# Patent

## Title
Phytase-containing animal food and method

## Abstract

A method is described for improving the nutritional value of a foodstuff comprising a source of myo-inositol hexakisphosphate by feeding the foodstuff in combination with a phytase expressed in yeast. The method comprises the step of feeding the animal the foodstuff in combination with a phytase expressed in yeast wherein the phytase can be selected from the group consisting of AppA1, AppA2 and a site-directed mutant of AppA. The invention also enables reduction of the feed to weight gain ratio and an increase bone mass and mineral content of an animal. A foodstuff and a feed additive comprising AppA2 or a site-directed mutant of AppA are also described.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C § 119(e) to U.S. Provisional Application Ser. No. 60/335,303, filed on Oct. 31, 2001.


FIELD OF THE INVENTION
The present invention is related to a method of improving the nutritional value of a foodstuff and to an improved foodstuff. More particularly, the invention relates to a method of improving the nutritional value of a foodstuff comprising myo-inositol hexakisphosphate by feeding the foodstuff to an animal in combination with a phytase expressed in yeast.
BACKGROUND AND SUMMARY OF THE INVENTION
Phytases are myo-inositol hexakisphosphate phosphohydrolases that catalyze the stepwise removal of inorganic orthophosphate from phytate (myo-inositol hexakisphosphate). Phytate is the major storage form of phosphate in plant feeds, including cereals and legumes. Because monogastric animals such as pigs, poultry, and humans have little phytase in their gastrointestinal tracts nearly all of the ingested phytate phosphate is indigestible. Accordingly, these animals require supplementation of their diets with phytase or inorganic phosphate. In contrast, ruminants have microorganisms in the rumen that produce phytases and these animals do not require phytase supplementation of their diets.
The unutilized phytate phosphate in monogastric animals creates additional problems. The unutilized phytate phosphate is excreted in manure and pollutes the environment. Furthermore, in monogastric animals phytate passes largely intact through the upper gastrointestinal tract where it chelates essential minerals (e.g., calcium and zinc), binds amino acids and proteins, and inhibits enzyme activities. Accordingly, phytase supplementation of the diets of monogastric animals not only decreases requirements for supplementation with inorganic phosphate, but also reduces pollution of the environment caused by phytate, diminishes the antinutritional effects of phytate, and increases the nutritional value of the feed.
There are two types of phytases including a 3-phytase (EC.3.1.3.8) which removes phosphate groups at the 1 and 3 positions of the myo-inositol ring, and a 6-phytase (EC.3.1.3.6) which first frees the phosphate at the 6-position of the ring. Plants usually contain 6-phytases and a broad range of microorganisms, including bacteria, filamentous fungi, and yeasts, produce 3-phytases. Two phytases, phyA and phyB from Aspergillus niger, have been cloned and sequenced. PhyA has been expressed in Aspergillus niger and the recombinant enzyme is available commercially for use in supplementing animal diets.
Phytase genes have also been isolated from Aspergillus terreus, Myceliophthora thermophila, Aspergillus fumigatus, Emericella nidulans, Talaromyces thermophilus, Escherichia coli (appA), and maize. Additionally, phytase enzymes have been isolated and/or purified from Bacillus sp., Enterobacter sp., Klebsiella terrigena, and Aspergillus ficum. 
The high cost of phytase production has restricted the use of phytase in the livestock industry as phytase supplements are generally more expensive than the less environmentally desirable inorganic phosphorous supplements. The cost of phytase can be reduced by enhancing production efficiency and/or producing an enzyme with superior activity.
Yeast expression systems can be used to effectively produce enzymes, in part, because yeast are grown in simple and inexpensive media. Additionally, with a proper signal sequence, the expressed enzyme can be secreted into the culture medium for convenient isolation and purification. Some yeast expression systems are also accepted in the food industry as being safe for the production of food products unlike fungal expression systems which may in some cases be unsafe, for example, for human food manufacturing.
Thus, one aspect of this invention is a method of improving the nutritional value of a foodstuff by supplementing the foodstuff with a yeast-expressed phytase with superior capacity to release phosphate from phytate in foodstuffs. The invention is also directed to a foodstuff with improved nutritional value comprising the yeast-expressed phytase. The phytase can be efficiently and inexpensively produced because the yeast-expressed phytase of the present invention is suitable for commercial use in the feed and food industries with minimal processing.
In one embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by a monogastric animal by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the animal the foodstuff in combination with less than 1200 units of a phytase expressed in yeast per kilogram of the foodstuff, wherein the phytase is Escherichia coli-derived AppA2, and wherein the bioavailability of phosphate from phytate is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell.
In another embodiment, a method is provided of reducing the feed to weight gain ratio of a monogastric animal by feeding the animal a foodstuff wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the animal the foodstuff in combination with a phytase expressed in yeast, wherein the phytase is selected from the group consisting of Escherichia coli-derived AppA2 and a site-directed mutant of Escherichia coli-derived AppA, and wherein the feed to weight gain ratio of the animal is reduced.
In an alternate embodiment, a method of improving the nutritional value of a foodstuff consumed by a monogastric animal by increasing the bone mass and mineral content of the animal wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the animal the foodstuff in combination with a phytase expressed in yeast wherein the phytase is selected from the group consisting of Escherichia coli-derived AppA2 and a site-directed mutant of Escherichia coli-derived AppA, and wherein the bone mass and mineral content of the animal is increased.
In yet another embodiment, a feed additive composition for addition to an animal feed is provided. The feed additive composition comprises a yeast-expressed phytase and a carrier for the phytase wherein the concentration of the phytase in the feed additive composition is greater than the concentration of the phytase in the final feed mixture.
In still another embodiment, a foodstuff is provided. The foodstuff comprises the above-described feed additive composition wherein the concentration of the phytase in the final feed mixture is less than 1200 units of the phytase per kilogram of the final feed mixture.
In another embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the steps of spray drying a phytase selected from the group consisting of Escherichia coli-derived AppA2 and a site-directed mutant of Escherichia coli-derived AppA, mixing the phytase with a carrier for the phytase and, optionally, other ingredients to produce a feed additive composition for supplementing a foodstuff with the phytase, mixing the feed additive composition with the foodstuff, and feeding the animal the foodstuff supplemented with the feed additive composition.
In an alternate embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by an avian species by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with less than 1200 units of a phytase expressed in yeast per kilogram of the foodstuff, wherein the bioavailability of phosphate from phytate is increased by at least 1.5-fold compared to the bioavailability of phosphate from phytate obtained by feeding to a non-avian species the foodstuff in combination with the phytase expressed in yeast.
In yet another embodiment, a method is provided of reducing the feed to weight gain ratio of an avian species by feeding the avian species a foodstuff wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with a phytase expressed in yeast wherein the feed to weight gain ratio of the animal is reduced.
In still another embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by an avian species by increasing the bone mass and mineral content of the avian species wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with a phytase expressed in yeast wherein the bone mass and mineral content of the avian species is increased.
In another embodiment, a method is provided of improving the nutritional value of a foodstuff consumed by an avian species wherein the foodstuff comprises myo-inositol hexakisphosphate. The method comprises the step of feeding to the avian species the foodstuff in combination with a phytase expressed in yeast wherein the number of eggs laid and the weight of the eggs laid by the avian species is increased.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid and nucleotide sequences of AppA2.
FIG. 2 shows the amino acid and nucleotide sequences of Mutant U.
FIG. 3 shows the percent increase in bioavailable phosphate in vivo in chickens fed an animal feed supplemented with Natuphos®, Mutant U, AppA or AppA2.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of improving the nutritional value of a foodstuff consumed by an animal wherein the foodstuff comprises myo-inositol hexakisphosphate, the substrate for the phytase enzymes of the invention. The method comprises the step of feeding to an animal the foodstuff in combination with a phytase expressed in yeast wherein the bioavailability of phosphate from phytate is increased, the feed to weight gain ratio is reduced, the bone mass and mineral content of the animal is increased or, for avian species, additionally the egg weight or number of eggs laid is increased. The phytase can be selected from the group consisting of Escherichia coli-derived AppA2 and a site-directed mutant of Escherichia coli derived-AppA. In an alternative embodiment, for avian species, the phytase can be any phytase, including phytases selected from the group consisting of Escherichia coli-derived AppA, Escherichia coli-derived AppA2, and a site-directed mutant of Escherichia coli-derived AppA. In some embodiments, the bioavailability of phosphate from phytate, the feed to weight gain ratio, and bone mass and mineral content are improved by at least 2-fold, for example, in an avian species, such as poultry, compared to the improvement in nutritional value obtained by feeding the foodstuff in combination with the same weight percent of a phytase expressed in a non-yeast host cell. The bioavailability of phosphate from phytate is also increased by at least 1.5-fold in porcine species compared to the improvement in nutritional value obtained by feeding the foodstuff in combination with the same weight percent of a phytase expressed in a non-yeast host cell. Additionally, the bioavailability of phosphate from phytate and the bone mass and mineral content obtained by feeding an avian species the foodstuff in combination with the phytase expressed in yeast is increased by at least 1.5-fold compared to the bioavailability of phosphate from phytate and the bone mass and mineral content obtained by feeding a non-avian species the foodstuff in combination with the yeast-expressed phytase.
As used herein “improving nutritional value” or “increased nutritional value” means an improvement in the nutritional value of a foodstuff as reflected by an increase in the bioavailability of phosphate from phytate, a reduction in the feed to weight gain ratio, an increase in bone mass and mineral content, an increase in the bioavailability of inositol from phytate, an increase in the bioavailability from phytate of minerals such as magnesium, manganese, calcium, iron and zinc in an animal fed the foodstuff, or an increase in egg weight or number of eggs laid for an avian species fed the foodstuff (e.g., for laying hens in the first or subsequent round of laying eggs).
As used herein an increase in the “bioavailability of phosphate from phytate” means an increase in availability of phosphate from phytate as reflected by an increase in weight gain or bone ash weight.
As used herein the term “non-yeast host cell” includes a fungal cell.
As used herein, the term “phytase” means an enzyme capable of catalyzing the removal of inorganic phosphate from myo-inositol hexakisphosphate.
As used herein, the term “phytate” means a composition comprising myo-inositol hexakisphosphate.
In accordance with the invention, the feed to weight gain ratio is calculated by dividing weight gain by feed intake. An increase in bone mass or mineral content is reflected by an increase in the dry weight of tibia or fibula bones or by an increase in ash weight.
A variety of phytase genes may be expressed to produce phytase for use in accordance with the invention. Exemplary of genes that can be used in accordance with the invention are phytase genes derived from bacteria, filamentous fungi, plants, and yeast, such as the appA (Gene Bank accession number M58708) and appA2 (Gene Bank accession number 250016) genes derived from Escherichia coli (E. coli) and the phyA and phyB genes derived from the fungus Aspergillus niger, or any site-directed mutant of these genes that retains or has improved myo-inositol hexakisphosphate phosphohydrolase activity.
Phytase genes can be obtained from isolated microorganisms, such as bacteria, fungus, or yeast, that exhibit particularly high phytase activity. As described below, the appA2 gene was cloned from such an E. coli isolate, and it is exemplary of such a phytase gene.
The expressed phytase gene can be a heterologous gene, or can be a homologous gene. A heterologous gene is defined herein as a gene originating from a different species than the species used for expression of the gene. For example, in the case of expression of a heterologous phytase gene, a phytase gene derived from E. coli or another species of bacteria can be expressed in a yeast species such as Saccharomyces cerevisiae or Pichia pastoris. A homologous gene is described herein as a gene originating from the same species used for expression of the gene. In the case of expression of a homologous phytase gene, a phytase gene derived from Saccharomyces cerevisiae can be expressed, for example, in the same yeast species.
Exemplary genes for use in producing phytase for use in accordance with the invention are appA, appA2, and site-directed mutants of appA or appA2. Substituted, deleted, and truncated phytase genes, wherein the resulting expressed phytase, or a fragment thereof, retains substantially the same phytase activity as the phytases specifically exemplified herein, are considered equivalents of the exemplified phytase genes and are within the scope of the present invention.
The appA gene was isolated from E. coli (see U.S. Pat. No. 6,451,572, incorporated herein by reference). The appA2 gene was isolated from a bacterial colony that exhibited particularly high phytase activity obtained from the colon contents of crossbred Hampshire-Yorkshire-Duroc pigs (see U.S. patent application Ser. No. 09/540,149, incorporated herein by reference). The AppA2 protein product exhibits a pH optimum between about 2.5 and about 3.5. The amino acid sequence of AppA2 is as shown in SEQ ID Nos.: 2, 3, and 10. FIG. 1 shows the amino acid and nucleotide sequences of AppA2. The untranslated region is indicated by lowercase letters. The underlined sequences are the primers used to amplify appA2 (Pfl: 1-22, and K2: 1468-1490), appA2 (E2: 243-252, and K2: 1468-1490). Potential N-glycosylation sites are boxed. The sequence of appA2 has been transmitted to Genebank data library with accession number 250016. The nucleotide sequence of AppA2 is as shown in SEQ ID No.: 1.
Several site-directed mutants of appA have been isolated (see PCT Publication No. WO 01/36607 A1 (U.S. patent application Ser. No. 60/166,179, incorporated herein by reference)). These mutants were designed to enhance glycosylation of the AppA enzyme. The mutants include A131N/V134N/D207N/S211N, C200N/D207N/S211N (Mutant U), and A131N/V134N/C200N/D207N/S211N (see Rodriguez et al., Arch. of Biochem. and Biophys. 382: 105-112 (2000), incorporated herein by reference). Mutant U has a higher specific activity than AppA, and, like AppA2, has a pH optimum of between about 2.5 and about 3.5. The C200N mutation in Mutant U is in a gapped region and C200 is involved with C210 in forming a unique disulfide bond in AppA. FIG. 2 shows the amino acid and nucleotide sequences of Mutant U. The amino acid sequence of Mutant U is shown in SEQ ID No.: 5, and the nucleotide sequence of Mutant U is shown in SEQ ID No.: 4.
Any yeast expression system or other eukaryotic expression system known to those skilled in the art can be used in accordance with the present invention. For example, various yeast expression systems are described in U.S. patent application Ser. No. 09/104,769 (now U.S. Pat. No. 6,451,572), U.S. patent application Ser. No. 09/540,149, and in U.S. patent application Ser. No. 60/166,179 (PCT Publication No. WO 01/36607 A1), all incorporated herein by reference. Any of these yeast expression systems can be used. Alternatively, other eukaryotic expression systems can be used such as an insect cell expression system (e.g., Sf9 cells), a fungal cell expression system (e.g., Trichoderma), or a mammalian cell expression system.
A yeast expression system can be used to produce a sufficient amount of the phytase being secreted from the yeast cells so that the phytase can be conveniently isolated and purified from the culture medium. Secretion into the culture medium is controlled by a signal peptide (e.g., the phyA signal peptide or yeast α-factor signal peptide) capable of directing the expressed phytase out of the yeast cell. Other signal peptides suitable for facilitating secretion of the phytase from yeast cells are known to those skilled in the art. The signal peptide is typically cleaved from the phytase after secretion.
If a yeast expression system is used, any yeast species suitable for expression of a phytase gene can be used including such yeast species as Saccharomyces species (e.g., Saccharomyces cerevisiae), Kluyveromyces species, Torulaspora species, Schizosaccharomyces species, and methylotrophic yeast species such as Pichia species (e.g., Pichia pastoris), Hansenula species, Torulopsis species, Candida species, and Karwinskia species. In one embodiment the phytase gene is expressed in the methylotrophic yeast Pichia pastoris. Methylotrophic yeast are capable of utilizing methanol as a sole carbon source for the production of the energy resources necessary to maintain cellular function, and contain a gene encoding alcohol oxidase for methanol utilization.
Any host-vector system known to the skilled artisan (e.g., a system wherein the vector replicates autonomously or integrates into the host genome) and compatible with yeast or another eukaryotic cell expression system can be used. In one embodiment, the vector has restriction endonuclease cleavage sites for the insertion of DNA fragments, and genetic markers for selection of transformants. The phytase gene can be functionally linked to a promoter capable of directing the expression of the phytase, for example, in yeast, and, in one embodiment, the phytase gene is spliced in frame with a transcriptional enhancer element and has a terminator sequence for transcription termination (e.g., HSP150 terminator). The promoter can be a constitutive (e.g., the 3-phospho-glycerate kinase promoter or the α-factor promoter) or an inducible promoter (e.g., the ADH2, GAL-1-10, GAL 7, PHO5, T7, or metallothionine promoter). Various host-vector systems are described in U.S. patent application Ser. No. 09/104,769 (now U.S. Pat. No. 6,451,572), U.S. patent application Ser. No. 09/540,149, and in U.S. patent application Ser. No. 60/166,179 (PCT Publication No. WO 01/36607 A1), all incorporated herein by reference.
