WO2001059141A2 - Methods and compositions that utilize barley as a foodstuff for animals - Google Patents

Abstract

In one aspect, the present invention provides foodstuffs comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (1,3-1,4)-β-glucanase portion. In another aspect, the present invention provides methods of utilizing barley grains as a foodstuff for an animal (such as chickens), the methods comprising the step of feeding to an animal a foodstuff comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (1,3-1,4)-β-glucanase portion.

Description

METHODS AND COMPOSITIONS THAT UTILIZE BARLEY AS A FOODSTUFF FOR ANIMALS

Field of the Invention This invention relates to methods and compositions that utilize barley, and barley malt, as a foodstuff for animals such as chickens.

Background of the Invention

Corn is the principal cereal used as feed for raising broiler chickens. Barley is cheaper than corn but is not acceptable as chicken feed because of its low nutritional value for poultry. The main reason why chickens are unable to efficiently utilize barley as an energy source is because chickens do not possess an enzyme in their gut that depolymerizes β-D-glucan which is one of the major carbohydrates present in the barley endosperm. The undigested β-D-glucan results in high viscosity of the barley feed in the intestine, a limited uptake of nutrients, a reduced rate of growth of the chicken, and the production of unhygienic, sticky, droppings which adhere to the chicken and to the floor of the production cages.

Consequently, there is a need for methods and compositions that improve the nutritional value of barley as a foodstuff for chickens and other animals.

Summary of the Invention In one aspect, the present invention provides methods of utilizing barley grains as a foodstuff for an animal (such as chickens), the methods comprising the step of feeding to an animal a foodstuff comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate- degrading enzyme comprising a (l,3-l,4)-β-glucanase portion. In another aspect, the present invention provides foodstuffs comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β-glucanase portion. In one embodiment, the recombinant carbohydrate-degrading enzyme consists of a (l,3-l,4)-β-glucanase enzyme that is at least 95% identical to a (1,3- 1 ,4)-β-glucanase enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1. Representative values for the ratio by weight of barley feed to barley malt are less than or equal to 9:1, or less than or equal to 5:1. Representative values for the concentration of the recombinant carbohydrate-degrading enzyme in the foodstuff are from 0.5 μg/g to 2.0 μg/g, or from 0.75 μg/g to 1.0 μg/g. The foodstuffs of the invention are useful, for example, in the practice of the methods of the invention, and in any situation where it is desired to utilize barley as a component of a foodstuff for animals.

In yet another aspect, the present invention provides methods of making a foodstuff, the methods comprising the step of mixing barley feed with transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β-glucanase portion.

In a further aspect, the present invention provides barley cells and barley plants comprising a vector comprising a nucleic acid molecule that encodes a (l,3-l,4)-β-glucanase comprising the amino acid sequence set forth in SEQ ID NO:l, wherein the nucleic acid molecule is operably linked to a promoter comprising the nucleic acid sequence set forth in SEQ ID NO:2.

Brief Description of the Drawings The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 shows a map of plasmid pJH271 described in Example 1. FIGURE 2 shows the weight gain over a 21 day period of chickens fed a diet of: corn; barley plus 6.2% transgenic (TL) malt; barley plus 6.2% non-transgenic, Golden Promise (GP) malt; and barley alone.

FIGURE 3 shows a bar graph that shows the number of chickens, on a given diet, with sticky droppings adhering to their down over the trial period described in Example 3 herein. The chicken diets were: corn; barley plus 6.2% transgenic (TL) malt; barley plus 6.2% non-transgenic, Golden Promise (GP) malt; and barley alone. FIGURE 4 shows the amounts of soluble and insoluble (l,3-l,4)-β-glucans in different parts of the gastrointestinal tract and in the excrements of chicks raised on the following diets: barley diet with added transgenic malt including recombinant

(l,3-l,4)-β-glucanase; and barley diet without added transgenic malt including recombinant (1,3-1 ,4)-β-glucanase.

FIGURE 5 shows the presence and amount of recombinant thermotolerant (l,3-l,4)-β-glucanase activities in different parts of the gastrointestinal tract and in the excrement of chicks fed barley plus barley malt including the recombinant thermotolerant (l,3-l,4)-β-glucanase. Detailed Description of the Preferred Embodiment

Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Press, Plainsview, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art.

As used herein, the term "barley feed" refers to any form of barley grains suitable for incorporation into a foodstuff. For example, the term "barley feed" includes barley meal made by physically grinding barley grains. The term "barley feed" also includes whole barley grains and barley that has been ground into pellets.

As used herein, the term "barley malt" refers to a material made from barley grains by soaking the grains intermittently in water, or an aqueous solution, allowing the grains to germinate in humid air, then drying the germinated grains in a kiln.

Typically, the dried grains are ground to form a powder which may be pressed with other components to form pellets.

As used herein, the term "transgenic barley malt" has the same definition as "barley malt" except that transgenic barley malt includes a nucleic acid molecule that was introduced (such as by genetic transformation) into the barley, from which the malt was produced, and that expresses a recombinant carbohydrate-degrading enzyme. Transgenic barley malt therefore contains one or more recombinant carbohydrate-degrading enzymes.

The term "recombinant carbohydrate-degrading enzyme" refers to an enzyme that is (a) capable of degrading one or more types of carbohydrate molecules, (b) that is expressed in barley grains, and (c) is encoded by, and expressed from, a nucleic acid molecule that was introduced (such as by genetic transformation) into the barley grains. The recombinant carbohydrate-degrading enzyme can be an enzyme that is normally found in barley, or can be an enzyme that is not normally found in barley.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).

