way of saying "reduced mortality rate".

[0238] As used herein, the term "carcass yield" means the amount of carcass as a percentage of live body weight following a commercial or experimental slaughter process. The term "carcass" means the body of an animal that has been slaughtered for consumption and from which the head, viscera, limbs, and feathers or skin have been removed. As used herein, the term "meat yield" means the amount of edible meat as a percentage of live body weight or the amount of a specified cut of meat as a percentage of live body weight.

[0239] The terms "carcass quality" and "meat quality" are used interchangeably and refer to compositional quality (lean-to-fat ratio) and palatability factors such as visual appearance, aroma, firmness, juiciness, tenderness, and flavor. For example, producing poultry without "woody breast." In the United States, a small percentage of chicken meats exhibit muscle abnormalities that lead to the quality problem of woody breast. This condition results in chicken breast that is difficult to handle and often has a pale color and poor texture. Woody breast does not pose any health or food safety risks to people, and the health of the chicken itself is not negatively affected.

[0240] "Weight gain" refers to the increase in body weight of animals fed a diet containing the feed additive composition compared to animals fed a diet without the feed additive composition.

[0241] The terms “animal feed composition,” “feed,” “feeding,” and “fodder” are used interchangeably and may contain one or more feed materials selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof) and / or large grains, such as corn or sorghum; b) by-products derived from cereals, such as corn gluten meal, dry distillers grains with solubles (DDGS) (particularly corn-based DDGS), wheat bran, whole wheat flour, wheat middlings, rice bran, rice husks, oat hulls, palm kernels, and citrus pomace; c) proteins derived from sources such as soybeans, sunflower seeds, peanuts, lupins, peas, broad beans, cotton, low-erucic acid rapeseed, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, dried coconut meat, and sesame; d) oils and fats derived from plant and animal sources; and / or e) minerals and vitamins.

[0242] Suitablely, premixes, as mentioned herein, can be compositions consisting of trace elements such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential components. Premixes are generally compositions suitable for blending into commercial diets.

[0243] As used herein, the term "contact" refers to the indirect or direct application of any engineered phytase polypeptide or fragment thereof (or a composition comprising any engineered phytase polypeptide or fragment thereof) to a product (e.g., feed). Examples of possible application methods include, but are not limited to: treating the product in a material containing the feed additive composition, applying directly by mixing the feed additive composition with the product, spraying the feed additive composition onto a product surface, or immersing the product in a formulation of the feed additive composition. In one embodiment, the feed additive composition of the present invention is preferably mixed with a product (e.g., feed). Alternatively, the feed additive composition may be contained in an emulsion or the original ingredients of the feed. For some applications, it is important that the composition is available or can be used on the surface of the product to be affected / treated. This allows the composition to impart performance benefits.

[0244] Any of the engineered phytase peptides or fragments thereof described herein (or a composition comprising such engineered phytase peptides or fragments thereof) may be used with a controlled amount of the enzyme for dispensing, coating and / or impregnation of products (e.g., diets or feeds or raw ingredients of feeds that are free of or substantially free of trace minerals).

[0245] On the other hand, the feed additive composition can be homogenized to produce a powder. The powder can be mixed with other components known in the art. The powder or a mixture containing the powder can be forced through a die, and the resulting filament can be cut into pellets of suitable different lengths.

[0246] Optionally, the pelleting step may include a steam treatment or conditioning stage prior to pellet formation. The mixture containing the powder may be placed in a conditioner, such as a mixer with steam injection. The mixture is heated in the conditioner to a specified temperature, such as 60°C–100°C, typically 70°C, 80°C, 85°C, 90°C, or 95°C. The residence time may range from a few seconds to several minutes. It should be understood that any of the engineered phytase peptides or fragments thereof described herein (or compositions containing any of the engineered phytase peptides or fragments thereof) is suitable for addition to any suitable feed material.

[0247] In other embodiments, pellets may be introduced into the feed pelleting process, wherein the feed pretreatment process may be carried out at a temperature between 70°C and 95°C, such as between 85°C and 95°C, for up to several minutes.

[0248] In some embodiments, based on pure enzyme protein, any one of the engineered phytase peptides or fragments thereof may be present in feed in the range of 1 ppb (parts per billion) to 10% (w / w). In some embodiments, the engineered phytase peptides or fragments thereof are present in feed in the range of 1-100 ppm (parts per million). A preferred dosage may be 1-20 g of engineered phytase peptides or fragments thereof per tonne of feed product or feed composition, or a final dosage of 1-20 ppm of engineered phytase peptides or fragments thereof in the final feed product.

[0249] Preferably, the engineered phytase peptide or fragment thereof present in the feed should be at least about 50–10,000 FTU / kg, which is equivalent to about 0.1 to 20 mg of protein of engineered phytase peptide or fragment thereof per kg.

[0250] The range may include, but is not limited to, any combination of the lower and upper limits discussed above.

[0251] Formulations and / or formulations containing any of the engineered phytase peptides or fragments thereof described herein and their compositions may be prepared in any suitable manner to ensure that the formulation contains active phytase. Such formulations may be liquids, dry powders, or granules, and may be uncoated / unprotected or may involve the use of a heat-protective coating, depending on the processing conditions. As mentioned above, engineered phytase peptides and fragments thereof can be inexpensively formulated on a solid carrier without particularly requiring a protective coating and remain active throughout conditioning and granulation. When certain areas where harsh conditions are used require or, if conditions permit (e.g., in the case of over-conditioned feed above 90°C), a protective coating that provides additional thermal stability when applied in solid form may be beneficial for achieving granulation stability.

[0252] The feed additive compositions described herein can be formulated as dry powder or granules, as described in WO 2007 / 044968 (referred to as TPT granules) or WO 1997 / 016076 or WO 1992 / 012645 (each of which is incorporated herein by reference).

[0253] In one embodiment, the feed additive composition may be formulated into pellets for use in a feed composition, the pellets comprising: a core; an active agent (e.g., phytase, such as any of the engineered phytase peptides disclosed herein); and at least one coating such that the active agent of the pellet retains at least 50%, at least 60%, at least 70%, or at least 80% activity after being subjected to one or more of the following conditions: a) a feed pelleting process, b) a steam-heated feed pretreatment process, c) storage, d) storage as an ingredient in an unpelleted mixture, and e) storage as an ingredient in a feed base mixture or feed premix comprising at least one compound selected from: trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds that produce acidic or alkaline feed base mixtures or feed premixes.

[0254] Regarding the particles, at least one coating may comprise a hydrating material comprising at least 55% w / w of moisture of the particles; and / or at least one coating may comprise two coatings. These two coatings may be a moisturizing coating and a moisture-proof layer. In some embodiments, the moisturizing coating may comprise 25% w / w to 60% w / w of the particles, and the moisture-proof layer may comprise 2% w / w to 15% w / w of the particles. The moisturizing coating may be selected from inorganic salts, sucrose, starch, and maltodextrin, and the moisture-proof layer may be selected from polymers, gums, whey, and starch.

[0255] In other embodiments, pellets may be introduced into the feed pelleting process, wherein the feed pretreatment process may be carried out at a temperature between 70°C and 95°C, such as between 85°C and 95°C, for up to several minutes.

[0256] The feed additive composition can be formulated into pellets for animal feed, the pellets comprising: a core; an active agent, the active agent of which retains at least 80% activity after storage and after a steam-heated pelleting process in which the pellet is an ingredient; a moisture barrier; and a moisturizing coating comprising at least 25% w / w of the pellet, the pellet having a water activity of less than 0.5 prior to the steam-heated pelleting process.

[0257] The granules may have a moisture barrier coating selected from polymers and resins, and the hydration material may be an inorganic salt. The hydration coating may comprise between 25% and 45% w / w of the granules, and the moisture barrier coating may comprise between 2% and 10% w / w of the granules.

[0258] Alternatively, the composition is in a liquid formulation suitable for consumption, preferably containing one or more of the following: buffer solution, salt, sorbitol and / or glycerol.