Yeast cells are transformed with a gene-vector construct comprising a phytase gene operatively coupled to a yeast expression system using procedures known to those skilled in the art. Such transformation protocols include electroporation and protoplast transformation.
The transformed yeast cells may be grown by a variety of techniques including batch and continuous fermentation in a liquid medium or on a semi-solid medium. Culture media for yeast cells are known in the art and are typically supplemented with a carbon source (e.g., glucose). The transformed yeast cells can be grown aerobically at 30° C. in a controlled pH environment (a pH of about 6) and with the carbon source (e.g., glucose) maintained continuously at a predetermined level known to support growth of the yeast cells to a desired density within a specific period of time.
The yeast-expressed phytase for use in accordance with the method of the present invention can be produced in purified form by conventional techniques (for example, at least about 60% pure, or at least about 70-80% pure). Typically, the phytase is secreted into the yeast culture medium and is collected from the culture medium. For purification from the culture medium the phytase can, for example, be subjected to ammonium sulfate precipitation followed by DEAE-Sepharose column chromatography. Other conventional techniques known to those skilled in the art can be used such as gel filtration, ion exchange chromatography, DEAE-Sepharose column chromatography, affinity chromatography, solvent-solvent extraction, ultrafiltration, and HPLC. Alternatively, purification steps may not be required because the phytase may be present in such high concentrations in the culture medium that the phytase is essentially pure in the culture medium (e.g., 70-80% pure).
In cases where the phytase is not secreted into the culture medium, the yeast cells can be lysed, for example, by sonication, heat, or chemical treatment, and the homogenate centrifuged to remove cell debris. The supernatant can then be subjected to ammonium sulfate precipitation, and additional fractionation techniques as required, such as gel filtration, ion exchange chromatography, DEAE-Sepharose column chromatography, affinity chromatography, solvent-solvent extraction, ultrafiltration, and HPLC to purify the phytase. It should be understood that the purification methods described above for purification of phytases from the culture medium or from yeast cells are nonlimiting and any purification techniques known to those skilled in the art can be used to purify the yeast-expressed phytase if such techniques are required to obtain a substantially pure phytase.
In one embodiment, the phytase is collected from the culture medium without further purification steps by chilling the yeast culture (e.g., to about 8° C.) and removing the yeast cells using such techniques as centrifugation, microfiltration, and rotary vacuum filtration. The phytase in the cell-free medium can be concentrated by such techniques as, for example, ultrafiltration and tangential flow filtration.
Various formulations of the purified phytase preparation may be prepared. The phytase enzymes can be stabilized through the addition of other proteins (e.g., gelatin and skim milk powder), chemical agents (e.g., glycerol, polyethylene glycol, EDTA, potassium sorbate, sodium benzoate, and reducing agents and aldehydes), polysaccharides, monosaccharides, lipids (hydrogenated vegetable oils), sodium phytate, and other phytate-containing compounds, and the like. Phytase enzyme suspensions can also be dried (e.g., spray drying, drum drying, and lyophilization) and formulated as powders, granules, pills, mineral blocks, liquids, and gels through known processes. Gelling agents such as gelatin, alginate, collagen, agar, pectin and carrageenan can be used. The invention also extends to a feed innoculant preparation comprising lyophilized nonpathogenic yeast which can express the phytases of the present invention in the gastrointestinal tract of the animal when the animal is fed the preparation.
In one embodiment, the phytase in the cell-free culture medium is concentrated such as by ultrafiltration and spray drying of the ultrafiltration retentate. The spray dried powder can be blended directly with a foodstuff, or the spray dried powder can be blended with a carrier for use as a feed additive composition for supplementation of a foodstuff with phytase. In one embodiment, the phytase in the retentate is co-dried with a carrier and/or stabilizer. In another embodiment, the phytase is spray dried with an ingredient that helps the spray dried phytase to adhere to a carrier, or, alternatively, the phytase can loosely associate with the carrier. The feed additive composition (i.e., the phytase/carrier composition and, optionally, other ingredients) can be used for blending with the foodstuff to achieve more even distribution of the phytase in the foodstuff.
Exemplary feed additive compositions (i.e., phytase/carrier compositions and, optionally, other ingredients) can contain 600 units of phytase/gram of the carrier to 5000 units of phytase/gram of the carrier. These phytase/carrier compositions can contain additional ingredients. For example, the compositions can be formulated to contain rice hulls or wheat middlings as a carrier (25-80 weight percent), the phytase (0.5 to 20 weight percent), calcium carbonate (10 to 50 weight percent), and oils (1 to 3 weight percent). Alternatively, the feed additive composition can include the phytase and the carrier and no additional ingredients. The feed additive composition may be mixed with the feed to obtain a final feed mixture with from about 50 to about 2000 units of phytase/kilogram of the feed.
Thus, a foodstuff comprising a source of myo-inositol hexakisphosphate, a yeast-expressed phytase, and a carrier is also provided in accordance with the invention. Additionally, a method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff comprises myo-inositol hexakisphosphate is provided wherein the method comprises the steps of spray drying a phytase, including a phytase selected from the group consisting of Escherichia coli-derived AppA, Escherichia coli-derived AppA2, and a site-directed mutant of Escherichia coli-derived AppA, mixing the phytase with a carrier, and, optionally, other ingredients, to produce a feed additive composition for supplementing a foodstuff with the phytase, mixing the feed additive composition with the foodstuff, and feeding the animal the foodstuff supplemented with the feed additive composition.
In these embodiments, the carrier can be any suitable carrier for making a feed additive composition known in the art including, but not limited to, rice hulls, wheat middlings, a polysaccharide (e.g., specific starches), a monosaccharide, mineral oil, vegetable fat, hydrogenated lipids, calcium carbonate, gelatin, skim milk powder, phytate and other phytate-containing compounds, a base mix, and the like. A base mix typically comprises most of the ingredients, including vitamins and minerals, of a final feed mixture except for the feed blend (e.g., cornmeal and soybean meal). The phytase for use in the feed additive composition is preferably E. coli-derived AppA, E. coli-derived AppA2, or a site-directed mutant of E. coli-derived AppA.
The feed additive composition containing the spray dried phytase and a carrier and, optionally, other ingredients, is mixed with the final feed mixture to obtain a feed with a predetermined number of phytase units/kilogram of the feed (e.g., about 50 to about 2000 units phytase/kilogram of the feed). Before blending with the carrier, the spray dried phytase is assayed for phytase activity to determine the amount of dried powder to be blended with the carrier to obtain a feed additive composition with a predetermined number of phytase units/gram of the carrier. The phytase-containing carrier is then blended with the final feed mixture to obtain a final feed mixture with a predetermined number of phytase units/kilogram of the feed. Accordingly, the phytase concentration in the feed additive composition is greater than the phytase concentration in the final feed mixture.
In accordance with one embodiment of the invention the foodstuff is fed in combination with the yeast-expressed phytase to any monogastric animal (i.e., an animal having a stomach with a single compartment). Monogastric animals that can be fed a foodstuff in combination with a yeast-expressed phytase include agricultural animals, such as porcine species (e.g., barrows (i.e., castrated male pigs), gilts (i.e., female pigs prior to first mating) and any other type of swine), chickens, turkeys (poults (i.e., first several weeks post-hatching) and older animals), ducks, and pheasants, any other avian species, marine or fresh water aquatic species, animals held in captivity (e.g., zoo animals), or domestic animals (e.g., canine and feline).
Agricultural monogastric animals are typically fed animal feed compositions comprising plant products which contain phytate (e.g., cornmeal and soybean meal contain phytate (myo-inositol hexakisphosphate)) as the major storage form of phosphate, and, thus, it is advantageous to supplement the feed with phytase. Accordingly, the foodstuffs that can be supplemented with phytase in accordance with the invention include feed for agricultural animals such pig feed and poultry feed, and any foodstuff for avian species or marine or fresh water aquatic species (e.g., fish food). In addition, humans can be fed any foodstuff, such as a cereal product, containing phytate in combination with the yeast-expressed phytase of the present invention.
In the case of an animal feed fed to monogastric animals, any animal feed blend known in the art can be used in accordance with the present invention such as rapeseed meal, cottonseed meal, soybean meal, and cornmeal, but soybean meal and cornmeal are particularly preferred. The animal feed blend is supplemented with the yeast-expressed phytase, but other ingredients can optionally be added to the animal feed blend. Optional ingredients of the animal feed blend include sugars and complex carbohydrates such as both water-soluble and water-insoluble monosaccharides, disaccharides and polysaccharides. Optional amino acid ingredients that can be added to the feed blend are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HCl, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cysteine ethyl HCl, and analogs, and salts thereof. Vitamins that can be optionally added are thiamine HCl, riboflavin, pyridoxine HCl, niacin, niacinamide, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, and vitamins A, B, K, D, E, and the like. Minerals, protein ingredients, including protein obtained from meat meal or fish meal, liquid or powdered egg, fish solubles, whey protein concentrate, oils (e.g., soybean oil), cornstarch, calcium, inorganic phosphate, copper sulfate, salt, and limestone can also be added. Any medicament ingredients known in the art can be added to the animal feed blend such as antibiotics.
The feed compositions can also contain enzymes other than the yeast-expressed phytase. Exemplary of such enzymes are proteases, cellulases, xylanases, and acid phosphatases. For example, complete dephosphorylation of phytate may not be achieved by the phytase alone and addition of an acid phosphatase may result in additional phosphate release. A protease (e.g., pepsin) can be added, for example, to cleave the yeast-expressed phytase to enhance the activity of the phytase. Such a protease-treated phytase may exhibit enhanced capacity to increase the bioavailability of phosphate from phytate, to reduce the feed to weight gain ratio, to increase bone mass and mineral content, and to increase the egg weight or number of eggs laid for an avian species compared to intact yeast-expressed phytase. Additionally, combinations of phytases can be used, such as any combinations that may act synergistically to increase the bioavailability of phosphate from phytate, or proteolytic fragments of phytases or combinations of proteolytic fragments can be used. In this regard, the phytase gene expressed in yeast could be used to produce a truncated product directly for use in the method of the present invention.
Antioxidants can also be added to the foodstuff, such as an animal feed composition, to prevent oxidation of the phytase protein used to supplement the foodstuff. Oxidation can be prevented by the introduction of naturally-occurring antioxidants, such as beta-carotene, vitamin E, vitamin C, and tocopherol or of synthetic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, tertiary-butylhydroquinone, propyl gallate or ethoxyquin to the foodstuff. Compounds which act synergistically with antioxidants can also be added such as ascorbic acid, citric acid, and phosphoric acid. The amount of antioxidants incorporated in this manner depends on requirements such as product formulation, shipping conditions, packaging methods, and desired shelf-life.
In accordance with one method of the present invention, the foodstuff, such as an animal feed, is supplemented with amounts of the yeast-expressed phytase sufficient to increase the nutritional value of the foodstuff. For example, in one embodiment, the foodstuff is supplemented with less than 2000 units (U) of the phytase expressed in yeast per kilogram (kg) of the foodstuff. This amount of phytase is equivalent to adding about 34 mg of the phytase to one kg of the foodstuff (about 0.0034% w/w). In another embodiment, the foodstuff is supplemented with less than 1500 U of the phytase expressed in yeast per kg of the foodstuff. This amount of phytase is equivalent to adding about 26 mg of the phytase to one kg of the foodstuff (about 0.0026% w/w). In another embodiment, the foodstuff is supplemented with less than 1200 U of the phytase expressed in yeast per kg of the foodstuff. This amount of phytase is equivalent to adding about 17 mg of the phytase to one kg of the foodstuff (about 0.0017% w/w). In another embodiment the foodstuff, such as an animal feed composition, is supplemented with about 50 U/kg to about 1000 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 14.3 mg/kg or about 0.00007% to about 0.0014% (w/w)). In yet another embodiment the foodstuff is supplemented with about 50 U/kg to about 700 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 10 mg/kg or about 0.00007% to about 0.001% (w/w)). In still another embodiment the foodstuff is supplemented with about 50 U/kg to about 500 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 7 mg/kg or about 0.00007% to about 0.007% (w/w)). In yet another embodiment, the foodstuff is supplemented with about 50 U/kg to about 200 U/kg of the yeast-expressed phytase (i.e., about 0.7 to about 2.9 mg/kg or about 0.00007% to about 0.0003% (w/w)). In each of these embodiments it is to be understood that “kg” refers to kilograms of the foodstuff, such as the final feed composition in the case of an animal feed blend (i.e., the feed in the composition as a final mixture). In addition, one unit (U) of phytase activity is defined as the quantity of enzyme required to produce 1 μmol of inorganic phosphate per minute from 1.5 mmol/L of sodium phytate at 37° C. and at a pH of 5.5.
The yeast-expressed phytase can be mixed with the foodstuff, such as an animal feed (i.e., the feed composition as a final mixture), prior to feeding the animal the foodstuff or the phytase can be fed to the animal with the foodstuff without prior mixing. For example, the phytase can be added directly to an untreated, pelletized, or otherwise processed foodstuff, such as an animal feed, or the phytase can be provided separately from the foodstuff in, for example, a mineral block, a pill, a gel formulation, a liquid formulation, or in drinking water. In accordance with the invention, feeding the animal the foodstuff “in combination with” the phytase means feeding the foodstuff mixed with the phytase or feeding the foodstuff and phytase separately without prior mixing.
The yeast expressed-phytase can be in an unencapsulated or an encapsulated form for feeding to the animal or for mixture with an animal feed blend. Encapsulation protects the phytase from breakdown and/or oxidation prior to ingestion by the animal (i.e., encapsulation increases the stability of the protein) and provides a dry product for easier feeding to the animal or for easier mixing with, for example, an animal feed blend. The yeast-expressed phytase can be protected in this manner, for example, by coating the phytase with another protein or any other substances known in the art to be effective encapsulating agents such as polymers, waxes, fats, and hydrogenated vegetable oils. For example, the phytase can be encapsulated using an art-recognized technique such as a Na2+-alginate encapsulation technique wherein the phytase is coated with Na2+-alginate followed by conversion to Ca2+-alginate in the presence of Ca2+ ions for encapsulation. Alternatively, the phytase can be encapsulated by an art-recognized technique such as prilling (i.e., atomizing a molten liquid and cooling the droplets to form a bead). For example, the phytase can be prilled in hydrogenated cottonseed flakes or hydrogenated soy bean oil to produce a dry product. The phytase can be used in an entirely unencapsulated form, an entirely encapsulated form, or mixtures of unencapsulated and encapsulated phytase can be added to the foodstuff, such as an animal feed composition, or fed directly to the animal without prior mixing with the foodstuff. Any phytase for use in accordance with the method of the present invention can be similarly treated.
In accordance with the method of the present invention, the phytase-containing foodstuff can be administered to animals orally in a foodstuff, such as an animal feed, or in a mineral block or in drinking water, but any other effective method of administration known to those skilled in the art can be utilized (e.g., a pill form). The foodstuff containing yeast-expressed phytase can be administered to the animals for any time period that is effective to increase the bioavailability of phosphate from phytate, to reduce the feed to weight gain ratio, or to increase the bone mass and mineral content of the animal. For example, in the case of a feed composition fed to a monogastric animal, the feed composition containing yeast-expressed phytase can be fed to the animal daily for the lifetime of the animal. Alternatively, the phytase-containing feed composition can be fed to the animal for a shorter time period. The time periods for feeding the phytase-containing foodstuff to animals are nonlimiting and it should be appreciated that any time period determined to be effective to enhance animal nutrition by administering the phytase-containing foodstuff can be used.
EXAMPLE 1 
Animal Feed Blend Composition
The composition of the animal feed blend for chicks and pigs (i.e., the feed composition without phytase) was as follows:







TABLE 1







Composition of the animal feed blend used in chick and


pig assays.