The term "vector" refers to a nucleic acid molecule, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a cDNA molecule. The term "vector" includes the T-DNA of the Ti vector.

In one aspect, the present invention provides a foodstuff comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β-glucanase portion. The foodstuffs of the invention are useful in any situation where it is desirable to degrade one or more types of carbohydrate molecules present in the barley feed. For example, the presence of a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β-glucanase portion in the barley malt facilitates digestion of the β-glucans present in the barley feed thereby enhancing the nutritional quality of the foodstuff for animals (such as broiler chickens) that are unable to digest the β-glucans.

The foodstuffs are made by any art-recognized means for combining barley feed and transgenic barley malt in a form suitable for consumption by an animal. For example, barley meal and powdered, transgenic, barley malt can be compressed (with or without heating) to form pellets, blocks or other shaped articles. The foodstuffs can optionally include any ingredient that provides a nutritional, dietary, physiological, or other benefit to an animal, such as vitamins, minerals, fats, proteins, carbohydrates and fiber.

The manufacture of barley malt is well known to those of ordinary skill in the art, and is extensively discussed, for example, in Briggs, D.E., et al., Malting and Brewing Science, Vol. 1 Malt and Sweet Wort, 2^nd edition (1981), Chapman and Hall. In one representative example, barley malt is prepared by steeping barley grains for 48 hours at 13°C/14°C (the grains are steeped for 8 hours in water, followed by 16 hours in humid air, followed by 24 hours in water) until the grain reaches a moisture content of 43 per cent. The grain is germinated at a temperature in the range of from 11°C to 13°C for 96 hours in humid air. The grain is then dried in a kiln for 12 hours (six hours at a temperature of 50°C to 55°C, rising thereafter to 80°C over a 2 hour period, and remaining at 80°C for 4 hours).

Typically, barley feed and barley malt are present in the foodstuffs of the invention in a ratio (by weight) of from 9: 1 to 5: 1.

Recombinant carbohydrate-degrading enzymes useful in the foodstuffs of the invention include (l,3-l,4)-β-glucanase. An exemplary (l,3-l,4)-β-glucanase enzyme useful in the practice of the present invention is disclosed in U.S. Patent Serial Number 5,470,725 to Borriss et al., which patent is incorporated herein by reference. SEQ ID NO:l herein discloses the amino acid sequence of the (l,3-l,4)-β- glucanase enzyme (SEQ ID NO:l) disclosed in U.S. Patent Serial Number 5,470,725 to Borriss et al. The (l,3-l,4)-β-glucanase enzyme having the amino acid sequence set forth in SEQ ID NO:l retains at least 50% of its activity after 10 minutes, preferably 15 minutes, more preferably 18 minutes, of incubation in 10 mM CaCl₂, 40 mM Na-acetate at pH 6.0 and 70°C, the incubated solution having a concentration range from 0.3 to 1 mg (l,3-l,4)-β-glucanase (SEQ ID NO:l) per ml, the activity of the (l,3-l,4)-β-glucanase (SEQ ID NO:l) being understood as the ability of the enzyme to hydrolyze-β-glycosidic linkages in (l,3-l,4)-β-glucans. The thermostability of the (l,3-l,4)-β-glucanase (SEQ ID NO:l) permits it to undergo the malting process without losing its glucanase activity.

Thus, some representative (l,3-l,4)-β-glucanase enzymes useful in the practice of the present invention are at least 95% (such as at least 99%) identical to the (l,3-l,4)-β-glucanase enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1. In this context, the term "percent identity" or "percent identical", when used in connection with (l,3-l,4)-β-glucanase enzymes useful in the practice of the present invention, is defined as the percentage of amino acid residues in a candidate protein sequence, that are identical with a subject protein sequence (such as the sequence of SEQ ID NO:l), after aligning the candidate and subject sequences to achieve the maximum percent identity. When making the comparison, the candidate protein sequence (which may be a portion of a larger protein sequence) is the same length as the subject protein sequence, and no gaps are introduced into the candidate protein sequence in order to achieve the best alignment.

Amino acid sequence identity can be determined in the following manner. The subject protein sequence is used to search a protein sequence database, such as the GenBank database (accessible at web site http://www.ncbi.nln.nih.gov/blast/), using the BLASTP program. The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity. The default parameters of BLASTP are utilized. Recombinant carbohydrate-degrading enzymes useful in the foodstuffs of the invention can include one or more carbohydrate-degrading activities in addition to (l,3-l,4)-β-glucanase activity. Such multifunctional enzymes can be constructed by fusing functional portions of different carbohydrate-degrading enzymes. For example, a nucleic acid molecule encoding a (l,3-l,4)-β-glucanase can be ligated to a nucleic acid molecule encoding a portion of a cellulase using standard DNA manipulation techniques, such as are disclosed in Sambrook et al. supra. Expression of the hybrid nucleic acid molecule yields a carbohydrate-degrading enzyme that possesses both (l,3-l,4)-β-glucanase and cellulase activities.