[0259] In addition, feed additive compositions can be formulated by applying enzymes (e.g., spraying) onto a carrier substrate (such as milled wheat).

[0260] In one embodiment, the feed additive composition may be formulated as a premix. For example only, the premix may contain one or more feed components, such as one or more minerals and / or one or more vitamins.

[0261] In one embodiment, a direct-feeding microorganism (“DFM”) and / or an engineered phytase peptide or fragment thereof is formulated with at least one physiologically acceptable carrier selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat fraction, sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate, and mixtures thereof.

[0262] It should be noted that any of the engineered phytase peptides and their fragments can be used in cereal applications, such as cereal processing for non-food / feed applications (e.g., ethanol production). Example

[0263] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY, Harper Perennial, New York (1991) provide general dictionaries for those skilled in the art of the use of many terms in this disclosure.

[0264] This disclosure is further defined in the examples below. It should be understood that these examples, while indicating certain embodiments, are given by way of illustration only. From the foregoing discussion and examples, those skilled in the art will be able to determine the essential features of this disclosure, and various changes and modifications can be made to suit various uses and conditions without departing from the spirit and scope of this disclosure.

[0265] Example 1: Novel concordant sequence bacterial 6-phytase variant in commercial corn-soybean meal containing normal levels of calcium The efficacy of replacing added trace minerals in feed

[0266] This example demonstrates the efficacy of broiler diets supplemented with a novel concordant sequence bacterial 6-phytase variant (PhyG; SEQ ID NO:26) without exogenous addition of TM (Zn, Fe, Cu, Mn, Se). When fed to broilers, these diets maintained growth performance, tibia ash, tibia weight, liver and plasma TM concentrations at levels comparable to or improved upon commercially available nutritionally adequate diets supplemented with TM.

[0267] Materials and Methods

[0268] This study was conducted in accordance with European Directive 2010 / 63 / EU and current Dutch regulations on the care and use of animals in research. The experimental protocol and procedures were evaluated and approved by the Animal Ethics Committee (Animal Experimentation Ethics Review) of the Schothorst Feed Research Institute (Leilestad, Netherlands).

[0269] Experimental Design, Poultry, and Housing: The experiment was conducted using a randomized complete block design with 12 dietary treatments and 8 replicates of floor-fenced enclosures (each enclosure being 2 m in size). 2 A total of 3,360 Ross 308 male broilers were assigned to individual pens on hatching day, ensuring each pen contained birds of roughly equal average weight. The pens were housed in broiler sheds with wood shavings as bedding. Temperature was initially maintained at 34°C, gradually decreasing to 22°C on day 24, then to 21°C on day 29, 20°C on day 32, and 19°C on day 35. The lighting program was LD 23:1h on day 1, and thereafter 3L:1D:12L:4D:3L:1D. Throughout all stages, the diet was fed ad libitum as pelleted feed (pelletizing temperature ≤ 80°C).

[0270] Feed treatment: The treated feed was formulated in three phases: the initial feeding phase for 1–10 days of age, the growth phase for 10–20 days of age, and the finishing phase for 20–35 days of age. The treatment included:

[0271] 1) CON1: Control diet, formulated to meet the nutritional requirements of broilers in the Netherlands (CVB, 2021), but without the addition of TM (Zn, Cu, Fe, Mn and Se).

[0272] 2) CON1 is supplemented with Zn, Cu, Fe, Mn in sulfate form and Se in Na2SeO3 form, added at 'low' levels (50% less than commercial levels) in all stages;

[0273] 3) CON1 is supplemented with Zn, Cu, Fe, Mn in sulfate form and Se in Na2SeO3 form, added at 'high' levels (commercial level) in all stages;

[0274] 4) CON1 is supplemented with Zn, Cu, Mn in oxide form and Se in Na2SeO3 form and Fe in sulfate form, added at 'low' levels in all stages;

[0275] 5) CON1, along with supplementary oxide-based Zn, Cu, Mn, and Na2SeO3-based Se and sulfate-based Fe, are added at 'high' levels in all stages;

[0276] 6) CON1 is supplemented with organic forms of Zn, Fe, Cu, Mn and Se in the form of Na2SeO3, added at 'low' levels in all stages;

[0277] 7) CON2: Control diet, the same as CON1, but with reduced Ca and digestible P (to account for the expected activity of PhyG phytase), and contains PhyG added at 2,000, 1,500 and 1,000 FTU / kg during the start-up, growth and finishing stages, respectively.

[0278] 8) CON2 is supplemented with Zn, Fe, Cu, Mn in sulfate form and Se in Na2SeO3 form, added at 'low' levels in all stages, and supplemented with PhyG, as shown in 7;

[0279] 9) CON2 is supplemented with Zn, Fe, Cu, Mn in sulfate form and Se in Na2SeO3 form, added at 'high' levels in all stages, and supplemented with PhyG, as shown in 7;

[0280] 10) CON2 is supplemented with Zn, Cu and Mn in the form of oxides and Se in the form of Na2SeO3 and Fe in the form of sulfates, added at 'low' levels in all stages, and supplemented with PhyG, as shown in 7;

[0281] 11) CON2 is supplemented with Zn, Cu and Mn in the form of oxides and Se in the form of Na2SeO3 and Fe in the form of sulfates, added at 'high' levels in all stages, and supplemented with PhyG, as shown in 7;

[0282] 12) CON2 is supplemented with organic forms of Zn, Cu, Fe and Mn and Se in the form of Na2SeO3, added at 'low' levels in all stages, and supplemented with PhyG, as shown in 7.

[0283] The concentration of a single TM added to each treatment is given in Table 1. The composition and calculated nutrient composition of the basal CON1 and CON2 diets are given in Table 2, and the analytical TM content of the diets is given in Table 3.

[0284] Table 1. Feed treatment details

[0285]

[0286] 1 The feed intake is 2,000, 1,500 and 1,000 FTU / kg for the start-up phase (days 1-10), the growth phase (days 10-20) and the fattening phase (days 20-35), respectively.

[0287] 2 The value for a single trace mineral represents the concentration expected to be present in the final feed at a rate of mg / kg.

[0288] 3 'Low': 50% lower than commercial supplement levels; 'High': Commercial supplement levels.

[0289] 4 For regulatory reasons, FeSO4 is used instead of FeO in treatments 4, 5, 10 and 11.

[0290] 5 In all treatments, Se was added in the form of Na2SeO3.

[0291] Table 2. Composition and calculated nutrient composition of the basal diet in Example 1

[0292]

[0293]

[0294] 1 Formulated to meet the nutritional requirements of broiler chickens suitable for the Netherlands (CVB, 2021), but without the addition of trace minerals.

[0295] 2 Compared to CON1, Ca and digestible P are reduced (to account for the expected Ca and digestible P contribution from the added phytase), and there is no micromineral supplement, but phytase is added (at 2,000, 1,500 and 1,000 FTU / kg during the starter, growth and fattening stages, respectively).

[0296] 3Supplement per kilogram of feed with: Vitamin A (retinyl acetate 3a672a, 10,000 IU), Vitamin D3 (3a671, 2,500 IU), Vitamin E (dl-α-tocopherol 3a700, 50 IU), Vitamin K3 (menaquinone 3a711, 1.5 mg), Vitamin B1 (thiamine nitrate 3a821, 2.0 mg), Riboflavin (7.5 mg), D-pantothenic acid (12 mg), Vitamin B6 (3.5 mg), Vitamin B12 (0.020 mg), Niacin (35 mg), Folic acid (1.0 mg), Biotin (0.20 mg), Choline (choline chloride, 460 mg), and I (KI, 0.8 mg).

[0297] Table 3: Trace mineral content of the experimental diets analyzed in Example 1.

[0298]

[0299] *Not reported.

[0300] 2 The feed intake is 2,000, 1,500 and 1,000 FTU / kg for the start-up phase (days 1-10), the growth phase (days 10-20) and the fattening phase (days 20-35), respectively.