Ingredient
Chick Assays
Pig Assay







Cornstarch
to 100.0
to 100.0



Corn
50.89
61.35



Soybean meal, dehulled
39.69
31.19



Soybean oil
5.00
3.00



Limestone, ground
1.67
1.06



Salt
0.40
—



Chick vitamin mix
0.20
—



Pig vitamin mix
—
0.20



Chick trace mineral mix
0.15
—



Pig trace vitamin mix
—
0.35



Choline chloride (60%)
0.20
—



Pig antibiotic premix (CSP)
—
0.50



Bacitracin premix
0.05
—



Copper sulfate
—
0.08



L-Lysine HCl, feed grade
—
0.17



DL-Methionine, feed grade
0.20
0.05










EXAMPLE 2 
Phytase Preparation
Yeast seed cultures were inoculated in growth medium with Pichia pastoris X33 transformed with either AOX1-appA, pGAP-appA2, or AOX1-Mutant U. The seed cultures were grown at 30° C. for about 24 hours until an OD600 of about 50 was reached. The seed cultures were then used to inoculate fermentors (batch process) containing sterile FM-22 growth medium containing 5% glucose. The 24-hour seed cultures were diluted about 1:25 to about 1:50 into the FM-22 growth medium. The yeast cultures were incubated aerobically in the fermentors at 30° C. with pH control at 6.0 (using NH2OH) and with continuous glucose feed until the cultures reached an OD600 of about 400 (about 36 hours).
To collect the phytases from the culture medium, the yeast cultures were rapidly chilled to 8° C. The cells were separated from the culture medium by centrifugation and by microfiltration. The phytases were 70-80% pure in the culture medium and were prepared for blending with a carrier as a feed additive as follows.
The cell-free media containing the secreted phytases were concentrated by ultrafiltration (10,000 MW exclusion limit). The ultrafiltration retentates (7-5% solids) were transferred to sterile containers for spray drying. The retentates were spray dried using standard techniques known in the art and the resulting powder was collected (4-6% moisture).
Microbiological testing of the powder was performed and the powder was assayed for phytase activity. The phytase activity of the powder (units of phytase activity/mg of powder) was used to determine the amount of dried powder to be blended with wheat middlings (i.e., the carrier) to obtain a phytase/carrier mixture with a predetermined number of phytase units/gram of the carrier. The dried phytase powder was mixed with the wheat middlings and packaged in moisture-proof containers. The phytase-containing wheat middlings were mixed with an animal feed blend as needed to obtain a final feed mixture with a predetermined number of phytase units/kg of the feed (about 400 to about 1000 U/kg).
EXAMPLE 3 
Feed Additive Composition
The following compositions are exemplary of feed additive compositions that may be mixed with an animal feed blend, such as the animal feed blend described in Example 1, to obtain a final feed mixture with, for example, about 50 U of phytase/kilogram of the final feed mixture to about 2000 U of phytase/kilogram of the feed. The feed additive compositions described below are nonlimiting and it should be appreciated that any phytase-containing feed additive composition determined to be effective to enhance the nutritional value of animal feed may be used. Exemplary feed additive compositions are shown for a feed additive composition containing 600 units of phytase/gram of the feed additive composition or 5000 units of phytase/gram of the feed additive composition.













600 phytase units/gram
5000 phytase units/gram



(weight percent)
(weight percent)

















Rice hulls
82.64
76.35


Calcium carbonate
15.00
15.00


Oil
1.5
1.5


Enzyme
0.86
7.15


Wheat middlings
82.64
76.35


Calcium carbonate
15.00
15.00


Oil
1.5
1.5


Enzyme
0.86
7.15









EXAMPLE 4 
Feeding Protocol 
Chicks were fed using the protocol described in Biehl, et al. (J. Nutr. 125:2407-2416 (1995)). Briefly, assays were conducted with male and female chicks from the cross of New Hampshire males and Columbian females and were conducted in an environmentally controlled laboratory room with 24 hour fluorescent lighting. From day 0 to day 7 posthatching, chicks were fed a basal diet of 23% crude protein, methionine-fortified corn-soybean meal as described above in Example 1. On day 8, chicks were weighed, wingbanded and assigned randomly to experimental treatments. Five pens of three or four chicks per pen received each dietary treatment for a 13-day experimental feeding period, and the chicks had an average initial weight of 80 to 100 grams.
Throughout the 13-day feeding period, chicks were confined in thermostatically controlled stainless-steel chick batteries, and stainless-steel feeders and waterers were also used. These steps were taken to avoid mineral contamination from the environment. Diets and distilled deionized water were freely available throughout the feeding period.
Pigs were fasted for 12 hours before the beginning of each assay, were fed the experimental diets for 23 days, and were fasted for 12 hours after each assay was completed. Ten pigs were used per treatment group and the pigs averaged about 8-120 kg at the initiation of the assay. Pigs were housed in individual pens that contained a stainless-steel feeder, a stainless-steel waterer, and galvanized round-bar fencing.
All of the chicks in each treatment group and the five median-weight pigs of each treatment group were euthanized for testing. Body weight gain was measured and tibia (chicks) or fibula (pigs) bones were harvested for bone ash analysis as a reflection of bone mass and mineral content.
EXAMPLE 5 
Measurement of Inorganic Phosphate and Bioavailable Phospate
Total phosphate in the feed samples used to generate a standard curve was quantified calorimetrically according to AOAC (1984) as described in Biehl et al. Monobasic potassium phosphate (KH2PO4) served as the standard. A standard curve was generated by measuring inorganic phosphate levels in basal feed supplemented with KH2PO4 (X-axis) and determining tibia ash weight (mg) or weight gain (g) (Y-axis) for animals fed basal feed supplemented with various levels of KH2PO4. The bioavailability of phosphate from phytate was then determined for animals fed basal feed supplemented with phytase by comparison of tibia ash weight and weight gain in these animals to the standard curve.
EXAMPLE 6 
Bone Ash Analysis
At the end of each experiment, chicks or pigs were euthanized, and right tibia or fibula bones were removed quantitatively from chicks or pigs, respectively. The bones were pooled by replicate pen and, after removal of adhering tissue, were dried for 24 hours at 100° C. and were weighed. After weighing, the bones were dry ashed for 24 hours at 600° C. in a muffle furnace. Ash weight was expressed as a percentage of dry bone weight and also as ash weight per bone.
EXAMPLE 7 
Phytase Expression in Yeast
In accordance with the present invention, any phytase gene may be expressed in yeast, and any yeast expression system may be used according to methods known to those skilled in the art. Yeast expression systems are described for exemplary phytase genes, such as the E. coli-derived appA and appA2 genes, and for a site-directed mutant of E. coli-derived AppA, in U.S. patent application Ser. No. 09/104,769 (now U.S. Pat. No. 6,451,572), U.S. patent application Ser. No. 09/540,149, and in U.S. patent application Ser. No. 60/166,179 (PCT Publication No. WO 01/36607 A1), all incorporated herein by reference. Exemplary yeast expression systems for expressing the AppA and AppA2 enzymes and a site-directed mutant of AppA are described briefly below.
Expression of the appA gene in Saccharomyces cerevisiae. 
The appA gene was expressed in Saccharomyces cerevisiae linked to the signal peptide of the phyA gene (phytase gene from Aspergillus niger). The appA gene was obtained from the ATCC, P.O. Box 1549, Manassas, Va. 20108, where it was deposited pursuant to the requirements of the Budapest Treaty, under ATCC accession number 87441. The appA gene (1.3 kb) was transformed into E. coli strain BL21 using the pappA1 expression vector (Ostanin et al., J. Biol. Chem., 267:22830-36 (1992)). To prepare the appA-phyA signal peptide construct, the polymerase chain reaction (PCR) was used. Two primers were synthesized and the 5′ primer was 80 base pairs in length and contained the phyA signal peptide sequence, a KpnI restriction enzyme cut site, and sequence complementary to the template as follows: 5′ GGG GTA CCA TGG GCG TCT CTG CTG TTC TAC TTC CTT TGT ATC TCC TGT CTG GAG TCA CCT CCG GAC AGA GTG AGC CGG AG 3′ (SEQ. ID No.: 6). The 3′ primer was 24 base pairs in length and contained an EcoRI site and sequence complementary to the template as follows: 5′ GGG AAT TCA TTA CAA ACT GCA GGC 3′ (SEQ. ID No.: 7). The PCR reaction was run for 25 cycles with 1 minute of denaturation at 95° C., 1 minute of annealing at 58° C., and 1 minute of chain extension at 72° C.
A 1.3 kb fragment was amplified by PCR, and was digested with KpnI and EcoRI and ligated into pYES2, a vector for expression in Saccharomyces cerevisiae. The pYES2-appA-phyA signal peptide construct was transformed into the yeast (INVScI, Invitrogen, San Diego, Calif.) by the lithium acetate method.
Selected transformants were inoculated into YEPD medium and expression was induced with galactose after an OD600 of 2 was reached. The cells were harvested 15-20 hours after induction. The AppA phytase enzyme was isolated from the culture supernatant and was the major protein present eliminating the need for a tedious purification.
Expression of the appA or appA2 gene in Pichia pastoris. 
appA. The template for the PCR reaction was as described above. The 5′ primer used for the PCR reaction was as follows: 5′ GGA ATT CCA GAG TGA GCC GGA 3′ (SEQ ID No.: 8). The 3′ primer was as follows: 5′ GGG GTA CCT TAC AAA CTG CAC G 3′ (SEQ ID No.: 9). The amplification reaction included 1 cycle at 94° C. (3 min.), 30 cycles at 94° C. (0.8 min), 30 cycles at 54° C. (1 min.), 30 cycles at 72° C. (2 min.), and 1 cycle at 72° C. (10 min). The product was first inserted into the pGEM T-easy vector (Promega), and E. coli strain TOP10F′ was used as the host to amplify the construct. The construct was then inserted into the yeast expression vector pPIcZαA (Invitrogen) at the EcoRI site, and E. coli strain TOP10F′ was again used as the host to amplify the construct.
The PIcZα vector containing appA was transformed into Pichia pastoris strain X33 by electroporation. The transformed cells were plated into YPD-Zeocin agar medium and positive colonics were incubated in minimal media with glycerol (BMGY) for 24 hours. When an OD600 of 5 was reached, the cells were centrifuged and were resuspended in 0.5% methanol medium (BMMY) for induction. Methanol (100%) was added every 24 hours to maintain a concentration of 0.5-1%. The cells were harvested at 192 hours after induction and the AppA protein was purified by ammonium sulfate precipitation and DEAE-Sepharose column chromatography.
appA2. The appA2 gene was isolated (see U.S. patent application Ser. No. 09/540,179) from a bacterial colony that exhibited particularly high phytase activity obtained from the colon contents of crossbred Hampshire-Yorkshire-Duroc pigs. To isolate a bacterial colony exhibiting high phytase activity the colon contents sample was diluted in an anaerobic rumen fluid glucose medium, was shaken vigorously for 3 minutes, and was serially diluted. The diluted samples were cultured at 37° C. for 3 days on a modified rumen fluid-glucose-cellobiose-Agar medium containing insoluble calcium phytate. Colonies with a clear zone were assayed for phytase activity using sodium phytate as a substrate. The colony identified as producing the highest phytase activity was identified as an E. coli strain. Accordingly, the appA2 gene was isolated using the primers as described above for appA expression in Pichia pastoris (SEQ. ID Nos. 8 and 9). The appA2 gene was cloned into the PIcZα vector and Pichia pastoris strain X33 was transformed with the PIcZα-appA2 construct as described above for appA expression in Pichia pastoris. The AppA2 enzyme was expressed as described above for AppA, and the AppA2 protein was collected from the yeast culture supernatant.
AppA Site-Directed Mutants.
Site-directed mutants of appA were prepared as described in U.S. patent application Ser. No. 06/166,179 (PCT Publication No. WO 01/36607 A1), incorporated herein by reference. Briefly, the E. coli appA mutants were constructed using the megaprimer site-directed mutagenesis method (Seraphin, B. et al., Nucleic Acids Res. 24:3276-77 (1996); Smith, A. M. et al., Biotechniques 22: 438-39 (1997), which are hereby incorporated by reference).
The template for mutagenesis was obtained from ATCC, and the gene (1.3 kb) was transformed into E. coli strain BL21 (No. 87441) using the pappA1 expression vector (Ostanin et al., J. Biol. Chem., 267:22830-36 (1992)). The template was amplified as described above for appA expressed in Pichia pastoris using the primers used above for appA expression in Pichia pastoris (SEQ. ID Nos.: 8 and 9). The amplification reaction included 1 cycle at 94° C. (3 min.), 30 cycles at 94° C. (0.5 min), 30 cycles at 54° C. (1 min.), 30 cycles at 72° C. (1.5 min.), and 1 cycle at 72° C. (10 min).
The mutagenesis PCR reaction was performed as described above using the primers as follows:









(SEQ ID No.: 10)













5′CTGGGTATGGTTGGTTATATTACAGTCAGGT3′
A131N





V134N









(SEQ ID No.: 11)













5′CAAACTTGAACCTTAAACGTGAG3′
C200N










(SEQ ID No.: 12)













5′CCTGCGTTAAGTTACAGCTTTCATTCTGTTT3′
D207N





S211N






The mutagenic PCR reactions incorporated appropriate primers to make the A131N/V134N/D207N/S211N, C200N/D207N/S211N (Mutant U), and A131N/V134N/C200N/D207N/S211N mutants of appA. The first mutagenic PCR reaction (100 μl) was performed as described above, using 4 μl of the intact appA PCR reaction mixture and the appropriate modified primers listed above. All megaprimer PCR products were resolved in a 1.5% low melting agarose gel. The expected fragments were excised and eluted with a GENECLEAN II kit. The final mutagenic PCR reaction (100 μl) was set up as described above, using 4 μl of the appA PCR product and varying concentrations of the purified megaprimer (50 ng to 41 μg), depending on its size. Five thermal cycles were set up at 94° C. for 1 minute and 70° C. for 2 minutes. While at 70° C., 1 μmol of forward primer and 2 U of AmpliTaq DNA polymerase were added and gently mixed with the reaction mixture, and thermal cycling continued for 25 cycles at 94° C. for 1 minute and 70° C. for 1.5 minutes.
The genes encoding the site-directed mutants were expressed in Pichia pastoris as described above for the appA2 gene. The protein products were expressed as described above for AppA, and the site-directed mutants were purified from the yeast culture supernatant by ammonium sulfate precipitation and DEAE-Sepharose chromatography.
EXAMPLE 8 
In Vivo Effects of Yeast—Expressed Phytases Fed to Chicks
To evaluate their potential as animal feed supplements, the yeast-expressed phytases AppA and AppA2, were dried and added to the animal feed blend (23% crude protein) described above in Example 1 using wheat middlings as a carrier. Chicks (four chicks per pen; average initial weight of 97 grams) were fed phytase-supplemented feed compositions as described above in Example 4. The treatment groups included various level of KH2PO4 to construct the standard curve, 500 U/kg of Natuphos®, a commercially available (Gist-Brocades) phytase expressed in the fungus Aspergillus niger, 500 U/kg of AppA expressed in Pichia pastoris or in E. coli, and various levels of AppA2/p (AppA2 expressed in Pichia pastoris using the constitutive pGAP promoter for gene expression) as follows:
Treatment Groups:













1.
Basal Diet (0.10% P, 0.75% Ca)


2.
Same as 1 + 0.05% P from KH2PO4


3.
Same as 1 + 0.10% P from KH2PO4


4.
Same as 1 + 0.15% P from KH2PO4


5.
Same as 1 + 500 U/kg AppA (yeast)


6.
Same as 1 + 500 U/kg AppA (E. coli)


7.
Same as 1 + 500 U/kg AppA2/p


8.
Same as 1 + 1000 U/kg AppA2/p


9.
Same as 1 + 1500 U/kg AppA2/p


10.
Same as 1 + 500 U/kg Natuphos ®









For the various treatment groups weight gain, feed intake, the feed to weight gain ratio, dry tibia weight, tibia ash weight, tibia ash weight as a percent of dry tibia weight, and the percentage of bioavailable phosphate based on both tibia ash weight and weight gain were determined. The results are expressed below as a mean for the four chicks for each of the five pens (R1, R2, R3, R4, and R5), and the mean for the five pens was also calculated (labeled “mean” in the tables). The treatment groups are labeled T1-T10 in the tables, and “g/c/d” indicates weight gain or feed intake in grams/chick/day.