Thus, for example, recombinant carbohydrate-degrading enzymes useful for inclusion in the foodstuffs of the invention can include cellulase activity, such as cellulase activity provided by the cellulase enzyme from Erwinia carotovora. This multi enzyme has been shown to depolymerize the consecutive (l,4)-β-linked glucose units that result from the action of the (l,3-l,4)-β-glucanase (SEQ ID NO:l) on the mixed linked barley β-glucan (Olsen, O., et al. (1996) Biotechnology 14:71-76). Again by way of example, recombinant carbohydrate-degrading enzymes useful for inclusion in the foodstuffs of the invention can include a (l,4)-β-xylanase activity, such as the (l,4)-β-xylanase activity provided by the (l,4)-β-xylanase enzyme disclosed in Ay, J., et al., Proc. Natl. Acad. Sci. USA 95:6613-6618 (1998); α- amylase; β-amylase and β-glucosidase. Preferred recombinant carbohydrate-degrading enzymes useful in the practice of the present invention do not lose their carbohydrate-degrading enzymatic activity during the malting process. Thus, preferred recombinant carbohydrate-degrading enzymes are not inactivated by exposure to temperatures of from 55°C to 80°C for a period of from four hours to six hours. Recombinant carbohydrate-degrading enzymes that possess the foregoing thermostability properties can be readily produced, for example by the technique of generating polynucleotides having desired characteristics by iterative selection and recombination, as disclosed in U.S. Patent Serial No. 6,180,406 to Stemmer, which patent is incorporated by reference herein. The recombinant carbohydrate-degrading enzymes possessing the desired thermostability properties can be identified, for example, by expressing the mutated nucleic acid molecules (produced, for example, by the foregoing iterative selection and recombination) in a population of host cells, such as E. coli cells, or a yeast cells, lysing the cells, and assaying the cell lysate for the presence of a carbohydrate-degrading enzyme that retains its enzymatic activity when incubated at a desired temperature for a specified time period. For example, the β-glucanase assay set forth in Example 2 herein can be used to identify the presence, in a cell extract, of a l,3-l,4)-β-glucanase having desired thermostability properties.

The foodstuffs of the invention include barley malt that is produced from barley plants that are genetically modified to include one or more nucleic acid molecules encoding one or more recombinant carbohydrate-degrading enzymes. Examples 1 and 4 herein describe transgenic barley lines that include a transgene that encodes a thermostable (l,3-l,4)-β-glucanase (SEQ ID NO:l) under the control of either a D hordein gene promoter, or an α-amylase gene promoter. Barley plants can be genetically modified to include one or more nucleic acid sequences encoding a carbohydrate-degrading enzyme by any art-recognized technique. These methods include, but are not limited to, (1) direct DNA uptake, such as particle bombardment or electroporation (see, Klein et al., Nature 327:70-73 [1987]; U.S. Pat. No. 4,945,050), and (2) Agrσbαcteπ^'wm-mediated transformation (see, e.g., U.S. Patent Serial Numbers: 6,051,757; 5,731,179; 4,693,976; 4,940,838; 5,464,763; and 5,149,645). Within the cell, the transgenic sequences may be incorporated within the chromosome. The skilled artisan will recognize that different independent insertion events may result in different levels and patterns of gene expression (Jones et al., EMBO J. 4:2411-2418 [1985]; De Almeida et al., MGG 218:78-86 [1989]), and thus that multiple events may have to be screened in order to obtain lines displaying the desired expression level and pattern.

Transgenic plants can be obtained, for example, by transferring vectors that include a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into Agrobacterium tumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature, 303:179-181 (1983) and culturing the Agrobacterium cells with leaf slices, or other tissues or cells, of the plant to be transformed as described by An et al., Plant Physiology, 81:301-305 (1986).

Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, for example, kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.

In addition to the methods described above, several methods are known in the art for transferring cloned DNA into a wide variety of plant species, including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick and Thompson, eds., Methods in Plant Molecular Biology, CRC Press, Boca Raton, Florida [1993], incorporated by reference herein). Representative examples include electroporation-facilitated DNA uptake by protoplasts in which an electrical pulse transiently permeabilizes cell membranes, permitting the uptake of a variety of biological molecules, including recombinant DNA (Rhodes et al., Science, 240:204-207 [1988]); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular Biology, 13:151-161 [1989]); and bombardment of cells with DNA-laden microprojectiles which are propelled by explosive force or compressed gas to penetrate the cell wall (Klein et al., Plant Physiol. 91:440-444 [1989] and Boynton et al., Science, 240(4858): 1534-1538 [1988]). Further, plant viruses can be used as vectors to transfer genes to plant cells. Examples of plant viruses that can be used as vectors to transform plants include the Cauliflower Mosaic Virus (Brisson et al., Nature 310:511-514 (1984); Other useful techniques include: site-specific recombination using the Crel-lox system (see, U.S. Patent Serial No. 5,635,381); and insertion into a target sequence by homologous recombination (see, U.S. Patent Serial No. 5,501,967). Additionally, plant transformation strategies and techniques are reviewed in Birch, R.G., Ann Rev Plant Phys Plant Mol Biol, 48:297 (1997); Forester et al., Exp. Agric, 33:15-33 (1997).

Example 4 herein sets forth two representative protocols for stably introducing a nucleic acid molecule into the genome of a barley plant.