[0301] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0302] 4 S, Start feeding (1 to 10 days old); G, Growth (10 to 20 days old); F, Fattening (20 to 35 days old).

[0303] Sampling, Measurement, and Statistical Analysis: Four birds / penns were euthanized at each of the 10, 20, and 35-day-old periods. Blood was collected from two birds / penns, plasma was extracted, and one of the two samples from each pen was subjected to TM analysis. Liver was collected from two birds / penns, weighed, and one of the two samples from each pen was subjected to TM analysis. Tibiae (left) were extracted from four birds / penns, combined, defatted, ashed, and subjected to TM analysis. Trace minerals in the liver were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES), and trace minerals in the plasma were analyzed by atomic absorption spectrometry (AAS), both performed in duplicate. Data were analyzed by two-way ANOVA (2 x 6 factorial design with two levels of phytase supplementation and six levels of trace mineral supplementation). Tukey's HSD test was used to distinguish treatment means. P < 0.05 was considered statistically significant. 0.05 ≤ P < 0.1 was considered a trend.

[0304] result

[0305] Growth performance: Results for body weight (BW), BW gain (BWG), feed intake (FI), and feed conversion ratio (FCR) by stage are presented in Tables 4-6.

[0306] Even without TM supplementation, the growth performance levels maintained by supplementing CON 2 with Phytase (treatment 7) were statistically comparable to or improved compared to those maintained by CON 1 treatments (treatments 2–6) with TM supplementation. This effect was observed in all performance metrics (BW, BWG, FI, and FCR) throughout all individual phases and overall (1–35 days of age, data not shown) (Tables 4, 5, and 6). The improvement in FCR in the case of phytase (unrelated to the effect of TM supplementation) (significant throughout all individual phases) indicates that the weight gain increase in poultry supplemented with phytase was not solely due to increased FI, but also to increased TM utilization per unit of feed intake.

[0307] In the absence of phytase, TM supplementation improved all growth energy measures (increased BW, increased BW, FI, and decreased FCR) during the start-up phase (1–10 days of age), but its effect was less pronounced during the growth phase (10–20 days of age) and the finishing phase (20–35 days of age). During the growth phase, TM supplementation increased BW, BWG, and FI, but had no effect on FCR, while during the finishing phase, TM supplementation had no effect on BWG, FI, or FCR, although most treatments supplemented with TM (except for the low treatment and oxide-based TM treatment 4) had higher final BW at 35 days of age compared to the unsupplemented CON1 diet (treatment 1). The effect of TM supplementation on growth performance was evident regardless of the source or dosage level of TM. In the presence of phytase, TM supplementation did not provide additional benefits in terms of growth performance (all measures, all phases) because phytase had a dominant beneficial effect (treatments 8–12, compared to treatment 7 in Tables 4, 5, and 6).

[0308] The mortality rate was generally low (less than 2.6% for 1–35 days old, average 1.3%) and was unaffected by treatment.

[0309] Table 4: Effects of supplements containing trace minerals (low or high), phytase, or both on growth performance during days 1–10; Two-factor ANOVA

[0310]

[0311] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0312] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 2).

[0313] 2 The feed intake is 2,000, 1,500 and 1,000 FTU / kg for the start-up phase (days 1-10), the growth phase (days 10-20) and the fattening phase (days 20-35), respectively.

[0314] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0315] Table 5: Effects of supplements containing trace minerals (low or high), phytase, or both on growth performance during days 10–20; Two-factor ANOVA

[0316]

[0317] a,b,c,d,e Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0318] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 1).

[0319] 2 The feed intake is 2,000, 1,500 and 1,000 FTU / kg for the start-up phase (days 1-10), the growth phase (days 10-20) and the fattening phase (days 20-35), respectively.

[0320] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0321] Table 6: Effects of supplements containing trace minerals (low or high), phytase, or both on growth performance during days 20–35; Two-factor ANOVA

[0322]

[0323] a,b Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0324] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 1).

[0325] 2 The intake is 2,000, 1,500 and 1,000 FTU / kg for the initial feeding stage (days 0-10), the growth stage (days 10-20) and the fattening stage (days 20-35), respectively.

[0326] Bone mineralization: In the absence of exogenous phytase, removal of TM negatively impacted tibial Zn (treatment 1 vs. treatments 2–6). Even without TM supplementation, phytase supplementation in CON 2 (treatment 7) improved tibial Zn compared to any of the CON 1 treatments (i.e., with or without TM supplementation, treatments 1–6; Table 7); in the absence of TM, tibial Zn in CON 2 + phytase (treatment 7) was 77% higher than in treatment 1 and 7%–25% higher than in treatments 2–6. As seen in growth performance, TM supplementation did not provide additional benefits to tibial Zn beyond or above phytase delivery, as indicated by the significant interaction between the effects of phytase and TM supplementation on tibial Zn in Table 7 and the differences between the corresponding means for treatments 7–12.

[0327] Table 7: Effects of supplements containing trace minerals (low or high), phytase, or both on tibial ash and trace mineral content at 20 days of age; two-factor ANOVA

[0328]

[0329] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0330] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 1).

[0331] 2 The feed intake is 2,000, 1,500 and 1,000 FTU / kg for the start-up phase (days 1-10), the growth phase (days 10-20) and the fattening phase (days 20-35), respectively.

[0332] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0333] Trace minerals in the liver: Phytase or trace mineral supplements had no effect on liver weight at 20 days of age (Table 8). Even without TM supplementation, liver Zn and Fe levels maintained by phytase supplementation in CON2 (treatment 7) were improved (relative to CON1) or comparable to those in CON1 treatment with TM supplementation (relative to treatments 2–6; Table 8).

[0334] For liver Zn and Fe, the observed interactions were similar to those observed for tibial Zn; TM supplementation did not produce additional benefits for liver Zn or Fe beyond or above phytase delivery (treatments 8–12, compared to treatment 7), while in the absence of phytase, there was some evidence of increased liver Zn in the presence of TM supplementation (e.g., significantly higher liver Zn in treatment 3 compared to treatment 1, while increased liver Zn and Fe values ​​were observed in treatments 2–6 compared to treatment 1, Table 8).

[0335] Table 8: Effects of supplements containing trace minerals (low or high), phytase, or both on liver trace mineral content at 20 days of age; two-factor ANOVA

[0336]

[0337] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0338] 1Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 1).

[0339] 2 The feed intake is 2,000, 1,500 and 1,000 FTU / kg for the start-up phase (days 1-10), the growth phase (days 10-20) and the fattening phase (days 20-35), respectively.

[0340] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0341] Trace minerals in plasma: Apart from plasma Se, phytase or trace mineral supplements have less effect on plasma mineral concentrations than on tibial or liver trace mineral concentrations (Table 9).

[0342] As before, even without TM supplementation, the plasma Zn, Fe, Cu, and Mn levels maintained by supplementing CON2 with phytase (treatment 7) were comparable to or improved compared to the plasma Zn, Fe, Cu, and Mn levels maintained by any of the CON1 treatments (treatments 2-6; Table 9) supplemented with TM.

[0343] For plasma Zn, the observed interactions were similar to those observed for tibial Zn; micromineral supplementation did not produce additional benefits for hepatic Zn or Fe beyond phytase delivery (treatments 8–12, compared to treatment 7), while in the absence of phytase, there was some evidence of increased plasma Zn and Se in the presence of TM supplementation (treatments 2–6, compared to treatment 1).

[0344] For plasma Se, unlike other trace minerals, TM supplementation showed a very significant enhancing effect (treatments 2–6 vs. treatment 1, +757% to +893%; Table 9), while phytase supplementation had no independent effect. However, interactions existed, thus the response of plasma Se to TM in the presence of phytase appeared to be slightly lower than that in the absence of phytase (treatments 8–12 vs. treatment 7, +652% to +723%; Table 9).

[0345] Table 9: Effects of supplements containing trace minerals (low or high) or phytase or both on plasma trace mineral levels at 20 days of age; two-factor ANOVA

[0346]

[0347] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0348] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 1).