Weight gain (g/c)


















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10





R1
185
282
315
321
314
284
334
352
334
269


R2
219
286
315
336
317
322
315
326
348
274


R3
234
277
327
335
321
312
318
321
342
267


R4
234
291
309
311
316
308
326
342
333
276


R5
223
278
303
332
316
268
313
336
361
294


Mean
219g
283ef
314cd
327bc
317c
299de
321bc
335ab
344a
276f


g/c/d
 16.8
 21.8
 24.2
 25.2
 24.4
 23.0
 24.7
 25.8
 26.5
 21.2





Pooled SEM = 6


LSD = 16
















13-d Feed intake (g/c)


















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10





R1
303
392
434
434
426
389
450
474
465
397


R2
330
462
448
454
429
430
425
449
472
396


R3
336
391
445
458
446
425
428
445
464
397


R4
350
416
432
424
432
420
441
464
449
386


R5
335
388
421
467
425
389
453
461
483
420


Mean
331f
410e
436c
447abc
432cd
411de
439bc
459ab
467a
399e


g/c/d
 25.5
 31.5
 33.5
 34.4
 33.2
 31.6
 33.8
 35.3
 35.9
 30.7





Pooled SEM = 7


LSD = 21
















Gain/feed (g/kg)


















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10





R1
611
718
726
740
738
729
742
742
720
678


R2
665
618
703
741
738
749
741
727
738
692


R3
696
708
736
730
719
736
743
722
736
671


R4
668
700
715
733
731
733
738
738
743
715


R5
665
717
721
710
742
688
691
730
749
710


Mean
661c
692b
720a
731a
734a
727a
731a
732a
737a
691b





Pooled SEM = 10


LSD = 28
















Dry tibia weight (mg/c)


















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10





R1
659
804
883
 981
892
787
914
1059
1106
757


R2
655
769
891
 977
907
918
873
 997
1083
759


R3
713
751
878
1008
901
820
905
 964
1065
726


R4
740
742
931
 925
823
809
923
1083
1096
729


R5
714
714
866
 942
841
809
931
1036
1132
764


Mean
698g
756f
890d
 967c
873de
829e
909d
1028b
1096a
747f





Pooled SEM = 16


LSD = 45
















Tibia Ash (mg/c)


















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10





R1
232
307
406
492
434
345
439
590
617
278


R2
215
315
415
507
445
435
420
546
600
305


R3
259
300
406
520
435
382
451
523
604
284


R4
237
297
442
462
392
372
454
590
616
267


R5
242
277
396
471
432
373
471
548
642
316


Mean
237h
299g
413e
490c
428de
381f
447d
559b
616a
290g





Pooled SEM = 10


LSD = 28
















Supplemental P Intake (g)














T1
T2
T3
T4

















R1
0
0.196
0.434
0.651



R2
0
0.231
0.448
0.680



R3
0
0.196
0.445
0.687



R4
0
0.208
0.432
0.636



R5
0
0.194
0.421
0.701



Mean
 0d
0.205c
0.436b
0.671a







Pooled SEM = 0.007



LSD = 0.022
















Tibia ash (%)


















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10





R1
35.15
38.22
46.03
50.12
48.64
43.78
47.98
55.64
55.79
36.78


R2
32.86
40.92
46.53
51.86
49.06
47.31
48.07
54.69
55.35
40.15


R3
36.30
39.96
46.22
51.61
48.31
46.62
49.85
54.23
56.69
39.14


R4
32.01
40.02
47.47
49.96
47.70
45.96
49.19
54.44
56.23
36.68


R5
33.45
38.82
45.74
49.95
51.40
46.11
50.54
52.86
56.71
41.32


Mean
33.95g
39.59f
46.40e
50.70c
49.02d
45.96e
49.13cd
54.37b
56.15a
38.81f





Pooled SEM = 0.57


LSD = 1.62






Phosphorus Equivalency Estimates













Tibia Ash Weight











KH2PO4 Standard Curve:
Y = tibia ash (mg)



X = supplemental or equivalent P intake (g)


Y = 232.0 + 389.9X


r2 = 0.97










For 500 U/kg Phytase activity


(example calculations using tibia ash treatment means)









% Bioavailable P













AppA (yeast):
(428−232.0)/
0.116%



389.9 = 0.503 g P from 432 g FI =


AppA (E. coli):
(381 − 232.0)/
0.093%



389.9 = 0.382 g P from 411 g FI =


AppA2/p:
(447 − 232.0)/
0.126%



389.9 = 0.551 g P from 439 g FI =


Natuphos ®:
(290 − 232.0)/
0.037%



389.9 = 0.149 g P from 399 g FI =





** Results from ANOVA (calculation performed for each pen of four birds; treatment legend on previous page)
















Bioavailable P (%)














T5
T6
T7
T8
T9
T10





R1
0.122
0.075
0.118
0.194
0.212
0.030


R2
0.127
0.121
0.113
0.179
0.200
0.047


R3
0.117
0.091
0.131
0.168
0.206
0.034


R4
0.095
0.085
0.129
0.198
0.219
0.023


R5
0.121
0.093
0.135
0.176
0.218
0.051


Mean
0.116c
0.093d
0.125c
0.183b
0.211a
0.037e





Pooled SEM = 0.005


LSD = 0.016






















Contrasts
Significance (P-value)







AppA (yeast) vs. AppA (E. coli)
0.006



AppA2/p linear
0.001



AppA2/p quadratic
0.039













Weight Gain














KH2PO4 Standard Curve:
X = weight gain (g)




Y = supplemental P intake (g)



Y = 234.1 + 157.2X



r2 = 0.84










Results from ANOVA (calculation performed for each pen of four birds; treatment legend on previous page)











Bioavailable P (%)














T5
T6
T7
T8
T9
T10





R1
0.119
0.082
0.141
0.158
0.137
0.056


R2
0.123
0.130
0.121
0.130
0.154
0.064


R3
0.124
0.117
0.125
0.124
0.148
0.053


R4
0.121
0.112
0.133
0.148
0.140
0.069


R5
0.123
0.055
0.111
0.141
0.167
0.091


Mean
0.122b
0.099d
0.126b
0.140ab
0.149a
0.067d





Pooled SEM = 0.007


LSD = 0.021




















Contrasts
Significance (P-value)



















AppA (yeast) vs. AppA (E. coli)
0.038



AppA2/p linear
0.036



AppA2/p quadratic
0.768










Supplementation of the animal feed blend with increasing amounts of KH2PO4 resulted in linear (p<0.001)increases in weight gain, tibia ash. Supplementation of the animal feed blend with Natuphos® resulted in linear increases (p<0.001) in weight gain, tibia ash, and % bioavailable phoshate. At 500 U/kg the yeast-expressed enzymes (AppA and AppA2/p) were more effective than E. coli-expressed AppA or Natuphos® at improving each of the in vivo responses tested, including the feed to weight gain ratio, tibia weight, and % bioavailable phosphate. In fact, AppA and AppA2/p were 2-6 times more effective at increasing the level of bioavailable phosphate than Natuphos®, depending on whether tibia ash weight or weight gain was used to calculate the percent of bioavailable phosphate.
EXAMPLE 9 
In Vivo Effects of Yeast-Expressed Phytases Fed to Chicks
The procedure was as described in Example 8 except that the chicks had an average initial weight of 91 grams, and the treatment groups were as follows:
Treatment Groups:













1.
Basal Diet (0.10% P, 0.75% Ca)


2.
Same as 1 + 0.05% P from KH2PO4


3.
Same as 1 + 0.10% P from KH2PO4


4.
Same as 1 + 300 U/kg Natuphos ® phytase


5.
Same as 1 + 500 U/kg Natuphos ® phytase


6.
Same as 1 + 700 U/kg Natuphos ® phytase


7.
Same as 1 + 900 U/kg Natuphos ® phytase


8.
Same as 1 + 1100 U/kg Natuphos ® phytase


9.
Same as 1 + 1300 U/kg Natuphos ® phytase


10.
Same as 1 + 1500 U/kg Natuphos ® phytase


11.
Same as 1 + 500 U/kg Ronozyme ® phytase


12.
Same as 1 + 300 U/kg Mutant U phytase


13.
Same as 1 + 500 U/kg Mutant U phytase


14.
Same as 1 + 500 U/kg AppA phytase


15.
Same as 1 + 500 U/kg AppA2 phytase









The Ronozyme® (Roche) phytase is a phytase expressed in fungus. Mutant U is the site-directed mutant of AppA described above. The tables are labeled as described in Example 8. The in vivo effects of phytase supplementation described in Example 8 were measured and the results were as follows:










Weight gain (g/c)























T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15





R1
287
295
318
269
295
271
301
305
304
317
256
324
340
319
343


R2
271
291
342
288
297
282
313
295
323
327
231
289
349
325
342


R3
268
302
326
286
278
267
298
309
308
327
274
332
337
348
336


R4
256
282
317
255
304
280
294
295
289
310
287
310
338
324
330


R5
215
279
310
292
270
290
302
270
295
306
284
316
329
319
331


Mean
259
290
323
278
289
278
302
295
304
317
266
314
339
327
336


g/c/d
 18.5
 20.7
 23.1
 19.9
 20.6
 19.9
 21.6
 21.1
 21.7
 22.6
 19.0
 22.4
 24.2
 23.4
 24.0





Pooled SEM = 6


LSD = 18
















Feed intake (g/c)























T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15





R1
463
450
489
428
450
435
466
479
445
489
422
500
487
483
503


R2
424
439
565
443
427
454
470
469
490
489
394
459
518
459
519


R3
425
446
526
444
417
425
444
480
483
485
427
522
496
520
535


R4
406
437
472
398
450
437
462
442
425
505
439
478
499
496
491


R5
381
443
478
421
423
438
447
423
455
452
437
463
496
476
519


Mean
420
443
506
427
433
438
458
459
460
484
424
484
499
487
513


g/c/d
 30.0
 31.6
 36.1
 30.5
 30.9
 31.3
 32.7
 32.8
 32.9
 34.6
 30.3
 34.6
 35.6
 34.8
 36.6





Pooled SEM =10


LSD = 27
















Gain/feed (g/kg)























T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15

























R1
620
656
650
627
655
623
645
636
683
648
605
648
699
661
681


R2
639
662
606
651
696
621
665
629
659
668
587
629
672
709
659


R3
630
677
619
644
666
628
671
644
637
673
641
635
680
669
629


R4
631
645
671
641
675
642
636
668
679
614
654
649
678
652
671


R5
564
630
649
694
639
662
675
639
648
678
649
683
663
669
638


Mean
617
654
639
651
666
635
658
643
661
656
627
649
678
672
656





Pooled SEM = 10


LSD = 28
















Dry tibia weight (mg/c)























1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

























R1
970
1010
1146
952
1028
1011
1007
995
1027
1131
—
1150
1169
1160
1234


R2
920
1029
1175
974
937
1047
1013
993
1077
1077
828
995
1251
1130
1209


R3
1038
872
1147
981
932
917
1049
1072
1030
1137
1029
1151
1238
1178
1215


R4
890
944
1125
957
1008
964
1073
1005
961
1100
919
1116
1273
1177
1128


R5
882
970
1078
954
976
1004
961
963
1065
1101
937
1046
1172
1141
1145


Mean
940
965
1134
964
976
989
1021
1006
1032
1109
928
1092
1221
1157
1186





Pooled SEM = 22


LSD = 61
















Supplemental P intake (g)













1
2
3
















R1
0
0.225
0.489



R2
0
0.220
0.565



R3
0
0.223
0.526



R4
0
0.219
0.472



R5
0
0.222
0.478



Mean
 0c
0.222b
0.506a




















Tibia ash (mg/c)























1
2
3
4
5
6
7
8
9
10
11
12
13
14
15





R1
284
333
437
279
303
305
328
324
340
369
—
401
428
453
457


R2
270
318
447
298
290
336
336
325
383
363
226
355
481
441
470


R3
291
271
398
302
278
263
326
357
345
403
293
410
479
420
455


R4
234
305
398
281
314
297
341
317
324
364
264
406
500
447
413


R5
243
327
388
287
279
309
302
305
352
368
279
354
447
424
443


Mean
264
311
414
289
293
302
327
326
349
373
266
385
467
437
448





Pooled SEM = 10


LSD = 28
















Tibia ash (%)























1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

























R1
29.30
32.94
38.15
29.25
29.48
30.18
32.55
32.54
33.10
32.60
—
34.90
36.63
39.07
37.06


R2
29.29
30.97
38.03
30.61
30.99
32.06
33.21
32.73
35.51
33.67
27.33
35.66
38.48
39.04
38.89


R3
28.03
31.08
34.70
30.81
29.79
28.71
31.12
33.26
33.45
35.49
28.50
35.63
38.73
35.63
37.48


R4
26.30
32.33
35.34
29.35
31.17
30.80
31.73
31.55
33.71
33.11
28.74
36.38
39.29
38.00
36.63


R5
27.52
33.76
35.98
30.13
28.60
30.81
31.44
31.67
33.09
33.41
29.77
33.81
38.14
37.18
38.70


Mean
28.09
32.21
36.44
30.03
30.00
30.51
32.01
32.35
33.77
33.65
28.58
35.28
38.25
37.78
37.75





Pooled SEM = 0.49


LSD = 1.39






Phosphorus Equivalency Estimates














KH2PO4Standard Curve:
Y = tibia ash (mg)



X = supplemental or equivalent P intake (g)


Y = 257.1 + 299.0X


r2 = 0.88





For 500 U/kg Phytase activity



















(example calculations using tibia ash treatment mean)
% Bioavailable P












Natuphos ®:
(293 − 257.1)/299.0 = 0.120 g
0.030%



P from 433 g FI =


Ronozyme ®:
(266 − 257.1)/299.0 = 0.030 g
0.007%



P from 424 g FI =


Mutant U:
(467 − 257.1)/299.0 = 0.702 g
0.141%



P from 499 g FI =


AppA:
(437 − 257.1)/299.0 = 0.602 g
0.124%



P from 487 g FI =


AppA2:
(448 − 257.1)/299.0 = 0.638 g
0.124%



P from 513 g FI =









Results from ANOVA (calculation performed for each pen of four birds; treatment legend on previous page)