Nucleic acid molecules encoding one or more carbohydrate-degrading enzymes are typically introduced into barley cells as part of a vector. Vectors useful in this aspect of the invention typically include regulatory sequences, such as promoters, translation leader sequences, introns, and polyadenylation signal sequences. "Promoter" refers to a DNA sequence involved in controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The term "promoter" includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Typically, promoters useful in this aspect of the invention direct gene expression in barley endosperm cells. Representative examples of promoters that are useful in this aspect of the invention include: Bl hordein gene promoter (Brandt, A.A., et al., Carlsberg Res. Comm. 50: 335-345 (1985); C hordein gene promoter (Entwhistle, J., Carlsberg Res. Comm. 53: 247-258 (1988); γ hordein gene promoter (Cameron-Mills, V. and Brandt A., Plant Mol Biol. 11: 449-461 (1988).

The plant vectors can be constructed using conventional techniques well known to those skilled in the art. The choice of vector is dependent upon the method that will be used to transform host plants and the desired selection markers. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the vector (for details of an exemplary expression vector for transformation of barley, see Example 1 herein).

The construction of plant vectors is facilitated by the use of a shuttle vector which can be manipulated and selected in both plant and a convenient cloning host such as a prokaryote. Such shuttle vectors thus can include a gene for selection in plant cells (e.g., kanamycin resistance) and a gene for selection in a bacterial host (e.g., actinomycin resistance). Such shuttle vectors also contain an origin of replication appropriate for the prokaryotic host used and preferably at least one unique restriction site or polylinker containing unique restriction sites to facilitate vector construction. Examples of such shuttle vectors include pMON530 (Rogers et al., Methods in Enzymology 153:253-277 [1988]) and pCGN1547 (McBride et al., Plant Molecular Biol. 14:269-276 [1990]).

The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the DNA of interest utilize standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press [1989]). The vectors may be prepared by manipulating the various elements to place them in proper orientation. Thus, adapters or linkers may be employed to join the DNA fragments. Other manipulations may be performed to provide for convenient restriction sites, removal of restriction sites or superfluous DNA. These manipulations can be performed by art-recognized methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press [1989]).

In another aspect, the present invention provides methods of utilizing barley grains as a foodstuff for animals (such as chickens), the methods comprising feeding to an animal a foodstuff comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β-glucanase portion. The foodstuffs of the invention are useful in the practice of the methods of the invention. The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.

EXAMPLE 1 This example describes the construction of transgenic barley plants that express a recombinant, thermostable, (l,3-l,4)-β-glucanase (SEQ ID NO:l) under the control of either a D hordein gene promoter (SEQ ID NO:2) or an α-amylase promoter. Malt from the transgenic barley plants that expressed a recombinant, thermostable, (l,3-l,4)-β-glucanase (SEQ ID NO:l) under the control of an α- amylase promoter were used in the experiments reported in Examples 2 and 3.

(a) Construction of transgenic barley plants that express a recombinant, thermostable, (l,3-1.4>β-glucanase (SEO ID NO:l under the control of a D hordein gene promoter.

Plasmid Constructions: Methods used for PCR and DNA manipulations were as described (Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY)). Plasmid pJH 271 (15.003 kb) was constructed in the binary cloning vector pJH2600, which is derived from pBIN 19 (Bevan, M. (1984) Nucleic Acids Res. 12:8711-8721). Plasmid pJH2600 was assembled by cloning a H dlll-Smαl fragment containing the bar gene 3' to the ubiquitin promoter and a Smal-EcoRl fragment with the nos terminator from pUBARN (Jensen, L.G., et al. (1998) Hereditas 129:215-225) into H ndlll-EcoRI-digested derivative of the pBIN 19 plasmid by a three-way ligation.

Plasmid pJΗ270: Plasmid pHor-H(A12-M)Y13-GC-NOS contained a translational fusion between the 434-bp D hordein gene promoter (SEQ ID NO:2) the ATG initiation codon (Sørensen, M.B., Mϋller, M., Skerritt, J. & Simpson, D. (1996) Mol. Gen. Genet. 250:750-760), and the heat-stable codon-optimized (l,3-l,4)-β- glucanase gene encoding the (l,3-l,4)-β-glucanase set forth in SEQ ID NO:l (Jensen, L.G., et al., (1996) Proc. Natl. Acad. Sci. USA 93:3487-3491). The D hordein gene promoter fragment (SEQ ID NO:2) was PCR amplified from a HorS-1 genomic clone (Sørensen, M.B., Mϋller, M., Skerritt, J. & Simpson, D. (1996) Mol. Gen. Genet. 250:750-760) by using the universal forward primer (5'- GTAAAACGACGGCCAGTG-3') (SEQ ID NO:3) and primer dhor3 (5'CGGTCTGCATCTCGGTGGACTGTC-3^*) (SEQ ID NO:4). The heat-stable β- glucanase gene was PCR amplified from plasmid pEII-αH (A12-M)ΔY13-GC-NOS (Jensen, L.G., et al., (1998) Hereditas 129:215-225) by using primer hor/glu (5'- CGAGATGCAGACCGGCGGCAGCTTC-3') (SEQ ID NO:5) and the M13 reverse primer (S'-GGTTTTCCCAGTCACGAC-S') (SEQ ID NO:6). These two PCR fragments were spliced together by overlap extension (SOE) (Jensen, L.G., Olsen, O., Kops, O., Wolf, N., Thomsen, K.K. & von Wettstein, D. (1996) Proc. Natl. Acad. Sci. USA 93:3487-3491). The resulting fragment was digested with BamHL and EcoRl and cloned into pUC18 digested with the same two enzymes. Plasmid pJH271: Plasmid pHsig-H(A12-M)ΔY13-GC-NOS is similar to plasmid pJH270 but also contained the coding sequence for the D hordein signal peptide (NH-MAKRLVLFVAVTVALVALTTA-COO)(SEQ ID NO:7). The D hordein gene fragment including the promoter, 5' untranslated leader region, and the signal peptide-coding region was amplified from the Hor3-l genomic clone (Sørensen, M.B., Mϋller, M., Skerritt, J. & Simpson, D. (1996) Mol. Gen. Genet. 250:750-760) by using the universal forward primer (SEQ ID NO:3) and primer B (5'-CGGTCTGAGCGGTGGTGAGAGCCAC-3') (SEQ ID NO: 8). The heat-stable β-glucanase gene was PCR amplified from plasmid pEII-αH(A12-M)Δ13-GC-NOS by using primer C (5'-CACCGCTCAGACCGGCGGGCAGCTTC-3') (SEQ ID NO:9) and the M13 reverse primer (SEQ ID NO:6). The two PCR fragments were spliced together by SOE and cloned into pUC18. FIGURE 1 shows a map of pJH271.