[0349] 2 The feed intake is 2,000, 1,500 and 1,000 FTU / kg for the start-up phase (days 1-10), the growth phase (days 10-20) and the fattening phase (days 20-35), respectively.

[0350] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0351] Tibial Trace Mineral Content at 10 Days Old: Results regarding the effects of trace mineral (TM) supplementation, phytase supplementation, or both on TM content at 10 days old are shown in Table 14. There was an interaction between TM and phytase for Zn and Mn concentrations in the tibia, and a tendency for interaction for Fe in the tibia.

[0352] The addition of TM significantly increased tibial Zn (64% to 117% increase in treatments 2–6 compared to treatment 1), with higher response levels in the TM 'high' treatments than in the TM 'low' treatments, and also higher response levels in oxidative-based treatments compared to sulfate-based or organic-based TM treatments. The addition of phytase without TM also significantly increased tibial Zn compared to no phytase or TM (treatment 1, +138%) or TM without phytase (treatments 2–6, +10% to +46%). Adding TM on top of phytase did not further increase tibial Zn.

[0353] Except for treatment 5 (“high” for oxide-based), TM supplements (without phytase) did not increase tibial Mn. Phytase significantly increased tibial Mn (treatment 7 vs. 1, +29%), and the addition of TM on top of phytase (regardless of TM source or dose level) further improved tibial Mn (treatments 8–12 vs. 7, +24% to +44%).

[0354] In phytase treatments, except for one TM treatment (a sulfate-based 'low' TM), supplemental TM moderately reduced tibial Fe (-4.4% to -7.2%) in all TM treatments. Phytase increased tibial Fe content (+7% in TM treatments relative to no phytase).

[0355] Table 14: Effects of supplements containing trace minerals (low or high) or phytase or both on tibial trace mineral content at 10 days of age in Example 1; Two-factor ANOVA

[0356]

[0357] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0358] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 2 of the patent application for the complete composition).

[0359] 2 The intake is 2,000, 1,500 and 1,000 FTU / kg for the initial feeding stage (days 0-10), the growth stage (days 10-20) and the fattening stage (days 20-35), respectively.

[0360] 3 The levels of individual trace minerals added to each treated diet and their sources are given in Table 1 of the patent application.

[0361] Trace minerals in the liver at 10 days of age: Results regarding the effects of TM supplementation, phytase supplementation, or both on liver weight and trace mineral concentrations are shown in Table 15. Interactions with liver Zn concentrations: Adding TM without phytase had no effect on liver Zn compared to not adding TM or phytase, while adding phytase without TM increased liver Zn (treatment 7 increased by 17% compared to 1). Regardless of the source or dosage of TM, trace mineral supplementation on the basis of phytase did not lead to a further increase in liver Zn (treatments 8–12 compared to 7).

[0362] Phytase increased liver Fe (+42% relative to no phytase), but TM supplementation did not increase liver Fe, although liver Fe in the sulfate-based 'high' treatment was higher than that in the oxide-based or organic-based 'low' treatment. Liver Cu concentration was not affected by TM supplementation but decreased by phytase supplementation (2.48 mg / kg relative to 2.57 mg / kg, or -3.5% relative to no phytase), which may be related to increased Zn uptake in the phytase-supplemented treatment, possibly due to competition from absorption transporters (17.4 mg / kg for liver Zn with phytase, compared to 16.6 mg / kg for liver Zn without phytase, +4.8%). Phytase supplementation increased liver Mn (+19% relative to no phytase), and to a lesser extent and only in certain treatments, TM supplementation also increased liver Mn (+7.4% to +18% relative to no TM).

[0363] Table 15: Effects of supplements containing trace minerals (low or high) or phytase or both on liver trace mineral content at 10 days of age in Example 1; Two-factor ANOVA

[0364]

[0365] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0366] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 2 of the patent application for the complete composition).

[0367] 2 The intake is 2,000, 1,500 and 1,000 FTU / kg for the initial feeding stage (days 0-10), the growth stage (days 10-20) and the fattening stage (days 20-35), respectively.

[0368] 3 The levels of individual trace minerals added to each treated diet and their sources are given in Table 1 of the patent application.

[0369] Conclusion: Supplementation of corn-soybean meal-based diets with PhyG phytase, which reduced calcium and digestible phosphorus while maintaining normal dietary calcium levels, completely replaced supplemented TM (Zn, Fe, Cu, Mn, and Se) throughout all growth stages and overall (1 to 35 days of age). Growth performance, liver and plasma TM levels were maintained at levels comparable to those produced by a nutritionally adequate control diet (supplemented with TM but without phytase). These results suggest that PhyG phytase can be used as a substitute for TM supplementation in monogastric animal diets.

[0370] Example 2: Novel shared sequence bacterial 6-phytase variant in corn-soybean meal-based diets containing high levels of calcium The efficacy of replacing added trace minerals

[0371] This study was conducted at the same research institution (Schothorst Feed Research Institute, Lelistad, Netherlands) as the study in Example 1, using the same experimental design, treatments, conditions, methods, and statistical analysis procedures, except for the use of higher levels of calcium (Ca) in the basal diet, as is found in some commercial diets (Petricevic et al., 2002). The composition of the basal diet and the calculated nutrient composition are presented in Table 10 (the Ca in the CON1 diet was 1.14% during the start-up phase (1–10 days of age) and 1.06% during the growth phase (10–20 days of age). In contrast, the NRC (1994) recommends 1.0% Ca during the start-up and growth phases. The concentrations of the single TM added to each treatment are shown in Table 1, while the mineral and TM contents analyzed in the diets are given in Table 11.

[0372] Table 10. Composition and calculated nutrient composition of the basal diet in Example 2

[0373]

[0374]

[0375] 1 Formulated to meet the nutritional requirements of broiler chickens suitable for the Netherlands (CVB, 2021), but without adding TM.

[0376] 2 Compared to CON1, Ca and digestible P are reduced (to account for the expected Ca and digestible P contribution from the added phytase), and there is no micromineral supplement. Phytase is added at 2,000, 1,500 and 1,000 FTU / kg during the starter, growth and fattening stages, respectively.

[0377] 3 Supplement per kilogram of feed with: Vitamin A (retinyl acetate 3a672a, 10,000 IU), Vitamin D3 (3a671, 2,500 IU), Vitamin E (dl-α-tocopherol 3a700, 50 IU), Vitamin K3 (menaquinone 3a711, 1.5 mg), Vitamin B1 (thiamine nitrate 3a821, 2.0 mg), Riboflavin (7.5 mg), D-pantothenic acid (12 mg), Vitamin B6 (3.5 mg), Vitamin B12 (0.020 mg), Niacin (35 mg), Folic acid (1.0 mg), Biotin (0.20 mg), Choline (choline chloride, 460 mg), and I (KI, 0.8 mg).

[0378] Table 11. Trace mineral content of the experimental diets analyzed in Example 2

[0379]

[0380] 2 The intake is 2,000 and 1,500 FTU / kg for the initial feeding stage (days 1-10) and the growth stage (days 10-20), respectively.

[0381] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0382] 4 Start feeding: 1 to 10 days old; Growth: 10 to 20 days old.

[0383] result

[0384] Growth performance: BW, BWG, FI, and FCR results by stage are presented in Tables 12–13. During the start-up phase (1–10 days old; Table 12), supplementation of CON2 with Phytase (treatment 7) improved growth performance (BWG and FI) in poultry, reaching levels higher than those achieved with CON1 treatments supplemented with 'low'™ (regardless of TM source; treatments 2, 4, and 6), equivalent to the growth performance response of poultry fed CON1 treatments supplemented with 'high'™ (treatments 3 and 5). By 10 days old, poultry fed CON2 (treatment 7) supplemented with phytase but without TM had higher BW than poultry fed CON1 diets supplemented with TM (regardless of TM dosage level). In the absence of phytase (treatments 2–6), CON1 diets supplemented with TM minerals improved all measures of growth performance (increased BW, BWG, FI, and decreased FCR) during the start-up phase. This effect was evident at all TM sources and dosage levels, but there is also some evidence of a dose-response effect at BWG and BW on day 10. However, no dose-response effect was observed with the addition of phytase (treatments 7–12); for any growth energy level, regardless of TM source or dosage level, TM supplementation did not provide any additional benefit beyond that achieved by phytase supplementation alone.