Bioavailable P (%)




















4
5
6
7
8
9
10
11
12
13
14
15






















R1
0.017
0.034
0.037
0.051
0.047
0.062
0.076
—
0.097
0.117
0.136
0.133


R2
0.031
0.026
0.058
0.057
0.049
0.086
0.072
−0.026
0.071
0.145
0.134
0.137


R3
0.034
0.017
0.005
0.052
0.069
0.061
0.101
0.028
0.098
0.150
0.105
0.124


R4
0.020
0.043
0.030
0.060
0.045
0.053
0.071
0.006
0.104
0.163
0.128
0.106


R5
0.024
0.018
0.040
0.034
0.038
0.071
0.082
0.017
0.070
0.128
0.117
0.120


Mean
0.025
0.027
0.034
0.051
0.050
0.066
0.080
0.006
0.088
0.140
0.124
0.124





Pooled SEM = 0.006


LSD = 0.018




















Significance


Contrasts
(P-value)





Linear response to Natuphos ® (treatment groups 5 (trt)
0.001


4–10)


Quadratic response to Natuphos ®
0.208


500 U/kg Natuphos ® (trt 5) vs 500 U/kg yeast-expressed
0.001


phytases (trt 13–15)


500 U/kg Natuphos ® (trt 5) vs 500 U/kg Ronozyme ®
0.031


(trt 11)


500 U/kg Ronozyme ® (trt 11) vs 500 U/kg yeast-
0.001


expressed phytases (trt 13–15)


300 U/kg Mutant U (trt 12) vs 500 U/kg Mutant U (trt 13)
0.001


500 U/kg Mutant U (trt 12) vs 500 U/kg AppA (trt 14)
0.074


500 U/kg Mutant U (trt 12) vs 500 U/kg AppA2 (trt 15)
0.074













Multiple Linear Regression:
Y = tibia ash (mg)




X = phytase intake (U)







Y = 263.462 + 0.144(Natuphos ®) + 0.014(Ronozyme ®) + 0.823


(Mutant U) + 0.711(AppA) + 0.718(AppA2)


R2 = 0.93













Ratio
Eq. To


Relative Phytase Activity
(%)
500 U/kg Natuphos ®













Ronozyme ®:
(0.014/
10
50



0.144) * 100 =


Mutant U:
(0.823/
572
2860



0.144) * 100 =


AppA:
(0.711/
494
2470



0.144) * 100 =


AppA2:
(0.718/
499
2495



0.144) * 100 =









At 500 U/kg, the yeast-expressed enzymes (Mutant U, AppA and AppA2) were more effective than Natuphos® or Ronozyme® (both enzymes are expressed in fungal expression systems) at improving the in vivo responses tested. For example, Mutant U, AppA and AppA2 were four times more effective than Natuphos® in releasing phosphate (see FIG. 3).
EXAMPLE 10 
In Vivo Effects of Yeast-Expressed Phytases Fed to Pigs
The procedure was as described in Example 8 except that pigs (average initial weight of 10 kg) were fed the phytase-supplemented feed composition. The treatment groups were as follows:
Treatment Groups:


    1) Basal diet (0.75 P; 0.60% Ca)
    2) Same as 1+0.05% P from KH2PO4 
    3) Same as 1+0.10% P from KH2PO4 
    4) Same as 1+0.15% P from KH2PO4 
    5) Same as 1+400 U/kg phytase from Natuphos®
    6) Same as 1+400 U/kg phytase from Mutant U phytase
    7) Same as 1+400 U/kg AppA phytase
    8) Same as 1+400 U/kg AppA2 phytase


For the various treatment groups weight gain, feed to weight gain ratio, fibula ash weight, fibula ash weight as a percentage of dry fibula weight, and the percentage of bioavailable phosphate based on fibula ash weight were determined. The results were as follows:







TABLE 3





Pig Assaya

















Fibula Composition









Bio-













Weight
G:F,
Ash,
Ash,
available


Treatment Groups
gain, g/d
g/kg
%
mg
P, %b





Basal Diet
369
533
29.31
666


Same as 1 + 0.05% P from KH2PO4
435
576
32.83
766


Same as 1 + 0.10% P from KH2PO4
446
618
36.62
972


Same as 1 + 0.15% P from KH2PO4
509
660
36.57
1123


Same as 1 + 400 U/kg Natuphos ® phytase
460
605
34.37
889
0.081


Same as 1 + 400 U/kg Mutant U phytase
458
645
35.45
961
0.116


Same as 1 + 400 U/kg AppA phytase
458
606
35.97
1035
0.136


Same as 1 + 400 U/kg AppA2 phytase
443
583
34.96
968
0.108











Contrast
Significance (P-value)















Natuphos ® (treatment group (trt) 5) vs. yeast-expressed
NS
NS
NS
0.05
0.048


phytases (trt 6–8)


Mutant U (trt 6) AppA vs. (trt 7) and AppA2 (trt 8)
0.10
0.10
NS
0.001
0.239





aData are means of ten replicates per treatment of individually housed pigs during a period of 23 days; average initial weight was 8.4 ± 0.2 kg.


bPercent bioavailable P calculations are estimates of P equivalency based on KH2PO4 standard curve (treatments 1–4). Calculations based on KH2PO4 standard curve where Y = fibula ash (mg) and X = supplemental or equivalent P intake (g): Y = 664.49 + 15.29X (r2 = 0.87).






At 400 U/kg, the yeast-expressed enzymes (Mutant U, AppA, and AppA2) were more effective than Natuphos® (expressed in fungus) at improving the responses tested.
EXAMPLE 11
In Vivo Effects of Yeast-Expressed Phytases in Chicks
The procedure was as described in Example 8 except that the chicks had an average initial weight of 83 grams, and the treatment groups were as follows:
Treatment Groups:













1.
Basal Diet (0.10% P; 0.75% Ca)


2.
Same as 1 + 0.05% P from KH2PO4


3.
Same as 1 + 0.10% P from KH2PO4


4.
Same as 1 + 0.15% P from KH2PO4


5.
Same as 1 + 500 U/kg Natuphos ® phytase (batch 1)


6.
Same as 1 + 500 U/kg Natuphos ® phytase (batch 2)


7.
Same as 1 + 1000 U/kg Natuphos ® phytase (batch 2)


8.
Same as 1 + 500 U/kg Ronozyme ® phytase (batch 1)


9.
Same as 1 + 500 U/kg Ronozyme ® phytase (batch 2)


10.
Same as 1 + 1000 U/kg Ronozyme ® phytase (batch 2)


11.
Same as 1 + 500 U/kg Mutant U phytase


12.
Same as 1 + 500 U/kg AppA phytase


13.
Same as 1 + 500 U/kg AppA2 phytase


14.
Same as 1 + 500 U/kg AppA2 + novel promoter phytase



(AppA2/p)









The tables are as labeled in Example 8. The in vivo effects of phytase supplementation described in Example 8 were measured and the results were as follows:










Weight gain (g/c)






















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
























R1
152
252
295
299
215
235
241
223
256
254
325
308
322
314


R2
199
238
290
332
206
210
256
237
212
243
312
316
309
306


R3
163
257
297
347
235
254
250
215
218
246
324
309
313
335


R4
176
262
288
330
242
250
288
228
195
223
329
318
310
317


R5
190
257
295
356
193
230
288
218
214
259
306
307
314
317


Mean
176
253
293
333
218
236
265
224
219
245
319
312
314
318


g/c/d
12.6
18.1
20.9
23.8
15.6
16.9
18.9
16.0
15.6
17.5
22.8
22.3
22.4
22.7





Pooled SEM = 7


LSD = 19
















Feed intake (g/c)






















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
























R1
279
373
431
424
347
386
352
359
284
321
435
429
447
424


R2
335
352
407
441
333
340
375
363
325
368
395
441
427
465


R3
291
374
419
472
369
400
381
330
348
367
439
459
440
472


R4
292
386
400
457
366
370
405
358
328
356
451
473
421
441


R5
344
374
427
483
341
370
450
336
344
394
425
420
449
445


Mean
308
372
417
455
351
373
393
349
326
361
429
444
437
449


g/c/d
22.0
26.6
29.8
32.5
25.1
26.6
28.1
24.9
23.3
25.8
30.6
31.7
31.2
32.1





Pooled SEM = 10


LSD = 28
















Gain/feed (g/kg)






















T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
























R1
544
674
684
705
618
608
684
621
676
793
747
718
719
742


R2
593
676
713
752
617
616
682
653
651
659
790
716
722
657


R3
558
686
709
736
636
635
656
650
626
671
737
673
713
708


R4
601
680
720
723
662
677
711
637
594
627
729
673
737
719


R5
551
685
690
737
565
622
641
649
624
658
721
732
700
712


Mean
569
680
703
731
620
632
675
642
634
682
745
702
718
708





Pooled SEM = 13


LSD = 37
















Dry tibia weight (mg/c)






















1
2
3
4
5
6
7
8
9
10
11
12
13
14
























R1
671
886
1048
1002
919
840
912
825
891
831
1117
1041
1057
1091


R2
815
—
1003
1298
752
765
906
865
756
916
1113
1152
1041
1070


R3
730
884
1036
1296
849
942
937
864
849
816
1154
1017
1130
1220


R4
698
911
931
1232
802
901
918
837
807
872
1163
1165
1047
1078


R5
773
929
1037
1266
793
845
867
768
825
962
1044
1054
1130
1140


Mean
737
903
1011
1219
823
859
908
832
826
879
1118
1086
1081
1120





Pooled SEM = 27


LSD = 77
















Supplemental P intake (g)














1
2
3
4

















R1
0
0.187
0.431
0.636



R2
0
0.176
0.407
0.661



R3
0
0.187
0.419
0.708



R4
0
0.193
0.400
0.685



R5
0
0.187
0.427
0.724



Mean
 0d
0.186c
0.417b
0.683a




















Tibia ash (mg/c)






















1
2
3
4
5
6
7
8
9
10
11
12
13
14

























R1
174
255
381
367
254
230
246
239
253
224
415
385
404
411


R2
197
279
343
483
199
198
244
242
200
249
395
425
357
353


R3
185
279
361
486
238
261
276
230
228
231
410
370
389
454


R4
175
287
315
459
220
262
282
222
210
244
416
455
371
396


R5
183
261
335
481
211
229
264
202
222
262
383
380
406
431


Mean
183
272
347
455
224
236
262
227
223
242
404
403
385
409





Pooled SEM = 12


LSD = 32
















Tibia ash (%)






















1
2
3
4
5
6
7
8
9
10
11
12
13
14

























R1
25.88
28.82
36.31
36.67
27.60
27.39
26.91
29.01
28.42
26.99
37.15
37.03
38.24
37.64


R2
24.20
—
34.21
37.18
26.51
25.89
26.86
27.97
26.44
27.16
35.49
36.90
34.33
32.98


R3
25.43
31.61
34.87
37.53
28.06
27.75
29.52
26.65
26.85
28.35
35.54
36.34
34.40
37.22


R4
25.04
31.53
33.85
37.22
27.42
29.15
30.73
26.46
26.03
27.97
35.76
39.09
35.43
36.71


R5
23.72
28.11
32.27
38.02
26.61
27.17
30.41
26.26
26.88
27.22
36.67
36.02
35.99
37.80


Mean
24.85
30.02
34.30
37.32
27.24
27.47
28.89
27.27
26.92
27.54
36.12
37.08
35.68
36.47





Pooled SEM = 0.57


LSD = 1.61


















Phosphorus Equivalency Estimates











KH2PO4 Standard Curve:
Y = tibia ash (mg)



X = supplemental or equivalent P intake (g)


Y = 187.9 + 393.4X


r2 = 0.95










For 500 U/kg Phytase activity









% Bioa-



vail-








(example calculations using tibia ash treatment means)
able P












Natuphos ® 1:
(224 − 187.9)/393.4 = 0.092 g P from
0.026%



351 g FI =


Natuphos ® 2:
(236 − 187.9)/393.4 = 0.122 g P from
0.033%



373 g FI =


Ronozyme ® 1:
(227 − 187.9)/393.4 = 0.099 g P from
0.028%



349 g FI =


Ronozyme ® 2:
(223 − 187.9)/393.4 = 0.089 g P from
0.027%



326 g FI =


Mutant U:
(404 − 187.9)/393.4 = 0.549 g
0.128%



P from 429 g FI =


AppA:
(403 − 187.9)/393.4 = 0.547 g P
0.123%



from 444 g FI =


AppA2:
(385 − 187.9)/393.4 = 0.501 g P from
0.115%



437 g FI =


AppA2/p:
(409 − 187.9)/393.4 = 0.562 g
0.125%



P from 449 g FI =





Results from ANOVA (calculation performed for each pen of four birds; treatment legend on previous page)
















Bioavailable P (%)


















5
6
7
8
9
10
11
12
13
14





















R1
0.048
0.028
0.042
0.036
0.058
0.029
0.133
0.117
0.123
0.138


R2
0.008
0.008
0.038
0.038
0.009
0.042
0.133
0.137
0.101
0.090


R3
0.035
0.047
0.059
0.032
0.029
0.030
0.129
0.101
0.116
0.143


R4
0.022
0.051
0.059
0.024
0.017
0.040
0.129
0.144
0.111
0.120


R5
0.017
0.028
0.043
0.011
0.025
0.048
0.117
0.116
0.123
0.139


Mean
0.026c
0.032bc
0.048b
0.028c
0.028c
0.038bc
0.128a
0.123a
0.115a
0.125a





Pooled SEM = 0.006


LSD = 0.018






At 500 U/kg, the yeast-expressed enzymes (Mutant U, AppA, AppA2, and AppA2/p) were more effective than Natuphos® or Ronozyme® at improving the in vivo responses tested including weight gain, feed to weight gain ratio, bone mass and mineral content, and percent bioavailable phosphate. The yeast-expressed enzymes were about four times more effective at increasing the level of bioavailable phosphate than either of the fungus-expressed enzymes.
EXAMPLE 12 
In Vivo Effects of Yeast-Expressed Phytases Fed to Post-Molt Laying Hens
The procedure was as described in Example 8 except post-molt laying hens were tested, egg production and egg weight was determined, and the treatment groups and basal diet were as follows:
Treatments:


    1. P-deficient corn-soybean meal basal diet (0.10% Pa; 3.8% Ca; 17% CP)
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+150 U/kg r-AppA2 phytase
    4. As 1+300 U/kg r-AppA2 phytase
    5. As 1+10,000 U/kg r-AppA2 phytase
















Basal Diet:




Ingredient
%



















Corn
63.65



Soybean meal, dehulled
25.65



Limestone, ground
9.80



Salt
0.40



Mineral premix
0.20



Vitamin premix
0.15



DL-methionine, feed-grade
0.10



Choline chloride
0.05







** Note:



Treatment 1 discontinued after week 4 due to egg production below 50%.