Plasmid pJH271 was introduced into barley variety Golden Promise by Agrobacterium-mediated transformation as described in Example 4. The amount of β-glucanase produced, on average in the maturing grain of 10 transgenic barley lines, was 54 μg/mg^"1 soluble protein. This high level of expression required that the β-glucanase gene was optimized to a C + G content of 63%, and that the β-glucanase protein had to be synthesized as a precursor with a signal peptide that delivers the protein to storage vacuoles. (b) Construction of transgenic barley plants that express a recombinant, thermostable, (l,3-l,4)-β-glucanase (SEQ ID NO:l) under the control of an - amylase gene promoter.

Plasmid constructions: Standard methods for PCR and DNA manipulations were as detailed by Sambrook et al. (supra). Plasmid pEmuGN, previously described by LAST et al. (Last DI, et al., Theor. Appl. Genet. 81: 581-588 (1991)), contains the reporter gene encoding β-glucuronidase (GUS) under control of the constitutive pEmu promoter with transcription termination directed by a 277-bp SαcI-EcoRI fragment of the 3' untranslated region and polyadenylation signal of the nopaline synthase gene (nos) from Agrobacterium tumefaciens (Bevan M, et al., Nucleic Acids Res. 11: 369-385 (1983)).

Plasmid pUBARN, used for selection of transformed barley tissue on medium containing bialaphos (Meiji Seika Kaisha, Tokyo, Japan), contains the open reading frame of the bar gene specifying phosphinothricin acetyltransferase (PAT) inserted between the maize ubiquitin Ubi-1 promoter/intron 1 and the nos terminator. The plasmid was constructed by first amplifying by PCR the sequence for PAT of plasmid pIJ4104, using primers

5'-TACTTCTGCAGATGAGCCCAGAACGACGCCCGGCCGAC-3' (SEQ ID NO: 10) (translational start codon underlined) and 5'-GGGGAGCTCTCAAATCTCGGTGACGGGCAGGAC-3' (SEQ ID NO: 11) (bases complementary to the TGA stop codon underlined). The product which contains a Pstl and a Sαcl site flanking the start and stop codon, respectively, was digested with Estl-Sαcl and inserted into pUC18 to give pBAR where an EcoRI site is located immediately downstream of the Sαcl site. pB AR was linearized with Sαcl- Ec RI and ligated with the nos terminator sequence of pΕmuGN, yielding pBARN. Fragments spanning bases -859 to +339 and +314 to +1147 of the Ubi-1 sequence (Christensen AH, et al., Plant Mol. Biol. 18: 675-689 (1992)) were amplified from maize genomic DNA (kindly provided by Michael Sørensen, Carlsberg Laboratory) using two sets of 26-base primers specifying the fragment ends. The PCR fragments were purified, combined, and fused in a second PCR using the outermost primers (Horton RM, et al., Gene 77: 61-68 (1989)). Insertion into Smαl-linearized pUC18 yielded plasmid pUBIpr, which subsequently was digested with Pstl to liberate a fragment spanning the vector polylinker sequence and promoter/intron 1 of Ubi-1. Finally, this fragment was inserted into Estl-linearized pBARN to give pUBARN. To direct tissue-specific expression of a secreted form of H(A12-M)ΔY13, an 860-bp fragment containing promoter and signal peptide coding region for a barley α-amylase high-pi isoform was amplified by PCR using genomic DNA of barley, cv Carlsberg II, with primers 5'-TAGAAACTTTCTGAATCTGCTGTGTCCAGT-3' (SEQ ID NO: 12) and 5'-GGTACATACAGAATCTGAAGATAGGACAAG-3' (SEQ ID NO:13) specifying bases -679 to -650 and +151 to +180 of the gene sequence (Khursheed B and Rogers JC, J. Biol. Chem. 263: 18953-18960 (1988)). The fragment which contains a unique Sfil restriction site close to the 3' end of the signal peptide coding region was inserted into Sraαl-linearized pUC18, giving pAMYss. Fusion of synthetic DNA fragments of -100 bp, each produced in amplification reactions with pair-wise combinations of overlapping oligonucleotides, as described in Rouwendal GJA, et al., Plant Mol. Biol. 33: 989-999 (1997), was used to produce a sequence encoding H(A12-M)ΔY13 where the codon usage matched that of the open reading frame for barley (l,3-l,4)-β-glucanase isoenzyme EII (Fincher GB, et al., Proc. Natl. Acad. Sci. USA. 83: 2081-2085 (1986), Jensen LG, et al., Proc. Natl. Acad. Sci. USA. 93: 3487-3491 (1996)). A final PCR introduced the sequence for barley high-pi α-amylase signal sequence, including the site for Sfϋ, in frame with that for H(A12-M)ΔY13, while a site for Sαcl was introduced immediately 3' to the stop codon. The amplified product was digested with Sαcl, and ligated with Smal- Sαcl linearized plasmid pUC18-wo_s, harboring the nos terminator Sacl-EcoRl fragment of pEmuGN, giving plasmid pUC18-αH(A12-M)ΔY13-GC-N. This was digested with BamΗl-Sjτl, and the large fragment ligated with the corresponding small Bamffl-Sfil fragment of pAMYss to produce the barley expression plasmid pAMY-αH(A12-M)ΔY13-GC-N. Transformation and identification of transgenic barley plants: Plasmid