[0385] Table 12: Effects of supplements containing trace minerals (low or high) or phytase or both on growth performance during days 1–10; Two-factor ANOVA

[0386]

[0387] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0388] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 2).

[0389] 2 The intake is 2,000 and 1,500 FTU / kg for the initial feeding stage (days 1-10) and the growth stage (days 10-20), respectively.

[0390] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0391] During the growth phase (10–20 days old, Table 13), the growth performance (BWG, FI, FCR) maintained by phytase without TM supplementation (treatment 7) was equivalent to, but not higher than, the growth performance maintained by all treatments (treatments 2–6) containing TM but without phytase supplementation. However, due to the additional beneficial effects of phytase occurring during the start-up phase, which outweighed the effects of TM supplementation, treatment 7 had a higher final BW at 20 days old than some treatments that supplemented with TM alone (higher than treatment 2, sulfate-based “low” TM, and treatment 6, organic compound “low” TM). As seen during the start-up phase, TM supplementation without phytase during the growth phase improved some measures of growth performance (BWG and FI) compared to no phytase or TM supplementation (treatment 1), but TM supplementation did not improve feed efficiency (FCR) during the growth phase. This suggests that the TM-mediated BWG improvement during the growth phase was primarily driven by increased FI rather than increased TM availability itself. In the case of phytase, the TM supplement did not provide any additional benefits to growth performance, consistent with the data generated in Example 1.

[0392] Mortality rates were low during both phases (average < 2.8%) and were unaffected by phytase or TM supplements.

[0393] Table 13: Effects of supplements containing trace minerals (low or high), phytase, or both on growth performance during days 10–20; Two-factor ANOVA

[0394]

[0395] a,b,c,d Within each column group, there were significant differences in the means with different superscripts, P < 0.05.

[0396] 1 Processes 1–6 are based on CON1, and processes 7–12 are based on CON2 (see Table 2).

[0397] 2 The intake is 2,000 and 1,500 FTU / kg for the initial feeding stage (days 1-10) and the growth stage (days 10-20), respectively.

[0398] 3 The levels of individual trace minerals added to each treatment diet and their sources are given in Table 1.

[0399] Overall Conclusion: In corn-soybean meal-based diets containing normal calcium levels (Example 1), regardless of TM source or dosage level, the addition of Phytase G to the diet in a phased, graded dosing regimen completely replaced supplemented TM throughout all growth phases and the overall period. When added to the same corn-soybean meal-based diet containing high calcium levels (1.19% and 1.09% in the diet without phytase during the start-up and growth phases, respectively, and 0.968% and 0.687% in the diet with phytase supplementation, respectively, analytical values; Example 2), the same phytase also completely replaced supplemented TM during both the start-up and growth phases. However, in high-Ca diets, there is also evidence of a dose-response effect of phytase; the average weight gain achieved by adding phytase to the diet without supplemented TM was equivalent to the average weight gain achieved by supplementing TM at a “high” dose and superior to the average weight gain achieved by supplementing TM at a “low” dose.

[0400] Example 3: Using a novel concordant sequence bacterial 6-phytase variant as an alternative to supplementation in commercial corn-soybean meal-based diets The effects of trace minerals on young broiler chickens raised in commercial environments

[0401] This case study evaluates whether a novel co-sequence bacterial 6-phytase variant (SEQ ID NO: 26) can completely replace added trace minerals supplemented into the diet at commercial dose levels in broiler chickens aged 0 to 21 days when raised in a commercial environment.

[0402] Materials and Methods

[0403] This study was conducted in accordance with European Directive 2010 / 63 / EU and current Dutch regulations on the care and use of animals in research. The experimental protocol and procedures were evaluated and approved by the Animal Ethics Committee (Animal Experimentation Ethics Review) of the Schothorst Feed Research Institute (Leilestad, Netherlands).

[0404] Experimental Design, Poultry, and Housing: The experiment was conducted using a randomized complete block design with four dietary treatments, each with eight replicate floor enclosures. Each replicate enclosure was an experimental unit containing 820 poultry (males and females, post-hatching). The stocking density was 17 poultry / m². 2 In each of the two stages, feed the diet as pelleted food (pelletizing temperature ≤ 80°C) on an ad libitum basis. Temperature and light should be determined according to the breeder's recommendations.

[0405] Feed treatment: The treated diet is formulated in two phases, focusing on the most sensitive stages in the broiler's life: the initial feeding stage (1–10 days old) and the growth stage (10–21 days old). Treatment includes:

[0406] 1) CON1: Control diet, nutritionally adequate, but without supplementation of Zn, Cu, Fe, and Mn;

[0407] 2) CON1+TM: Same as CON1, but contains supplemental trace minerals (Zn, Cu, Fe, Mn) from the addition of standard mineral premix, expected to provide 60 mg / kg Zn, 10 mg / kg Cu, 50 mg / kg Fe and 70 mg / kg Mn in the diet;

[0408] 3) CON2: CON1 was reformulated based on the mineral matrix of phytase, with reduced Ca and retainable P, and supplemented with a novel common sequence bacterial 6-phytase variant (PhyG) at 2,000 FTU / kg and 1,500 FTU / kg during the feeding and growth phases, respectively, without supplementation of Zn, Cu, Fe, and Mn.

[0409] 4) CON2+TM: CON1 is reformulated based on the mineral matrix of phytase, with reduced Ca and retainable P. PhyG is supplemented at 2,000 FTU / kg and 1,500 FTU / kg during the feeding and growth stages, respectively, and additional Zn, Cu, Fe and Mn are supplemented (as in CON1+TM).

[0410] Processing details are given in Table 16. The composition of the diet and the calculated nutrient composition are given in Table 17.

[0411] Table 16. Processing Overview

[0412]

[0413] 1 The commercial premixes from treatments 2 and 4 were added to provide vitamins and trace minerals, which are present in the diet at concentrations of 60, 10, 50, and 70 mg / kg for Zn, Cu, Fe, and Mn, respectively. The premixes from treatments 1 and 3 provided vitamins and other trace minerals besides the added Zn, Cu, Fe, and Mn (see Table 17).

[0414] 2 In treatments 3 and 4, dietary calcium and retainable phosphorus decreased by 2.26 and 1.86 g / kg, respectively, during the initial feeding phase and by 2.18 and 1.79 g / kg, respectively, during the growth phase.

[0415] Table 17. Dietary composition and calculated nutrient composition

[0416]

[0417]

[0418]

[0419] 1 Special vitamin-mineral premixes have the same chemical composition as standard vitamin-mineral premixes, except that they do not contain Zn, Cu, Fe, and Mn.

[0420] 2 The standard vitamin-mineral premix is ​​a commercial premix that provides per kilogram of feed: 9000 IU vitamin A, 2800 IU vitamin D3; 20 IU vitamin E; 2 mg vitamin K3; 2 mg vitamin B1; 5 mg vitamin B2; 10.9 mg Ca-D-pantothenic acid; 40 mg nicotinamide; 3 mg vitamin B6; 1 mg folic acid; 20 µg vitamin B1. 12 70 µg biotin; 197 mg betaine hydrochloride; 50 mg Fe from sulfate; 1 mg iodine; 10 mg Cu from sulfate; 70 mg Mn from oxide; 60 mg Zn from oxide; 0.2 mg Se from Na-selenium

[0421] SID, Standardized Ileal Digestibility.

[0422] Table 18. Calculated and (italicized) analysis of macrominerals (Ca and P; g / kg) and microminerals (TM: Fe, Cu, Mn, Zn; mg / kg) in the experimental diet of Example 3.

[0423]

[0424] 2 The intake is 2,000 and 1,500 FTU / kg for the initial feeding stage and the growth stage, respectively.