The following tables are labeled as described in Example 8 and some of the same responses as described in Example 8 were measured. The results show that AppA2 increases egg production and egg weight in post-molt laying hens.
Treatments:













1.
P-deficient corn-soybean meal basal diet (0.10% pa; 3.8% Ca; 17%



CP)


2.
As 1 + 0.10% Pi from KH2PO4


3.
As 1 + 150 U/kg r-AppA2 phytase


4.
As 1 + 300 U/kg r-AppA2 phytase


5.
As 1 + 10,000 U/kg r-AppA2 phytase



















Initial body weights (g; mean of 12 hens)













T1
T2
T3
T4
T5


















R1
1699
1792
1682
1790
1707



R2
1785
1698
1734
1855
1694



R3
1690
1665
1775
1724
1824



R4
1688
1745
1739
1823
1760



mean
1716
1725
1733
1798
1746







Pooled SEM = 26



LSD = 78
















4-wk body weights (g: mean of 12 hens)













T1
T2
T3
T4
T5


















R1
1566
1802
1763
1769
1748



R2
1558
1734
1816
1860
1723



R3
1633
1707
1744
1769
1850



R4
1615
1749
1762
1827
1757



mean
1593
1748
1771
1806
1770







Pooled SEM = 21



LSD = 64
















12-wk body weights (g: mean of 12 hens)













T1
T2
T3
T4
T5


















R1
—
1876
1831
1792
1781



R2
—
1791
1775
1856
1791



R3
—
1800
1765
1806
1933



R4
—
1853
1814
1876
1815



mean
—
1830
1796
1833
1830







Pooled SEM = 24



LSD = 74






Treatments:














1.
P-deficient corn-soybean meal basal diet (0.10% pa; 3.8% Ca; 17%



CP)


2.
As 1 + 0.10% Pi from KH2PO4


3.
As 1 + 150 U/kg r-AppA2 phytase


4.
As 1 + 300 U/kg r-AppA2 phytase


5.
As 1 + 10,000 U/kg r-AppA2 phytase



















Feed intake (g/h/d)1













T1
T2
T3
T4
T5


















R1
89
118
122
115
116



R2
92
125
114
122
119



R3
89
117
118
116
124



R4
94
123
119
115
123



mean
 91b
 121a
 118a
 117a
 121a







Pooled SEM = 2



LSD = 5



1Means are average daily feed intakes of hens for the first 4-wk period for treatment 1, and for the entire 12-wk period for treatments 2–5.
















Egg weights (g)1













T1
T2
T3
T4
T5


















R1
57.5
64.0
65.4
65.7
64.5



R2
63.5
64.7
64.3
66.0
65.5



R3
60.3
64.3
64.6
64.8
65.6



R4
62.8
63.3
62.2
65.3
63.7



mean
61.0b
64.1a
64.1a
65.5a
64.8a







Pooled SEM = 0.7



LSD = 2.2



1Means are average egg weights of hens for the first 4-wk period for treatment 1, and for the entire 12-wk period for treatments 2–5.






Treatments:

    1. P-deficient corn-soybean meal basal diet (0.10% pa; 3.8% Ca; 17% CP)
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+150 U/kg r-AppA2 phytase
    4. As 1+300 U/kg r-AppA2 phytase
    5. As 1+10,000 U/kg r-AppA2 phytase












Egg production by week(%)




















1
2
3
4
5
6
7
8
9
10
11
12























D1
75.3
55.7
36.0
22.9
—
—
—
—
—
—
—
—


D2
88.4
90.8
88.1
87.8
88.4
85.4
86.0
81.8
80.4
79.8
80.7
78.3


D3
84.5
85.1
83.3
85.1
83.3
82.1
83.6
79.2
77.4
77.4
79.5
76.5


D4
86.6
86.3
83.9
82.4
82.1
84.5
81.5
77.4
78.0
74.7
73.8
72.0


D5
82.4
83.3
83.6
84.8
80.7
81.3
82.7
78.6
80.1
78.9
76.8
72.6


SEM
3.0
3.2
3.4
3.5
3.3
3.2
3.2
4.1
3.4
4.5
3.5
3.9



















Egg production (%)1













T1
T2
T3
T4
T5
















R1
44.6
86.5
73.0
81.0
80.7


R2
60.1
85.7
78.1
81.7
74.7


R3
43.2
87.2
84.3
83.9
87.8


R4
42.0
80.9
90.8
74.6
85.3


mean
47.5
85.1
81.6
80.3
82.1


Least-squares means2
53.8b
81.2a
80.7a
77.8a
82.9a





Pooled SEM = 2.1


1Means are the average egg production of hens for the first 4-wk period for treatment 1, and for the entire i2-wk period for treatments 2–5.


2Due to variation in week 1 egg production (above), covariance was used to analyze overall egg production, with least-squares means showing the effect of the covariable.






EXAMPLE 13 
In Vivo Effects of Yeast-Expressed Phytases Fed to Finishing Pigs
The procedure was as described in Example 8 except finishing pigs (i.e., gilts and barrows) were tested and the basal diet and treatment groups were as follows:
Treatments:


    1. P-deficient corn-soybean meal basal diet
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+250 FTU/kg r-AppA2 phytase
    4. As 1+500 FTU/kg r-AppA2 phytase
    5. As 1+1,000 FTU/kg r-AppA2 phytase
    6. As 1+10,000 FTU/kg r-AppA2 phytase

Basal Diets:


















Weight range (kg)











Ingredient
50–80
80–120







Cornstarch
to 100
to 100



Corn
78.42
83.85



Soybean meal, dehulled
18.08
12.65



Limestone, ground
1.06
1.07



Dicalcium phosphate
0.16
—



Trace mineral premix
0.35
0.35



Vitamin premix
0.10
0.10



L-Lysine-HCl, feed-grade
0.16
0.11



L-threonine, feed-grade
0.02
—



Antibiotic premix
0.75
0.75



Calculated composition (NRC, 1998)



Crude protein, %
15.1
13.0



Lysine, total %
0.88
0.69



Calcium, %
0.50
0.45



Phosphorus, total %
0.38
0.32



Phosphorus, estimated bioavailable, %
0.09
0.05



ME, kcal/kg
3293
3295










The following tables are labeled as described in Example 8 and some of the responses described in Example 8 were measured. Gain/feed ratio is shown rather than a feed/gain ratio.
The results show that AppA2 was as effective as phosphate at increasing bone mass and mineral content, and at improving the gain/feed ratio.
Treatments:


    1. P-deficient corn-soybean meal basal diet
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+250 FTU/kg r-AppA2 phytase
    4. As 1+500 FTU/kg r-AppA2 phytase
    5. As 1+1,000 FTU/kg r-AppA2 phytase
    6. As 1+10,000 FTU/kg r-AppA2 phytase












Initial pig weights (kg)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
52.2
52.8
53.0
51.8
52.0
51.8
51.2
50.8
52.2
52.2
51.3
52.4


R2
51.0
51.1
51.7
50.3
51.6
50.4
49.6
49.6
50.3
50.0
50.4
50.8


R3
48.2
49.6
49.8
49.6
50.1
49.2
48.1
49.6
47.9
47.1
48.8
48.4


R4
46.4
46.5
46.5
46.9
47.4
47.9
45.9
45.4
44.3
46.6
46.5
45.7


R5
52.0
44.1
51.0
52.4
46.4
50.7
43.4
43.9
44.0
43.1
44.1
44.0


mean
50.0
48.8
50.4
50.2
49.5
50.0
47.6
47.9
47.7
47.8
48.2
48.3





Pooled SEM = 0.4
















Phase-switch pig weights (kg)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
78.8
83.0
85.0
76.0
79.6
79.2
79.7
80.1
88.6
84.1
83.1
89.6


R2
76.8
80.6
86.9
79.9
82.2
83.8
80.0
83.5
87.7
84.5
87.3
83.5


R3
73.7
79.8
77.1
79.1
79.0
75.9
77.1
77.6
81.3
79.9
82.6
82.4


R4
82.3
82.5
79.2
79.1
84.4
84.5
74.7
78.1
73.9
84.6
78.9
79.1


R5
84.5
78.5
84.7
83.2
85.3
85.2
83.3
80.5
84.4
87.2
81.7
82.5


mean
79.2
80.9
82.6
79.5
82.1
81.7
78.9
79.9
83.2
83.4
82.7
83.4





Pooled SEM = 0.8
















Final pig weights (kg)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
111.3
121.2
121.9
115.9
112.9
111.1
105.9
109.5
119.9
116.1
105.4
130.1


R2
111.5
119.6
132.7
111.9
121.3
116.3
105.8
115.6
118.3
115.3
123.3
112.5


R3
115.9
126.4
117.1
119.9
114.0
120.7
104.9
107.9
123.6
125.2
127.1
130.8


R4
116.6
117.9
110.0
110.0
119.7
122.1
120.2
121.6
117.9
127.7
109.3
123.0


R5
118.3
111.6
122.3
114.2
123.0
117.1
115.2
110.4
119.2
135.1
119.1
117.1


mean
114.7
119.3
120.8
114.4
118.2
117.5
110.3
113.0
119.8
123.9
116.8
122.7





Pooled SEM = 3.0 (Sex × Diet, P < 0.10)


Contrasts: Sex × Pi (2) vs Phytase (3–6), P < 0.05.






Treatments:

    1. P-deficient corn-soybean meal basal diet
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+250 FTU/kg r-AppA2 phytase
    4. As 1+500 FTU/kg r-AppA2 phytase
    5. As 1+1,000 FTU/kg r-AppA2 phytase
    6. As 1+10,000 FTU/kg r-AppA2 phytase












Weight gain, initial-switch (g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
1024
1163
1233
931
1061
1055
816
836
1040
911
909
1063


R2
993
1135
1353
1140
1178
1283
867
966
1068
986
1055
936


R3
979
1162
1048
1131
1109
1028
829
801
955
935
965
971


R4
1025
1028
936
921
1057
1046
822
936
847
1001
925
952


R5
931
983
963
881
1109
984
950
870
962
1048
895
918


mean
990
1094
1107
1001
1103
1079
857
882
974
976
950
968





Pooled SEM = 0.24


Contrasts: Barrows (Ba) vs Gilts (Gi), P < 0.01; 1 vs 2–6, P < 0.01
















Weight gain, switch-final(g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
832
979
945
1023
855
818
706
794
844
865
604
1095


R2
890
1000
1174
822
1001
833
688
874
835
830
974
784


R3
919
1013
870
889
762
974
545
593
829
888
871
949


R4
927
971
832
833
954
1015
893
854
861
904
596
862


R5
912
895
1018
836
1020
864
531
499
581
798
624
577


mean
896
972
968
881
918
901
673
723
790
857
734
853





Pooled SEM = 37


Contrasts: Ba vs Gi, P < 0.05
















Weight gain, overall (g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
909
1053
1060
986
937
913
760
814
940
887
752
1079


R2
931
1054
1246
949
1072
1013
775
919
948
906
1013
858


R3
941
1066
934
976
888
993
660
678
880
907
910
958


R4
975
1006
882
876
1004
1030
864
887
855
944
730
898


R5
922
938
992
858
1063
922
703
652
738
901
735
717


mean
935
1023
1023
929
993
974
752
790
872
909
828
902





Pooled SEM = 38 (Sex × diet, P < 0.10)


Contrasts: Sex × Pi (2) vs Phytase (3–6), P < 0.05






Treatments:

    1. P-deficient corn-soybean meal basal diet
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+250 FTU/kg r-AppA2 phytase
    4. As 1+500 FTU/kg r-AppA2 phytase
    5. As 1+1,000 FTU/kg r-AppA2 phytase
    6. As 1+10,000 FTU/kg r-AppA2 phytase












Feed intake, initial-switch (g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
2670
2733
2947
2271
2516
2448
2152
2029
2437
2074
2211
2579


R2
2541
2564
2940
2590
2484
2899
2425
2068
2543
2326
2363
1979


R3
2277
2499
2338
2385
2601
2066
2134
2020
2388
2168
2207
2093


R4
2371
2370
2311
2206
2457
2077
2104
2230
1919
2139
2260
2215


R5
2665
2312
2603
2308
2696
2366
2008
1289
2396
2237
1494
1866


mean
2505
2496
2628
2352
2551
2371
2165
1927
2337
2189
2107
2146





Pooled SEM = 67


Contrasts: Ba vs Gi, P < 0.01
















Feed intake, switch-final (g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
3181
3427
3559
3270
2962
2918
2443
2615
2890
2651
2094
3739


R2
2922
3039
3833
3011
3141
3147
2481
2652
2936
2796
3316
2565


R3
3087
3330
2847
2877
2904
2686
2241
2110
2849
2692
2820
2952


R4
2978
2945
2872
2646
3104
2876
2935
2946
2373
2685
2481
2836


R5
3244
2958
3549
3106
3517
3008
2307
1747
2728
2947
2224
2385


mean
3082
3140
3332
2982
3126
2926
2482
2414
2755
2754
2587
2895





Pooled SEM = 105


Contrasts: Ba vs Gi, P < 0.01
















Feed intake, overall (g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
2977
3149
3314
2870
2784
2726
2302
2330
2670
2370
2151
3175


R2
2770
2849
3476
2842
2878
3048
2454
2368
2745
2568
2853
2280


R3
2794
3030
2663
2699
2794
2462
2197
2073
2661
2479
2571
2603


R4
2683
2632
2599
2432
2790
2488
2597
2655
2188
2463
2391
2583


R5
2963
2644
3089
2718
3118
2696
2184
1559
2591
2654
1993
2171


mean
2837
2861
3028
2712
2873
2684
2347
2197
2571
2507
2378
2562





Pooled SEM = 81


Contrasts: Ba vs GI, P < 0.01






Treatments:

    1. P-deficient corn-soybean meal basal diet
    2. As 1+0. 10% Pi from KH2PO4 
    3. As 1+250 FTU/kg r-AppA2 phytase
    4. As 1+500 FTU/kg r-AppA2 phytase
    5. As 1+1,000 FTU/kg r-AppA2 phytase
    6. As 1+10,000 FTU/kg r-AppA2 phytase












Gain/feed, initial-switch (g/kg)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
383
425
418
410
421
431
379
412
427
439
411
412


R2
391
443
460
440
474
442
357
467
420
424
447
473


R3
430
465
448
474
427
498
388
396
400
431
437
464


R4
432
434
405
418
430
504
390
420
441
468
409
430


R5
349
425
370
382
411
416
473
675
402
469
599
492


mean
397
438
420
425
433
458
397
474
418
446
461
454





Pooled SEM = 12


Contrasts: 1 vs. 2–6, P < 0.01; 3 vs 4–6, P < 0.10
















Gain/feed, switch-final (g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
262
286
265
313
289
281
289
304
292
326
289
293


R2
305
329
306
273
319
265
277
329
285
297
294
306


R3
298
304
307
309
262
363
243
281
291
330
309
321


R4
311
329
290
315
307
353
304
290
363
337
240
304


R5
281
303
287
269
290
287
230
285
213
271
281
242


mean
291
310
291
296
293
310
269
298
289
312
283
293





Pooled SEM = 8
















Gain/feed overall (g/d)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
305
334
320
344
337
335
330
349
352
374
350
340


R2
336
370
358
334
372
332
316
388
345
353
355
376


R3
337
352
351
362
318
403
301
327
331
366
354
368


R4
363
381
339
361
360
414
333
334
391
383
305
348


R5
311
355
321
316
341
342
322
418
285
340
382
330


mean
331
358
338
343
346
365
320
363
341
363
349
352





Pooled SEM = 8


Contrasts: 1 vs 2–6, P < 0.01






Treatments:

    1. P-deficient corn-soybean meal basal diet
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+250 FTU/kg r-AppA2 phytase
    4. As 1+500 FTU/kg r-AppA2 phytase
    5. As 1+1,000 FTU/kg r-AppA2 phytase
    6. As 1+10,000 FTU/kg r-AppA2 phytase












Fibula Dry Weight (g)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
8.18
10.90
11.45
12.11
10.08
12.08
7.54
10.95
9.92
9.42
9.87
11.29


R2
8.84
11.74
8.66
10.98
11.21
11.66
7.96
8.81
9.33
11.41
10.70
12.73


R3
8.54
11.29
9.81
11.90
10.10
12.77
8.62
10.25
9.94
10.50
11.86
12.46


R4
9.82
10.69
9.06
10.22
11.05
12.40
8.26
11.61
9.67
10.92
10.91
10.49


R5
7.88
8.88
10.33
10.51
12.01
11.26
7.68
9.51
11.16
11.48
10.10
11.44


mean
8.65
10.70
9.86
11.14
10.89
12.03
8.01
10.23
10.00
10.75
10.69
11.68





Pooled SEM = 0.27


Contrasts: 1 vs 2–6, P < 0.01; 3 vs 4–6, P < 0.01
















Fibula Ash Weight (g)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
4.37
6.32
6.34
6.90
6.03
6.94
4.06
6.42
5.21
5.33
5.61
6.78