DNA for plant transformation was prepared by digesting pAMY-αH(A12-M)ΔY13- GC-N and pUBARN with BamHL which linearizes either plasmid in the polylinker immediately upstream of the plant promoter sequence, while EcøRI was used to linearize pEmuGN downstream of the nos terminator. Gold particles (1 μm diameter) were coated with linearized plasmid DNA and accelerated into immature embryos of barley, cv Golden Promise, using a particle gun (BioRad), as described in Example 4 herein. Thereafter, transgenic tissue was selected as described (Jensen LG, et al., Proc. Natl. Acad. Sci. USA. 93: 3487-3491 (1996); Wan Y and Lemaux PB, Plant Physiol. 104: 37-48 (1994)). Putative transgenic barley plants were screened by PCR for the presence or absence of transgene sequences. Genomic DNA for PCR analysis was purified from leaves of primary regenerated plants (defined as generation To) and offspring plants (generations Ti, T₂, T₃) using the method detailed by Edwards K, et al., Nucleic Acids Res. 19: 1349 (1991). Primer sets 5'-GCAGGAACCGCAGGAGTGGA-3' (SEQ ID NO: 14) and 5'-ATCTCGGTGACGGGCAGGAC-3' (SEQ ID NO: 15), 5'- ATGTTACGTCCTGTAGAAACCCCAACCC-3' (SEQ ID NO: 16) and 5'-GGCGTGGTGTAGAGCATTACGGTGCGATGG-3', (SEQ ID NO: 17) and 5'-CCTCTTCCTCGTCCTCCTTGGCC-3' (SEQ ID NO: 18) and 5'-GGGGGAATTCCCGATCTAGTAACATAGATGAC-3' (SEQ ID NO: 19) directed amplification of specific fragments from the transgenes encoding PAT, GUS, H(A12-M)ΔY13, respectively. Control amplifications included the relevant plasmid DNA, and the quality of genomic DNA for PCR was evaluated by amplifying a fragment within the promoter of the barley gene Amy6-4 (Khursheed B and Rogers JC, J. Biol. Chem. 263: 18953-18960 (1988)), specified by primer set 5'-TAGAAACTTTCTGAATCTGCTGTGTCCAGT-3' (SEQ ID NO:20) and 5'-GTACATACAGAATCTGAAGATAGGACAAG-3' (SEQ ID NO:21).

For segregation analyses, transgenic plants were allowed to self-pollinate. The transgene genetics were assessed by scoring the seedlings and grains by PCR analysis and enzyme assays, respectively, as described below.

EXAMPLE 2 This example sets forth the materials and methods used to conduct the experiments and generate the data set forth in Example 3 herein.

Experimental animals and conditions. The chicken trial was performed with 240 Hubbard High Yield broilers (Fors Farms Inc., Puyallup, WA). One day-old chicks were transferred to electrically heated Petersime Brood-units with raised floors (Petersime Incubator Co., OH). Each of the four experimental diets was randomly distributed among 12 pens and 5 birds randomly assigned to a pen. Feed and water were available ad libitum and 16-hour daylight was maintained. Diet composition and preparation. Chickens were fed 4 diets with the composition given in Table 1: com basal, barley basal (cv. Baronesse), barley diet with malt of Golden Promise containing no β-glucanase, and barley diet with malt of transgenic line 5607 containing heat-stable β-glucanase (SEQ ID NO:l). Malt was prepared using micro-scale malting equipment available at the Department of Crop and Soil Sciences, Washington State University, Pullman WA 99164. Kilning was performed at 45°C for 6 hr and 80°C for 4 hr in order to deactivate the endogenous β-glucanase present in germinated barley. Malt was added in amount of 6.2% of the total diet during mixing of feed ingredients. Table 1. Composition of diets in weight per cent

Additional ingredients: fishmeal, 5%; beef tallow, 5%; dicalcium phosphate, 1.60%; limestone, 1.70%; iodized sodium chloride, 0.20; DL- methionine, 0.20%; vitamin premix, 0.25; trace mineral mix, 0.05%, in each diet.

Golden Promise.