[0425] 4 Start feeding (1 to 10 days old); growth (10 to 21 days old).

[0426] Sampling, Measurement, and Statistical Analysis: Body weight was measured at the beginning and end of each phase of the study and used to calculate body weight gain (BWG). Feed loss was measured at each phase to determine feed conversion ratio (FI). Feed conversion ratio was calculated for each phase for each pen and corrected for mortality. At 20 days of age, two males and two females were randomly selected from each pen and euthanized with CO2. Left and right tibias were collected, pooled by each pen, and stored at -20°C. For tibia ash analysis, the left tibias were thawed, autoclaved, and washed. The cleaned tibias were dried using standard procedures and defatted with 100% petroleum ether according to the Soxhlet principle. The defatted pooled tibias were air-dried, oven-dried, and then incinerated in a muffle furnace, first at 500°C and then at 700°C for 18 h. Trace minerals were analyzed in duplicate by inductively coupled plasma optical emission spectrometry (ICP-OES).

[0427] Outlier analysis was performed prior to data analysis. Outliers were defined as residuals exceeding 2.5 times the standard error of the mean. If any of the growth performance response measures for each stage was identified as an outlier, all performance response measures for that stage were removed from the dataset. Data were analyzed using one-way ANOVA. Fences were experimental units. Tukey's HSD test was used to distinguish treatment means. P < 0.05 was considered statistically significant. 0.05 ≤ P < 0.1 was considered a trend.

[0428] result

[0429] Growth performance: BW, BWG, FI, and FCR results by stage and cumulative are presented in Table 19. Throughout the period (1 to 21 days of age), phytase (CON2) increased BW and BWG and decreased FCR in the absence of phytase and without TM supplementation (CON1), reaching levels comparable to those achieved with TM supplementation in CON1 (CON1+TM) in each case. Adding supplemental TM on top of supplemental phytase did not result in a further increase in BW, BWG, or FCR throughout the period. Micromineral supplementation and phytase supplementation had no effect on FI throughout the period or in any single stage. Overall mortality levels remained low (below 1%) and were unaffected by treatment (data not shown).

[0430] Table 19. Effects of supplements containing trace minerals or phytase or both on growth performance during 1–21 days of age; single-factor ANOVA.

[0431]

[0432] 1 The values ​​presented are the values ​​at the end of the relevant phase.

[0433] Bone mineralization: The effects of supplements containing trace minerals, phytase, or both on trace mineral concentrations in the tibia are presented in Table 20. In CON1, discontinuing the TM supplement in CON1 reduced bone Mn and Zn concentrations. In CON2, phytase supplementation without TM increased bone Mn and Zn (by 2.61 mg / kg or 47.6% and 61.1 mg / kg or 20.3%, respectively), compared to CON2. In CON2+TM, the addition of TM supplementation on top of phytase resulted in a further increase in tibial Mn (+2.65 mg / kg or 32.8%), but no further increase in tibial Zn.

[0434] Table 20. Effects of supplements containing trace minerals or phytase, or both, on bone mineral content in 20-day-old broilers; single-factor ANOVA

[0435]

[0436] Conclusions: In commercially raised 1- to 21-day-old broilers, supplementation with PhyG phytase in corn-soybean meal-based diets completely replaced commercially available levels of supplemented Zn, Fe, Cu, and Mn. Growth performance (all measures during the start-up and growth phases, and overall) and tibia TM concentration levels maintained by phytase administered at 2,000 FTU / kg during the start-up phase and 1,500 FTU / kg during the growth phase were comparable to those achieved by a nutritionally adequate diet supplemented with TM but without phytase (with some improvement at the Zn concentration level). These results indicate that PhyG phytase can be used to replace commercially available levels of TM (including Zn, Fe, Cu, and Mn) in the diets of young broilers.

[0437] Example 4: Using a novel concordant sequence bacterial 6-phytase variant as an alternative to supplementation in commercial corn-soybean meal-based diets The effects of trace minerals on piglets

[0438] This case study evaluated whether phytase (PhyG; SEQ ID NO:26) could completely replace trace minerals (TM; Zn, Cu, Fe, and Mn) supplemented at commercial dose levels in piglets with a weight of 7 to 25 kg.

[0439] Materials and Methods

[0440] The study was conducted at the Bingen University of Applied Sciences in Germany.

[0441] Experimental design, animals, and housing: The experiment was conducted using a randomized complete block design with three dietary treatments, each with 12 replicates. Each pen contained four piglets (1:1 male / female). The pen dimensions were 3 m x 3 m. The lighting program consisted of 16 hours of light and 8 hours of darkness.

[0442] This study used a total of 144 Danny x Pietrain weaned piglets (weaned at 28 days old) with an initial weight of 7.0 (±0.44) kg (on the day of weaning). The diet was based on corn and SBM and was fed in two phases (days 1-14 and 14-42).

[0443] Feed treatment: Treatment includes:

[0444] 1) CON1: Control diet, nutritionally adequate, but without supplementation of Zn, Cu, Fe, and Mn.

[0445] 2) CON1 + TM: Same as CON1, containing commercially typical levels of supplemental trace minerals (Zn 120 ppm, Cu 80 ppm, Fe 96 ppm, Mn 80 ppm).

[0446] 3) CON2: CON1 was reformulated without the addition of inorganic P and with reduced Ca content. A novel shared-sequence phytase (PhyG) was supplemented at 1500 and 1000 FTU / kg in stages 1 and 2, respectively. No Zn, Cu, Fe, or Mn supplements were added.

[0447] Processing details are given in Table 21. The composition of the diet and the calculated nutrient composition are given in Table 22.

[0448] Table 21. Processing Overview

[0449]

[0450] 1 The supplemental premix provides: 120 ppm Zn from ZnSO4, 80 ppm Cu from CuSO4, 96 ppm Fe from FeSO4, and 80 ppm Mn from MnSO4.

[0451] 2 In treatment 3, Ca was reduced and monocalcium phosphate (MCP) was eliminated.

[0452] Table 22. Feed formulations and calculated nutrients:

[0453]

[0454]

[0455]

[0456] 1 The standard vitamin-mineral premix is ​​a commercial premix that provides per kilogram of feed: 16,000 IU vitamin A, 2,000 IU vitamin D3; 100 mg vitamin E; 4 mg vitamin K3; 97.5 mg vitamin C; 250 mg vitamin H; 2 mg vitamin B1; 8 mg vitamin B2; 20 mg Ca-D-pantothenic acid; 30 mg nicotinamide; 6 mg vitamin B6; 2 mg folic acid; and 40 mg vitamin B1. 12 500 mg choline chloride; 96 mg Fe from sulfate; 2.5 mg iodine; 80 mg Cu from sulfate; 80 mg Mn from sulfate; 120 mg Zn from sulfate; 0.4 mg Se from Na-selenium.

[0457] 2 Special vitamin-mineral premixes have the same chemical composition as standard vitamin-mineral premixes, except that they do not contain Zn, Cu, Fe, and Mn.

[0458] SID, Standardized Ileal Digestibility.

[0459] The content of nutrients and trace minerals analyzed in the diet is given in Table 23.

[0460] Table 23. Nutrients and trace minerals analyzed in the diet (by feeding):

[0461]

[0462] Sampling, measurement, and statistical analysis:

[0463] At the end of the study (day 42), blood samples were collected from one piglet in each pen for trace mineral analysis. Body weight (BW) and feed intake were recorded at each stage and at the beginning and end of each feeding period. Pigs were weighed individually and the weight was recorded, and averages were calculated for each pen. Feed was weighed and pen consumption was calculated for each stage. Feed conversion ratio (FCR) was calculated based on average pen weight gain and average pen feed intake, adjusted for mortality.