R2
4.26
7.05
5.12
6.49
6.24
6.80
4.36
5.18
5.51
6.25
6.21
7.16


R3
4.51
6.54
5.78
7.17
5.81
7.49
4.35
5.91
6.11
5.79
6.93
7.23


R4
5.34
6.35
5.19
5.90
6.73
7.33
4.28
7.13
5.66
6.35
6.56
6.22


R5
4.37
5.22
6.02
6.34
7.06
6.64
3.91
5.64
7.02
6.25
5.88
6.93


mean
4.57
6.30
5.69
6.56
6.37
7.04
4.19
6.06
5.90
5.99
6.24
6.86





Pooled SEM = 0.17


Contrasts: Ba vs Gi, P < 0.10; 1 vs 2–6, P < 0.01; 3 vs 4–6, P < 0.01; 4 vs 5–6, P < 0.10; 5 vs 6, P < 0.01
















Fibula Ash Percent (%)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
53.42
57.98
55.37
56.98
59.82
57.45
53.85
58.63
52.52
56.58
56.84
60.05


R2
48.19
60.05
59.12
59.11
55.66
58.32
54.77
58.80
59.06
54.78
58.04
56.25


R3
52.81
57.93
58.92
60.25
57.52
58.65
50.46
57.66
61.47
55.14
58.43
58.03


R4
54.38
59.40
57.28
57.73
60.90
59.11
51.82
61.41
58.53
58.15
60.13
59.29


R5
55.46
58.78
58.28
60.32
58.78
58.97
50.91
59.31
62.90
54.44
58.22
60.58


mean
52.85
58.83
57.79
58.88
58.54
58.50
52.36
59.16
58.90
55.82
58.33
58.84





Pooled SEM = 0.65


Contrasts: 1 vs 2–6, P < 0.01






Treatments:

    1. P-deficient corn-soybean meal basal diet
    2. As 1+0.10% Pi from KH2PO4 
    3. As 1+250 FTU/kg r-AppA2 phytase
    4. As 1+500 FTU/kg r-AppA2 phytase
    5. As 1+1,000 FTU/kg r-AppA2 phytase
    6. As 1+10,000 FTU/kg r-AppA2 phytase












Metatarsal Dry Weight (g)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6























R1
11.42
14.03
15.84
15.36
14.43
13.85
11.77
15.90
16.00
15.48
12.65
15.05


R2
11.89
14.52
13.27
14.26
13.73
15.06
11.66
13.74
14.14
14.19
13.75
14.87


R3
14.01
14.45
13.20
13.99
14.91
17.43
10.52
12.20
11.95
16.31
17.53
17.13


R4
12.25
14.38
12.54
15.99
15.26
17.01
11.68
13.49
13.16
14.20
12.77
14.23


R5
12.55
13.30
14.30
14.36
17.79
14.29
11.26
12.76
12.47
16.93
12.78
13.10


mean
12.42
14.14
13.83
14.79
15.22
15.53
11.38
13.62
13.54
15.42
13.90
14.88





Pooled SEM = 0.44


Contrasts: 1 vs 2–6, P < 0.01; 3vs 4–6, P < 0.05
















Metatarsal Ash Weight (g)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
5.28
6.59
7.97
6.93
6.74
6.86
4.74
7.21
6.72
7.09
6.07
7.50


R2
6.81
7.10
5.94
6.74
6.32
7.44
4.84
6.28
6.40
6.55
6.71
7.07


R3
4.82
6.95
6.41
6.77
6.72
7.88
4.82
5.59
6.67
6.99
8.13
8.11


R4
4.83
6.81
6.26
7.73
7.88
7.48
4.86
7.27
5.92
7.15
6.97
7.13


R5
5.20
5.75
7.22
6.99
8.33
7.14
5.24
6.61
6.65
7.07
6.04
6.55


mean
5.39
6.64
6.76
7.03
7.20
7.36
4.90
6.59
6.47
6.97
6.78
7.27





Pooled SEM = 0.18


Contrasts: 1 vs 2–6, P < 0.01; 3 vs 4–6, P < 0.05
















Metatarsal Ash Percent (%)










Barrows
Gilts




















1
2
3
4
5
6
1
2
3
4
5
6






















R1
46.25
46.99
50.31
45.15
46.75
49.56
40.22
45.37
42.01
45.80
47.96
49.84


R2
39.90
48.90
44.75
47.16
46.02
49.39
41.50
45.72
45.27
46.18
48.80
47.55


R3
34.38
48.12
48.59
48.36
45.09
45.18
45.84
45.78
55.84
42.87
46.39
47.35


R4
39.44
47.27
49.89
48.36
51.65
43.98
41.63
53.93
44.97
50.32
54.59
50.11


R5
41.44
43.26
50.50
48.69
46.81
49.95
46.50
51.82
53.33
41.74
47.28
50.00


mean
40.28
46.91
48.81
47.54
47.26
47.61
43.14
48.52
48.28
45.38
49.00
48.97





Pooled SEM = 1.05


Contrasts: 1 vs 2–6, P < 0.01






EXAMPLE 14 
In Vivo Effects of Yeast-Expressed Phytases Fed to Pigs
The procedure was as described in Example 8 except that the treatment groups were as follows:










28-day period
















1.
Basal − .08% available phosphorus


2.
Basal + .05 phosphorus from monosodium phosphate


3.
Basal + .10 phosphorus from monosodium phosphate


4.
Basal + .15 phosphorus from monosodium phosphate


5.
Basal + 250 FTU/kg experimental phytase product


6.
Basal + 500 FTU/kg experimental phytase product


7.
Basal + 1,000 FTU/kg experimental phytase product


8.
Basal + 2,000 FTU/kg experimental phytase product


9.
Basal + Natuphos ® 500 FTU/kg









The results are shown in the following table. The results show that AppA2 increases bone mass and mineral content and improves the gain/feed ratio as effectively as phosphate.










Effect of phytase supplementation on pig growth performance and bone asha

















Added NaH2PO4H2Oh








500



Phytase units/gc
0.00
0.05
0.10
0.15
250
500
1,000
2,000
BASF
S.E.




















Daily gain, kgij
0.35g
0.39fg
0.46de
0.49d
0.38g
0.42el
0.47d
0.49d
0.42l
0.01


Daily feed intake, kgj
0.75f
0.75f
0.81def
0.85d
0.77ef
0.79def
0.83def
0.85d
0.85de
0.07


G:Fijk
0.48f
0.53de
0.57d
0.58d
0.50ef
0.54de
0.57d
0.57d
0.49ef
0.02


Fibula ash, gij
0.57h
0.65gh
0.77f
0.88c
0.59h
0.72f
0.85e
0.97d
0.70fg
0.03


Fibula ash, %ij
34.6h
36.0gh
37.8g
41.5de
33.9h
38.2fg
40.4ef
42.6d
38.5f
0.84


% Available Pl
18.43g
—
—
—
22.56g
38.31f
53.56e
66.71d
34.47f
4.07


aP Intake, g/dijl
0.58g
0.92g
1.45f
1.92c
0.68g
1.22f
1.81e
2.31d
1.17f
0.13


Supplemental aP Intake,
0.02h
0.34gh
0.84f
1.27e
0.12h
0.62fg
1.17e
1.64d
0.57fg
0.13


g/dijm





aSix replications of two pigs per pen for performance data; six replications of two pigs per pen for bone data except for the treatment with phytase added at 500 units/g, which has five replications.


bAdded P from monosodium phosphate (NaH2PO4H2O) to the basal diet.


cSupplemental phytase added to the basal diet.


defghMeans Within a row without common superscripts differ (P < 0.05).


iLinear effect of added P from monosodium phosphate (P < 0.001).


jLinear effect of supplemental phytase (P < 0.001).


kUF phytase v. BASF phytase (P < 0.07)


lAssumes that the P in corn, soybean meal, and monosodium phosphate is 11.25, and 100% available, respectively.


mAssumes that the P in monosodium phosphate is 100% available.






EXAMPLE 15 
In Vivo Effects of Yeast-Expressed Phytases Fed to Chicks and Pigs
The procedure for the studies summarized in the following tables was as described in Example 8. The treatment groups are shown in each table and the basal diet compositions are shown in the following table (see next page). The results show that AppA2 (ECP) is as effective as phosphate in improving the gain/feed ratio and in increasing bone mass and mineral content. The results also show that AppA2 is more effective than Natuphos® and Ronozyme® at increasing bioavailable phosphate. Furthermore, the results show that AppA2 increases egg weight and egg production in laying hens as effectively as phosphate.










Percentage composition of diets (as-fed basis).












Chick
Young pig
Finishing pig assay
Laying












Ingredient
assays
assay
50–80 kg
80–120 kg
hen assay





Cornstarch
to 100
to 100
to 100
to 100
—


Corn
50.89
60.85
78.42
83.85
63.65


Soybean meal, dehulled
39.69
31.19
18.08
12.65
25.65


Soybean oil
5.00
3.00
—
—
—


Limestone, ground
1.67
1.06
1.06
1.07
9.80


Salt
0.40
—
—
—
0.40


Dicalcium phosphate
—
—
0.16
—
—


Trace mineral premix
0.15a
0.35b
0.35b
0.35b
0.20a


Vitamin premix
0.20c
0.20d
0.10d
0.10d
0.15c


Choline Chloride (60%)
0.20
—
—
—
0.05


Antibiotic premix
0.05e
1.00f
0.75g
0.75g
—


Copper sulfate
—
0.08
—
—
—


L-Lysine HCI, feed grade
—
0.17
0.16
0.11
—


L-Threonine, feed grade
—
—
0.02
—
—


DL-Methionine, feed grade
0.20
0.05
—
—
0.10


Chemical composition


Crude protein, %h
22.6
20.8
15.1
13.0
17.0


Total phosphorus, %h
0.42
0.35
0.38
0.32
0.34


Available phosphorus, %i
0.10
0.075
0.09
0.05
0.07


Calcium, %i
0.75
0.60
0.50
0.45
3.8


ME, kcal/kgi
3123
3387
3293
3295
2758





aSupplied the following per kilogram of complete diet: Fe, 75 mg (FeSO4H2O); Zn, 100 mg (ZnO); Mn, 75 mg (MnO); Cu, 8 mg (CuSO4H2O); I, 0.35 mg (CaI2); Se, 0.3 mg (Na2SeO3); NaCL, 3 g.


bSupplied the following per kilogram of complete diet: Fe, 90 mg (FeSO4H2O); Zn, 100 mg (ZnO); Mn, 20 mg (MnO); Cu, 8 mg (CuSO4H2O); I, 0.35 mg (CaI2); Se,.0.3 mg (Na2SeO3); NaCl, 3 g.


cSupplied the following per kilogram of complete diet: retinyl acetate, 1,514 μg; cholecalciferol, 25 μg; DL-α-tocopheryl acetate, 11 mg; menadione sodium bisulfite complex, 2.3 mg; niacin, 22 mg; D-Ca-pantothenate, 10 mg; riboflavin, 4.4 mg; vitamin B12, 11 μg.


dSupplied the following per kilogram of complete diet; retinyl acetate, 2,273 μg; cholecalciferol, 16.5 μg; DL-α-tocopheryl acetate, 88 mg; menadione, 4.4 mg (menadione sodium bisulfite complex); niacin, 33 mg; D-Ca-pantothenate, 24.2 mg; riboflavin, 8.8 mg; vitamin B12, 35 μg; choline chloride, 319 mg.


eProvided 50 mg of bacitracin per kilogram of complete diet.


fProvided 55 mg of mecadox per kilogram of complete diet.


gProvided 38 mg of roxarsone per kilogram of complete diet.


hAnalyzed (AOAC, 1999).


iCalculated (NRC, 1994; NRC, 1998).
















Assessment of relative phosphorus bioavailability in chicks as affected by


two different phytase enzymes (Chick assay 1)a.












Weight
Gain/feed,
Tibia ash
Bioavailable












Diet
gain, g
g/kg
%
mg
P, %





1. Basal diet
259e
617d
28.1f
264f
—


2. As 1 + 0.05% Pi
290d
654c
32.2d
311e
—


  (KH2PO4)


3. As 1 + 0.10% Pi
323c
639cd
36.4c
414d
—


  (KH2PO4)


4. As 1 + 500 FTU/kg
289d
666c
30.0c
293c
0.027d


  Natuphos ®


5. As 1 + 500 FTU/kg
346c
656c
37.8c
448c
0.124c


ECP


Pooled SEM
 6
 10
 0.5
 10
0.006





aValues are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d post-hatching; average initial weight was 91 g.


bThe linear regression of tibia ash (mg) for Diets 1 to 3 as a function of supplemental P intake (g) was Y = 257.1 ± 9.8 + 299.0 ± 30.7X (r2 = 0.88); Bioavailable P concentrations (equivalent P yields) for Diets 4 and 5 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g), and multiplying by 100.


c,d,e,fMeans within a column with different superscripts are different, P < 0.05.
















Relative phosphorus bioavailability in chicks fed different


phytase enzymes (Chick assay 2)a.












Weight
Gain/feed,
Tibia ash
Bioavailable












Diet
gain, g
g/kg
%
mg
P, %





 1. Basal diet
176k
569k
24.9k
183k
—


 2. As 1 + 0.05% Pi
253hi
680h
30.0h
272h
—


   (KH2PO4)


 3. As 1 + 0.10% Pi
293g
703fgh
34.3g
347g
—


   (KH2PO4)


 4. As 1 + 0.15% Pi
333f
731ef
37.3e
455e
—


   (KH2PO4)


 5. As 1 + 500
218j
620j
27.2j
224j
0.026g


   FTU/kg


   Natuphos ®c


 6. As 1 + 500
236ij
632j
27.5ij
236ij
0.032fg


   FTU/kg


   Natuphos ®d


 7. As 1 + 1,000 FTU/
265i
675gh
28.9hi
262hi
0.048f


   kg Natuphos ®d


 8. As 1 + 500
219j
634j
26.9j
223j
0.028g


   FTU/kg


   Ronozyme ®


 9. As 1 + 1,000
245i
682gh
27.5ij
242hij
0.038fg


   FTU/kg


   Ronozyme ®


10. As 1 + 500
318ef
708fgh
36.5ef
409f
0.125e


FTU/kg ECP


Pooled SEM
 7
 10
 0.6
 12
0.006





aValues are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d posthatching; average initial weight was 83 g.


bThe linear regression of tibia ash (mg) for Diets 1 to 4 as a function of supplemental P intake (g) was Y = 187.9 ± 8.7 + 393.4 ± 21.2X (r2 = 0.95); Bioavailable P concentrations (equivalent P yields) for Diets 4–11 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g) of the pen, and multiplying by 100.


cEnzyme was from the same batch that was used for chick assay 1.


dEnzyme was from a different batch that was used for chick assay 1.


e,f,g,h,i,j,kMeans within a column with different superscripts are different, P < 0.05.
















The effect of activity level on the phosphorus-releasing


efficacy of E. coli phytase in chicks (Chick assay 3)a.