Data collection during trial. The 5 birds of each pen were weighed on days 0, 6, 13 and 20. Feed consumption by the individual pen was determined as difference between initial feed weight for each pen and feed weight on the day of determination. Cumulative feed efficiency per chicken was calculated as the ratio of weight gained to feed consumed. Dry matter of excreta was determined on days 5, 8, 11, 14, 17, and 20 by collecting droppings and drying them for 6 hr at 105°C. Samples of dried excreta were collected on days 8, 14, and 20. The number of chicks with sticky droppings adhering to down of the cloaca area was noted on the days for excreta collection. Analyses of experimental diets. The diets were ground in an UDY mill to flour with a 0.5 mm screen. Moisture and ash contents were determined according to AOAC methods 930.15 and 942.05, respectively (Association of Official Analytical Chemists (1990) Official methods of Analysis, 15^th ed. AOAC Inc., VA.). Protein content (N x 5.7) was determined with a Leco FP-428 nitrogen analyzer (Leco Corporation, St. Joseph, MI). Neutral and acid detergent soluble fiber, comprising nonstarch polysaccharides and Klason lignin was determined on an Ankom ²⁰⁰ fiber analyzer (Ankom Technology Corporation, Fairport, NY). Flour (0.5 g) was placed in filter bags and extracted sequentially in the reaction vessel with neutral and acid detergent solution under positive pressure at 99°C. Starch was digested with α-amylase in the rinsing solution after draining the neutral detergent. Fiber content is calculated as the difference between dry-weights before and after extraction. Activity of endogenous and heat-stable β-glucanase was measured with azo-β-glucan substrate (Megazyme, Australia). Soluble protein was determined with the detergent compatible Lowry phosphomolybdic reagent (D_c, Bio-Rad Laboratories, CA) and enzyme activity expressed as μg enzyme g^"1 soluble protein. Average β-glucanase activity for malts of Golden Promise and line 5607 were 0.054 μg g^"1 and 4.647 μg g^" ', respectively. Total β-glucan contents of diets and malts were estimated according to McCleary and Mugford (McCleary, V.B. & Mugford, D.C. (1997) /. AOAC Internat. 80:580-583) using the Megazyme kit. Water-soluble β-glucans were determined according to Jørgensen (Jørgensen, K.G. (1988) Carlsberg. Res. Commun. 53:277-285).

Collection of intestinal contents. On day 21 of the experiment 192 birds were euthanized with CO₂ and revival was prevented by cervical dislocation. After about 2 min. the abdomen was opened and various parts of the chick gastrointestinal tract were excised from 4 chicks per pen. Pooled contents of the glandular stomach, intestine and caeca were flash-frozen in liquid N₂ and stored at -20°C. All experiments with the animals were carried out with approval from the Washington State University Animal Care and Use Committee. The Committee follows the guidelines established by the Canadian Council on Animal Care of 1980 (Canadian Council on Animal Care (1980) Guide to care and use of experimental animals. Vol. 1. CCAC, Ottawa CN).

Viscosity and β-glucanase measurements. One gram of glandular stomach, small intestine, caeca content or excreta was weighed out into a centrifuge tube and one ml water added. The contents was mixed thoroughly and centrifuged at 18,000 rpm for 20 min. The supernatant was collected in 2 ml-Eppendorf tubes and recentrifuged (13,000 rpm, 5 min). Supematants free of particles were collected and used for measurements at 30°C with a Brookfield Viscometer fitted with the CP-40 cone (Brookfield Engineering Laboratories, Inc. Massachusetts). The samples were then analyzed for heat-stable (l,3-l,4)-β-glucanase activity as follows: the sample (50 μl) was mixed with buffer containing 40 mM sodium acetate and 40 mM sodium phosphate, pH 4.6, and incubated for 30 min at 65°C. An aliquot of this was used to monitor the hydrolysis of azo-β-glucan (Megazyme, Australia) at 65°C for 30 min. Soluble protein in the extract was measured with the Bio-Rad Dc method. Activity of recombinant enzyme was expressed in μg g^"1 soluble protein. Recombinant heat- stable β-glucanase activity in excreta was also measured as outlined above but the protein content of the excretal extract was measured with the Commassie Blue dye binding method of Bradford (Bio-Rad). Soluble and insoluble (l,3-l,4Vβ-glucans in the gastrointestinal tract and excreta. Contents (50 mg) from the glandular stomach, the small intestine, the caecum or of excreta were placed in 2 ml Eppendorf tubes and suspended in 1 ml 80% v/v) ethanol. With the lids tightly secured the tubes were incubated for 5 min in a boiling water bath. After cooling to room temperature and vigorous vortexing the insoluble material was collected by centrifugation at 12000 rpm for 10 min.

Soluble β-glucans were isolated by three extractions with water as follows: the pellet was suspended in 1 ml of water, vortexed vigorously and incubated in boiling water for 10 min. Contents were cooled to room temperature, vortexed, and centrifuged (6,000 rpm, 10 min). Supernatant was collected into graduated tube. Pellet was resuspended in another 1 ml water, vortexed, and centrifuged (6,000 rpm, 10 min). Supernatant was collected and added to the graduate tube. The pellet was resuspended in 0.5 ml water, vortexed, centrifuged (6,000 rpm, 10 min), the supernatant pooled to the graduate tube and the pellet saved. The volumes of supematants in the graduate tubes were adjusted to 2.5 ml with water and 20 μl of a 2 M sodium phosphate buffer (pH 6.5) was added. Insoluble β-glucans are retrieved from the pellet remaining after extraction of the soluble β-glucans. The pellet was suspended in 1 ml of 50 mM HC1, the lid secured tubes incubated for 10 min in a boiling water bath. After cooling and vortexing the suspension was centrifuged at 4000 rpm for 10 min and the supernatant collected in a graduated tube. The extraction was repeated and the combined supematants adjusted to 2 ml and 0.5 ml 2 M Na-PO₄-buffer (pH 6.5) added.