[0464] One-way ANOVA was performed on performance and blood trace mineral parameters using JMP (version 16.1). Using each pig as the experimental unit, with treatment as a fixed factor, initial body weight as the covariance, and blocks and sex as random factors, data on performance parameters (including body weight (BW) and average daily gain (ADG)) were analyzed. Using pens as the experimental unit and treatment as a fixed factor, average daily feed intake (ADFI) and feed rate (FCR) were analyzed. Tukey's HSD test was used to distinguish treatment means. P < 0.05 was considered statistically significant. 0.05 ≤ P < 0.1 was considered a trend.

[0465] result

[0466] Growth performance: BW, ADG, ADFI and FCR results by stage and cumulative are presented in Table 24.

[0467] Compared to the absence of phytase and the absence of TM supplementation (CON1), phytase (CON2) increased BW and overall ADG in the absence of TM supplementation, reaching levels comparable in each case to those achieved with TM supplementation in CON1 (CON1+TM). Micromineral supplementation and phytase supplementation had no effect on ADFI or FCR throughout the period or in any single phase. Mortality was low (< 5%) and there were no differences between treatments (data not shown).

[0468] Table 24. Effects of supplements containing trace minerals or phytase or both on growth performance of piglets; single-factor ANOVA.

[0469]

[0470] 1 In phases 1 and 2, a novel shared-sequence phytase (PhyG) was supplemented at 1500 and 1000 FTU / kg, respectively.

[0471] 2 It is supplemented with trace minerals, including 120 ppm Zn from ZnSO4, 80 ppm Cu from CuSO4, 96 ppm Fe from FeSO4, and 80 ppm Mn from MnSO4.

[0472] Blood Trace Mineral Content: The effects of supplements containing trace minerals, phytase, or both on the concentration of trace minerals in the blood are presented in Table 25. Compared to CON1 without TM, CON2 without TM supplementation showed an increase in the amount of Mn and Zn in phytase, but the difference was not statistically significant.

[0473] Table 25. Effects of supplements containing trace minerals or phytase, or both, on blood trace mineral concentrations at the end of the study (day 42), single-factor ANOVA

[0474]

[0475] 1 In phases 1 and 2, a novel shared-sequence phytase (PhyG) was supplemented at 1500 and 1000 FTU / kg, respectively.

[0476] 2 It is supplemented with trace minerals, including 120 ppm Zn from ZnSO4, 80 ppm Cu from CuSO4, 96 ppm Fe from FeSO4, and 80 ppm Mn from MnSO4.

[0477] Conclusion: In piglets with a body weight (BW) of 7–25 kg, supplementation with Phytase G in corn-soybean meal-based diets completely replaced commercially available levels of supplemented Zn, Fe, Cu, and Mn. The body weight levels maintained by phytase administered at 1500 FTU / kg during Phase 1 and 1000 FTU / kg during Phase 2 were comparable to those achieved with a nutritionally adequate diet supplemented with TM in the absence of phytase. These results indicate that Phytase G can be used to replace commercially available levels of TM (including Zn, Fe, Cu, and Mn) in piglet diets.

[0478] Example 5: The effect of novel concordant sequence bacterial 6-phytase variants included in formulated corn-soybean meal-based diets on the reduction of... Efficacy of low Campylobacter levels in broilers

[0479] This example demonstrates the effect of a specially formulated broiler diet free of exogenous iron and inorganic phosphate (IP-free) and supplemented with a novel concordant sequence bacterial 6-phytase variant (PhyG; SEQ ID NO:26) on the prevalence and load of Campylobacter in broilers. In this example, all poultry were challenged orally with Campylobacter jejuni via gavage at 14 days of age, and Campylobacter spp. in the cecum were counted at slaughter age (D42). When fed to broilers, the "test diet" without added iron and without IP, supplemented with PhyG, consistently maintained growth performance; more birds were Campylobacter-negative; fewer birds had high colonization (super-colonizers); and the average Campylobacter spp. load was reduced by approximately 1 Log. 10 .

[0480] Materials and Methods

[0481] This study was conducted at the Southern Poultry Research Group (SPRG) in Georgia, USA. The experimental protocol and procedures were evaluated and approved by the institute's Animal Ethics Committee and complied with local regulations regarding the use of animals in research.

[0482] Experimental Design, Poultry, and Housing: The experiment was conducted using a randomized design with two diet treatments, 10 replicate floor-fenced enclosures, and 25 birds / enclosures. A total of 500 Ross 308 male broilers were assigned to enclosures on hatch day to ensure that each enclosure contained birds of roughly equal average weight. The enclosures were housed in broiler housing with clean wood shavings as bedding. Temperature was maintained according to the breeder’s recommendations for the age of the birds. The birds were provided with 24 hours (24 hours) of light per day for the first 48 (48) hours, after which the light was reduced to 20 hours per day until the end of the study.

[0483] Feed treatment: The treated diet was formulated in three phases: the initial feeding phase for 1–14 days of age, the growth phase for 15–28 days of age, and the finishing phase for 29–42 days of age. The treatment included:

[0484] 1) The positive control diet for the attack; formulated with dicalcium phosphate and a premix containing added iron to meet the nutritional requirements of broilers;

[0485] 2) Test diets for attack; formulated to meet the nutritional needs of broilers, but without dicalcium phosphate and using oxide-based premixes without added Fe at all stages.

[0486] The composition of the diets and the calculated nutrient composition are given in Table 26, and the analyzed total Fe content is given in Table 27. Total Fe content is the sum of the Fe content added via the premix and the Fe content of the base feed portion. The measured total Fe levels confirmed that the deficiency of Fe added via the premix resulted in a significant reduction in the total Fe level in the test diets.

[0487] Table 26. Composition and calculated nutrient composition of the two diets used in Example 5

[0488]

[0489] Table 27. Total Fe mineral content of the experimental diets analyzed in Example 5.

[0490]

[0491] Campylobacter challenge: On the day of arrival, five additional birds from this batch were randomly euthanized, and their cecum, liver, and spleen were aseptically extracted and analyzed using a Campylobacter culture-dependent method to confirm a negative result for Campylobacter. On day 7, boot swab analysis confirmed a negative result for Campylobacter in the poultry house. On day 14, all birds in the experiment were orally administered 0.1 ml of approximately 1.0 x 10⁻⁶ spores. 6 CFU / ml (approximately 1.0 x 10⁻⁶) 5 Campylobacter jejuni strain JB (CFU / chicken). On day 42, poultry were treated as described below to determine the colonization characteristics of the Campylobacter genus.

[0492] Sampling, Measurement, and Statistical Analysis: Performance parameters, including body weight (BW), free flow rate (FI), free flow rate (FCR), and survival rate, were measured at the end of each feeding phase (days 14, 28, and 42). Performance results were analyzed using one-way ANOVA (JMP 16.0). P < 0.05 was considered statistically significant. 0.05 ≤ P < 0.1 was considered a trend. On day 42, 10 birds in each pen (100 birds per treatment) were euthanized via neck dislocation, and the cecum was aseptically removed and placed in a sterile sampling bag for Campylobacter spp. counting using a culture-dependent method. Briefly, aliquots of 1 ml gastrocecal samples (MRD culture medium) were transferred to 96-well plates. Serial dilutions were performed 10-fold, and the diluted solutions were spread onto Campy-Cefex agar plates and incubated at 42°C for 48 hours under microaerophilic conditions before counting. Kruskal-Wallis nonparametric analysis of variance (ANOVA) was used to assess cecal load. P < 0.05 was considered statistically significant.

[0493] result

[0494] Growth performance: Results for body weight (BW), BW gain (BWG), feed intake (FI), feed conversion ratio (FCR), and survival rate by stage and overall are presented in Table 28. Overall, the test diet (formulated as follows: iron-free, IP-free, and using oxide-based premix and PhyG) resulted in a higher BW value at day 42 (2655.3 g vs. 2524.9 g in PC), although this was not statistically significant (P = 0.11). Survival rate improved numerically from 94.60% to 96.80% at day 42.

[0495] Table 28: Effects of phytase supplementation on dietary stages and overall growth performance. Single-factor ANOVA 1 .

[0496]

[0497] 1 P < 0.05 is considered statistically significant. 0.05 ≤ P < 0.1 is considered a trend.