Bioa-




Gain/
Tibia
vail-



Weight
feed,
ash
able


Diet
gain, g
g/kg
mg
P, %b





1. Basal diet
219h
661c
237i
—


2. As 1 + 0.05% Pi(KH2PO4)
283fg
692d
299h
—


3. As 1 + 0.10% Pi(KH2PO4)
314c
720c
413g
—


4. As 1 + 0.15% Pi(KH2PO4)
327dc
731c
490e
—


5. As 1 + 500 FTU/kg ECP
321dc
731c
447f
0.125c


6. As 1 + 1,000 FTU/kg ECP
335cd
732c
559d
0.183d


7. As 1 + 1,500 FTU/kg ECP
344c
737c
616c
0.211c


8. As 1 + 500 FTU/kg Natuphos ®
276g
691d
290h
0.037f


Pooled SEM
 6
 10
 12
0.005





aValues are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d post-hatching; average initial weight was 97 g.


bThe linear regression of tibia ash (mg) for Diets 1 to 4 as a function of supplemental P intake (g) was Y = 232.0 ± 6.9 + 389.9 ± 16.7X (r2 = 0.97); Bioavailable P concentrations (equivalent P yields) for Diets 5 to 8 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g) of the pen, and multiplying by 100.


c,d,e,f,g,h.iMeans within a column with different superscripts are different, P < 0.05.
















Combining 3- and 6-phytases does not produce


synergistic effects on Pi-release in chicks fed


a corn-soybean meal diet(Chick assay 4)a.












Weight
Gain/feed,
Tibia ash
Bioavailable












Diet
gain, g
g/kg
%
mg
P, %b





 1. Basal diet
137g
610g
25.4g
134h
—


 2. As 1 + 0.05% Pi
191ef
678dc
29.0f
198fg
—


   (KH2PO4)


 3. As 1 + 0.10% Pi
225d
712d
32.8c
253e
—


   (KH2PO4)


 4. As 1 + 0.15% Pi
276c
762c
36.3d
339d
—


   (KH2PO4)


 5. As 1 + 500
192ef
624fg
28.0f
187g
0.041g


   FTU/kg


   Natuphos ®


 6. As 1 + 500
182f
655cf
27.7f
188g
0.047fg


   FTU/kg


   Ronozyme ®


 7. As 1 + 500 FTU/
272c
760c
37.0d
343d
0.153d


   kg ECP


 8. As 5 + 6
211de
693de
28.3f
212fg
0.064ef


 9. As 5 + 7
282c
763c
37.8d
360d
0.162d


10. As 1 + 1,000
217d
703d
29.0f
217f
0.067c


   FTU/kg


   Natuphos ®


11. As 1 + 1,000
201def
666ef
27.9f
194fg
0.050efg


   FTU/kg


   Ronozyme ®


12. As 1 + 1,000
292c
758c
41.1c
433c
0.206c


FTU/kg ECP


Pooled SEM
 9
 15
 0.6
 10
0.007





aValues are means of five pens of four male chicks fed the experimental diets during the period 8 to 22 d post-hatching; average initial weight was 68 g.


bThe linear regression of tibia ash (mg) for Diets 1 to 4 as a function of supplemental P intake (g) was Y = 138.6 ± 4.9 + 371.3 ± 14.7X (r2 = 0.97); Bioavailable P concentrations (equivalent P yields) for Diets 5 to 8 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g) of the pen, and multiplying by 100.


c,d,e,f,gMeans within a column with different superscripts are different, P < 0.05.
















Effect of E. coli phytase on performance of laying hens from week 1–4.a















Feed
Egg
Egg



Initial hen
4-wk hen
intake,
production,
weight,


Diet
weight, g
weight, g
g/d
%b
g





1. P-deficient
1716
1593
 90
54.0
61.0


  basal diet


2. As 1 +
1725
1748
122
84.8
64.2


  0.10% Pi


3. As 1 + 150
1733
1771
119
83.7
63.8


  FTU/kg


  ECP


4. As 1 + 300
1798
1806
119
82.3
65.4


  FTU/kg


  ECP


5. As 1 +
1746
1770
123
85.9
65.1


10,000 FTU/


kg ECP


Pooled SEM
 26
 21c
 2c
 1.6c
 0.7c





aData are means of four replicates of 12 hens for the first 4 weeks of the study period.


bEgg production (%) analyzed using covariance; data presented are least-squares means.


cDiet 1 vs diets 2–5, P < 0.01.
















Effect of E. coli phytase on performance of laying hens from


week 5–12a.













Feed
Egg




4-wk hen
intake,
production,
Egg


Diet
weight, g
g/d
%b
weight, g





2. As 1 + 0.10% Pi
1830
120
80.5
64.0


3. As 1 + 150 FTU/kg
1796
118
80.6
64.1


   ECP


4. As 1 + 300 FTU/kg
1833
116
77.2
65.5


   ECP


5. As 1 + 10,000 FTU/kg
1830
120
81.2
64.8


   ECP


Pooled SEM
 24
 2
 2.5
 0.5c





aData are means of four replicates of 12 hens for weeks 5 through 12 of the study period. Diet 1 was removed from study due to poor egg production.


bEgg production (%) analyzed using covariance; data presented are least-squares means.


cDiet 3 vs diets 4 and 5, P < 0.01.
















Effect of E. coli phytase on performance of laying hens from week 1–12a.













Feed
Egg
Egg



Hen weights
intake,
production,
weight,













Diet
Initial
4-wk
12-wk
g/d
%b
g





1. P-deficient
1716
1593
—
 90
53.8
61.0


  basal diet


2. As 1 +
1725
1748
1830
121
81.2
64.1


  0.10% PI


3. As 1 + 150
1733
1771
1796
118
80.7
64.1


  FTU/kg


  ECP


4. As 1 + 300
1798
1806
1833
117
77.8
65.5


  FTU/kg


  ECP


5. As 1 +
1746
1770
1830
121
82.9
64.8


10,000


FTU/kg ECP


Pooled SEM
 26
 21c
 24
 2c
 2.1c
 0.7c





aData are means of four replicates of 12 hens. Data are means for the first 4 weeks for diet 1, but for all 12 weeks for diets 2–5.


bEgg production (%) analyzed using covariance; data presented are least-squares means.


cDiet 1 vs diets 2–5, P < 0.01.
















Relative bioavailability of phosphorus in young pigs fed


different phrase enzymes (Pig assay 1).













Gain/





Weight
feed,
Fibula ash
Bioavailable












Diet
gain, g/da
g/kga
%
mg
P, %c





1. Basal diet
369f
533f
29.3g
 666i
—


2. As 1 +
435e
576ef
32.8f
 766hi
—


  0.05% Pi


  (KH2PO4)


3. As 1 +
476de
618de
36.6d
 972ef
—


  0.10% Pi


  (KH2PO4)


4. As 1 +
509d
660d
36.6d
1123d
—


  0.15% Pi


  (KH2PO4)


5. As 1 + 400
460c
605de
34.4def
 889fg
0.081de


  FTU/kg


  Natuphos ®


6. As 1 + 400
445e
565ef
33.5ef
 805gh
0.043f


  FTU/kg


  Ronozyme ®


7. As 1 + 400
443e
583ef
35.0def
 968ef
0.108d


FTU/kg ECP


Pooled SEM
 17
 21
 0.8
 38
0.016





aData are means of 10 individually-fed pigs over a 23-d feeding period; average initial weight was 8.4 kg.


bData are means of five individually-fed pigs, chosen from the median-weight blocks at the end of the 23-d feeding period.


cThe linear regression of fibula ash (mg) for Diets 1 to 4 as a function of supplemental P intake (g) was Y = 664.5 ± 25.5 + 15.3 ± 1.4X (r2 = 0.87); Bioavailable P concentrations (equivalent P yields) for Diets 5–7 were determined by calculating equivalent bioavailable P intake (g) from the standard curve, dividing that by the feed intake (g) of the pig, and multiplying by 100.


d,e,f,g,h,iMeans within a column with different superscripts are different, P < 0.05.
















Effect of E. coil phytase on growth performance of finishing pigs (Pig assay 2)a.










Dietary treatment
















P-deficient
As 1 +
As 1 + 250
As 1 + 500
As 1 + 1,000
As 1 + 10,000
Pooled


Response variable
basal diet
0.10% Pi
FTU/kg ECP
FTU/kg ECP
FTU/kg ECP
FTU/kg ECP
SEM

















Daily gain, gb









Barrows
935
1023
1023
929
993
974


Gilts
752
790
872
909
828
902


Mean
844
907
947
919
910
938
38


Daily feed, gc


Barrows
2837
2861
3028
2712
2873
2684


Gilts
2347
2197
2571
2507
2378
2562


Mean
2592
2529
2800
2610
2625
2623
81


Gain/fed, g/kgd


Barrows
331
358
338
343
346
365


Gilts
320
363
341
363
349
352


Mean
325
361
339
353
347
359
9





aData are means of five individually-fed pigs of each sex fed their experimental diets from 48.9 to 117.6 kg body weight.


bSex x diet interaction. P < 0.10; Sex x Pi vs phytase-supplemented dies, P < 0.05.


cBarrows vs gilts, P < 0.01.


dP-deficient vs Pi- and phytase-supplemented diets, P < 0.01.
















Effect of E. coil phytase on bone characteristics of finishing pigs (Pig assay 2)a.










Dietary treatment
















P-deficient
As 1 +
As 1 + 250
As 1 + 500
As 1 + 1,000
As 1 + 10,000
Pooled


Response variable
basal diet
0.10% Pi
FTU/kg ECP
FTU/kg ECP
FTU/kg ECP
FTU/kg ECP
SEM

















Fibula ash, % gb









Barrows
52.9
58.8
57.8
58.9
58.5
58.5


Gilts
52.4
59.2
58.9
55.8
58.3
58.8


Mean
52.6
59.0
58.3
57.3
58.4
58.7
0.7


Fibula ash, gbcdef


Barrows
4.57
6.30
5.69
6.56
6.37
7.04


Gilts
4.19
6.06
5.90
5.99
6.24
6.86


Mean
4.38
6.18
5.80
6.28
6.31
6.95
0.17


Metatarsal ash,


%b


Barrows
40.3
46.9
48.8
47.5
47.3
47.6


Gilts
43.1
48.5
48.3
45.4
49.0
49.0


Mean
41.7
47.7
48.5
46.5
48.1
48.3
1.1


Metatarsal ash,


gbd


Barrows
5.4
6.6
6.8
7.0
7.2
7.4


Gilts
4.9
6.6
6.5
7.0
6.8
7.3


Mean
5.1
6.6
6.6
7.0
7.0
7.3
0.2





aData are means of five individually-fed pigs of each sex fed their experimental diets from 48.9 to 117.6 kg body weight.


bP-deficient vs Pi- and phytase-supplemented diets, P < 0.01.


cBarrows vs gilts, P < 0.10.


d250 U/kg vs higher phytase activity levels, P < 0.01.


e500 U/kg vs 1,000 and 10,000 U/kg phytase, P < 0.10.


f1,000 U/kg vs 10,000 U/kg phytase, P < 0.01.

## Claims

1. A method of improving the nutritional value of a foodstuff consumed by a monogastric animal by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the step of feeding to the animal the foodstuff in combination with less than 1200 units of an E. coli 6-phytase expressed in yeast per kilogram of the foodstuff,
wherein the bioavailability of phosphate from phytate is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell.

2. The method of claim 1 wherein the animal is an avian species.

3. The method of claim 2 wherein the avian species is selected from the group consisting of a chicken, a turkey, a duck, and a pheasant.

4. The method of claim 1 wherein the animal a porcine species.

5. The method of claim 1 wherein the animal is a marine or a fresh water aquatic species.

6. The method of claim 1 wherein the animal is a domestic animal.

7. The method of claim 6 wherein the domestic animal is a canine species.

8. The method of claim 6 wherein the domestic animal is a feline species.

9. The method of claim 1 wherein the animal is a human.

10. The method of claim 4 wherein the foodstuff is pig feed.

11. The method of claim 3 wherein the foodstuff is poultry feed.

12. The method of claim 1 wherein the yeast is selected from the group consisting of Saccharomyces species, Pichia species, Kluyveromyces species, Hansenula species, and Candida species.

13. The method of claim 12 wherein the yeast is Saccharomyces cerevisiae.

14. The method of claim 12 wherein the yeast is Pichia pastoris.

15. The method of claim 1 wherein the animal is fed the foodstuff in combination with from about 50 to about 1000 units of the 6-phytase expressed in yeast per kilogram of the foodstuff.

16. The method of claim 1 wherein the animal is fed the foodstuff in combination with from about 50 to about 700 units of the 6-phytase expressed in yeast per kilogram of the foodstuff.

17. The method of claim 1 wherein the animal is fed the foodstuff in combination with from about 50 to about 500 units of the 6-phytase expressed in yeast per kilogram of the foodstuff.

18. The method of claim 1 wherein the animal is fed the foodstuff in combination with from about 50 to about 200 units of the 6-phytase expressed in yeast per kilogram of the foodstuff.

19. The method of claim 1 wherein the 6-phytase has an optimal activity at a pH of less than about 4.

20. The method of claim 1 wherein the 6-phytase expressed in yeast is cleaved with a protease to enhance the capacity of the 6-phytase to increase the bioavailability of phosphate from phytate compared to intact yeast-expressed 6-phytase.

21. A method of improving the nutritional value of a foodstuff consumed by a monogastric animal wherein the foodstuff comprises myo-inositol hexakisphosphate, the method comprising the steps of:
spray drying an E. coli 6-phytase;
mixing the phytase with a carrier for the phytase and, optionally, other ingredients to produce a feed additive composition for supplementing the foodstuff with the phytase;
mixing the feed additive composition with the foodstuff; and
feeding the animal the foodstuff supplemented with the feed additive composition.

22. A method of improving the nutritional value of a foodstuff consumed by an avian species by increasing the bioavailability of phosphate from phytate wherein the foodstuff comprises myo-inositol hexaikisphosphate, the method comprising the step of feeding to the avian species the foodstuff in combination with less than 1200 units of an E. coli 6-phvtase expressed in yeast per kilogram of the foodstuff, wherein the bioavailability of phosphate from phytate is increased by at least 1.5-fold compared to the bioavailability of phosphate from phytate obtained by feeding to a non-avian species the foodstuff in combination with the phytase expressed in yeast.

23. The method of claim 22 wherein the avian species is selected from the group consisting of a chicken, a turkey, a duck, and a pheasant.

24. The method of claim 22 wherein the foodstuff is poultry feed.

25. The method of claim 22 wherein the yeast is selected from the group consisting of Saccharomyces species, Pichia species, Kluyveromyces species, Hansenula species, and Candida species.

26. The method of claim 22 wherein the animal is fed the foodstuff in combination with 2000 units or less of the phytase expressed in yeast per kilogram of the foodstuff.

27. The method of claim 22 wherein the animal is fed the foodstuff in combination with 1500 units or less of the phytase expressed in yeast per kilogram of the foodstuff.

28. The method of claim 22 wherein the phytase has an optimal activity at a pH of less than about 4.

29. The method of claim 22 wherein the animal is fed the foodstuff in combination with from about 50 to about 1000 units of the phytase expressed in yeast per kilogram of the foodstuff.

30. The method of claim 22 wherein the animal is fed the foodstuff in combination with from about 50 to about 700 units of the phytase expressed in yeast per kilogram of the foodstuff.

31. The method of claim 22 wherein the animal is fed the foodstuff in combination with from about 50 to about 500 units of the phytase expressed in yeast per kilogram of the foodstuff.

32. The method of claim 22 wherein the animal is fed the foodstuff in combination with from about 50 to about 200 units of the phytase expressed in yeast per kilogram of the foodstuff.

33. The method of claim 22 wherein the phytase expressed in yeast is cleaved with a protease to enhance the capacity of the phytase to increase the bioavailability of phosphate from phytate compared to intact yeast-expressed phytase.

34. The method of claim 22 wherein the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the phytase expressed in yeast is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell.

35. The method of claim 21 wherein the foodstuff is supplemented with less than 1200 units of the phytase per kilogram of the foodstuff and wherein the bioavailability to the animal of phosphate from phytate is increased by at least 2-fold compared to the bioavailability of phosphate from phytate obtained by feeding the foodstuff in combination with the same units of a phytase expressed in a non-yeast host cell.