[0498] 2 FCRc = Weight-corrected FCR

[0499] Campylobacter was observed in poultry fed a test diet. The test diet showed 84% (84 out of 100) Campylobacter colonization in the cecum compared to 88% (88 / 100) in the PC diet. Both the positive control and the test diet showed varying degrees of Campylobacter load per cecum; however, the test diet, lacking IP, Fe, and formulated with oxide-based premixes, showed approximately 1 Log lower than the highest levels achieved. 10 CFU, overall, reduced the high sowing rate of Campylobacter spp. entering the environment (2.45 x 10⁻⁶ in PC and test diets). 10 Compared to 2.97 x 10 9 The CFU / g figure, although not statistically significant (P > 0.05), was likely due to high variability in recorded CFU levels. Additionally, compared to PC, the mean Campylobacter load in the test diet was significantly reduced by nearly 1 Log. 10 However, this was not statistically significant, possibly due to variations in overall colonization levels (PC and test diet were 3.78 x 10⁻⁶). 8 CFU / g relative to 4.90 x 10 7 CFU / g).

[0500] The effects of the test diet formulations on reducing Campylobacter colonization are visualized in Table 29. The test diets resulted in colonization rates higher than 1 x 10⁻⁶. 8 Fewer colonists had high CFU / g thresholds (2% of all sampled poultry compared to 6%). Furthermore, the tested diets resulted in a reduction of the second defining threshold for Campylobacter colonization to 10. 6 CFU / gram cecum. 30% of PC poultry have levels exceeding 10. 6 CFU / g, which is only 20% of the amount in the test diet feeding treatment.

[0501] Table 29. Campylobacter load in the cecum of broilers on day 42 (positive poultry %)

[0502]

[0503] Conclusion: Overall, diets formulated without added Fe, inorganic phosphates, and utilizing oxide-based premixes and PhyG resulted in a reduction in mean Campylobacter spp. load to below the recommended processing contamination threshold (1 x 10⁻⁶). 8 The level of CFU / g cecum was reduced; subsequent extended targets (1 x 10) were lowered. 6 CFU / g cecum) to further reduce the spread of Campylobacter in the processing plant; reduce super-colonizers and potential super-shedders (driving the spread of infection) while maintaining final weight (a key production indicator).

[0504] Effective control of Campylobacter is a multi-pronged approach. Reducing Campylobacter levels on farms will decrease transmission and contamination at slaughterhouses / processing plants, fundamentally reducing the likelihood of edible poultry tissues being contaminated with Campylobacter and lowering the risk of foodborne illnesses after human handling and consumption. References

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Claims

1. An animal diet comprising (i) an engineered phytase polypeptide or a fragment thereof containing phytase activity; and (ii) lacking one or more exogenously added trace minerals.

2. The feed as claimed in claim 1, wherein the one or more trace minerals are selected from the group consisting of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn) and selenium (Se).

3. The feed as claimed in claim 1 or claim 2, wherein the phytase polypeptide or a fragment thereof containing phytase activity has at least 82% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

4. The diet according to any one of claims 1-3, wherein the phytase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:3 ...9, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO NO:35, SEQ ID NO:36 and SEQ ID NO:

37.

5. The diet according to any one of claims 1-4, wherein the diet further comprises one or more of the following: (a) a direct-feeding microorganism containing at least one bacterial strain, (b) at least one other enzyme, and / or (c) essential oil.

6. The diet according to any one of claims 1-5, wherein the engineered phytase polypeptide or a fragment thereof is present in an amount of at least about 0.1 g / ton of feed.

7. The diet according to any one of claims 1-6, wherein the diet contains about 0.62% to 0.72% calcium in the starter diet, about 0.54% to 0.64% in the growing diet, and / or about 0.42% to 0.55% calcium in the finishing diet.

8. The feed as claimed in claim 7, wherein the fattening feed contains about 0.46% to 0.55% or about 0.42% to 0.50% calcium.

9. The diet according to any one of claims 1-8, wherein the diet contains about 1.18% to 1.22% amino acids in the starter diet, about 1.06% to 1.10% in the growing diet, and / or about 0.88% to 1% in the finishing diet.

10. The feed as claimed in claim 9, wherein the fattening feed contains about 0.96% to 1.0% or about 0.88% to 0.92% amino acids.

11. The diet of claim 9 or claim 10, wherein the amino acid comprises digestible lysine.

12. The diet according to any one of claims 1-11, wherein the diet contains about 2824 to 2950 kcal / kg in an initial feeding diet, about 2924 to 3050 kcal / kg in a growing diet, and / or about 2970 to 3120 kcal / kg in a finishing diet.

13. The diet of claim 12, wherein the fattening diet contains about 2970 to 3100 kcal / kg or about 2994 to 3120 kcal / kg of metabolizable energy.

14. The diet according to any one of claims 1-13, wherein the diet contains about 0.13% to 0.17% sodium.

15. The diet according to any one of claims 1-14, wherein the phytase is present at a dose between about 500 FTU / kg and about 2000 FTU / kg.

16. The diet according to any one of claims 1-15, wherein the diet contains a phytate source, the phytate source comprising one or more of corn, wheat, soybean meal, rapeseed, rice and / or wheat bran.

17. The feed as claimed in any one of claims 1-16, wherein the feed further comprises oat bran.

18. The diet according to any one of claims 1-17, wherein the diet is deficient in meat and / or bone meal.

19. The diet according to any one of claims 1-18, wherein the diet further comprises one or more additional feed enzymes selected from the group consisting of xylanase, protease, amylase and glucosylamylase.

20. The feed as claimed in any one of claims 1-19, wherein the feed is an starter feed.

21. The feed diet according to any one of claims 1-20, wherein the feed diet is a growth feed diet.

22. The feed diet according to any one of claims 1-21, wherein the feed diet is a fattening feed diet.

23. The feed as claimed in any one of claims 1-22, wherein the animal is poultry.

24. The feed as claimed in any one of claims 1-22, wherein the animal is a pig.

25. The feed as claimed in any one of claims 1-22, wherein the animal is a ruminant.

26. The feed of claim 25, wherein the ruminant is a calf.

27. The diet according to any one of claims 1-26, wherein the diet further contains no or substantially no exogenously added inorganic phosphates.

28. A method for improving animal performance in one or more indicators, the method comprising administering to the animal an effective amount of a diet as described in any one of claims 1-27.

29. The method of claim 28, wherein the one or more indicators are selected from the group consisting of: increased feed efficiency, increased weight gain, decreased feed conversion ratio, improved digestibility of nutrients or energy in feed, improved nitrogen retention, improved ability to avoid the negative effects of necrotizing enteritis, and improved immune response.

30. The method of claim 28 or claim 29, wherein the animal is poultry, pig, or ruminant.

31. The method of claim 30, wherein the poultry is selected from the group consisting of turkey, duck, chicken, goose, pheasant, quail and emu.

32. The method of claim 31, wherein the chicken is selected from the group consisting of laying hens and broilers.

33. A method for reducing pathogenic bacterial populations in an animal, the method comprising administering to the animal an effective amount of a diet as described in any one of claims 1-27.

34. The method of claim 33, wherein a) the one or more trace minerals are iron (Fe); and / or b) the diet is further free of or substantially free of exogenously added inorganic phosphates.

35. The method of claim 33 or claim 34, wherein the pathogenic bacterial community is one or more bacteria selected from the group consisting of: Actinobacillus, Bordetalla, Campylobacter, Clostridium, Corynebacterium, Escherichia coli, Globicatella, Listeria, Mycobacterium, Salmonella, Staphylococcus, and Streptococcus.

36. The method of any one of claims 34-35, wherein the animal is poultry, pig, or ruminant.

37. The method of claim 36, wherein the poultry is selected from the group consisting of turkey, duck, chicken, goose, pheasant, quail and emu.

38. The method of claim 36, wherein the chicken is selected from the group consisting of laying hens and broilers.

39. The method of claim 36, wherein the ruminant is a calf.

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