Digestion of soluble and insoluble β-glucans with lichenase (Megazyme) was performed by adding 30 ml (~1U) of lichenase to the graduated tubes, mixing contents well, and incubating tubes in a water bath with shaking at 50°C for 1 hr. After lichenase digestion, aliquots (0.1 ml) were accurately dispensed on the bottom of 3 Eppendorf tubes. Fifty μl of β-glucosidase was added to the two of these tubes and to the third, the blank, 50 μl of sodium acetate buffer (50 mM, pH 4.0) was added. Tubes were incubated at 50°C for 30 min, and 1 ml of glucose oxidase/peroxidase reagent (Megazyme) was added to all tubes and incubated for further 30 min. Blank (sodium acetate buffer+water) and glucose standards (15 μl and 30 μl) were included in each set of samples analyzed. Amount of β-glucans was estimated from absorbance of glucose at 510 nm. Calculations of amount of β- glucans were done according to McCleary and Glennie-Holmes (Association of Official Analytical Chemists (1990), Official Methods of Analysis, 15^th Ed., AOAC Inc., VA).

Western blot analyses with heat-stable (l,3-l,4)-β-glucanase specific antibody. Acetone powder was prepared from digesta of the glandular stomach, the intestine, the caeca and the excrements of chickens fed the barley diet with added transgenic malt. As controls were used acetone powders from caecum digesta of chickens fed corn diet, barley diet and barley diet with added normal Golden Promise barley malt. Material (200 mg) was weighed into 2 ml Eppendorf tubes, suspended in 2 ml acetone with a glass rod and centrifuged for 5 min at 13000 rpm. The acetone wash was repeated and the pellet dried in air. Urea was removed from the excrement samples by extraction with 2 ml aliquots of ethanol prior to acetone extraction. For isolation of heat-stable (l,3-l,4)-β-glucanase the air-dried pellets were suspended in 300 μl of 40 mM sodium acetate, 40 mM sodium dihydrogen phosphate (pH 4.6). (For excrements 600 μl were required). After incubation at 65°C for 30 min and centrifugation at 13000 rpm (10 min) protein content and heat- stable β-glucanase activity were determined in the supernatant. For total protein extraction the acetone powders were suspended in 300 μl of a solution containing 0.1 M Tris-HCl pH 8.8, 1% SDS, 0.1% β-mercaptoethanol and the capped tubes incubated for 5 min in a boiling water bath. After cooling and centrifugation at 13000 rpm (10 min) the supematants were collected. The proteins were separated by electrophoresis in a 15% polyacrylamide gel, containing 1% SDS, and transferred on to a nitrocellulose membrane using a BioRad semidry blotter and a solution containing 2.93 g 1^_1 glycine 5.81 g l^-1 Tris and 200 ml methanol. Electroblotting was performed for 45 min at 15V. The membrane was incubated for 1 h in a solution of 20 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.05% Tween-20 and 5% non-fat milk to block reacting groups. It was then incubated in the above solution overnight with an antibody raised against the heat-stable (l,3-l,4)-β-glucanase expressed in E. coli (dilution 1:2000). Excess antibody was removed by three successive washes with 20 mM Tris-HCl pH 7.5 containing 0.5 M NaCl, 0.05% Tween 20 and 5% non-fat milk. The blots were incubated with the secondary antibody (peroxidase-linked goat-antirabbit monoclonal IgG, Sigma Chemical Co., St. Louis, MO) at 1:20000 dilution in the non-fat milk solution, excess antibody removed and stained for peroxidase activity in a solution of 0.01% H₂O₂, 0.5 mg ml^"1 4-chloro-α-naphtol in Tris-HCl pH 7.5.

Statistical Analyses. Analysis of variance was conducted using the General Linear Models procedure of SAS software (SAS Institute Inc. (1986) SAS/STAT: Guide for Personal Computers SAS Institute, Cary, NC) and descriptive statistics package of Microsoft Excel.

EXAMPLE 3 This example shows the effect of malt made from genetically altered barley, that expresses a heat-stable (l,3-l,4)-β-glucanase (SEQ ID NO:l), on the nutritional quality of chicken feed made from barley.

Analyses of Diets. The protein content of the com diet, the diet with Golden Promise malt and that with malt from the transgenic line 5607 containing the heat- stable (l,3-l,4)-β-glucanase was very similar and amounted to about 22% (Table 2). The barley diet had a 3% higher protein content. The two malt additives contained 12 and 10% protein. The fiber fractions consist of the nonstarch polysaccharides and Klason lignin. Nonstarch polysaccharides of barley are (l,3-l,4)-β-D-glucans, arabinoxylans and cellulose. Com contains pentosans instead of (1,3-1, 4)-β-glucans. The barley diet had considerable more fiber extractable with neutral and acid detergent than the corn diet and the difference is accentuated by the addition of malt. The amount of heat-stable (l,3-l,4)-β-glucanase in the malt of the transgenic line was 4.28 μg g^"1 soluble protein, which resulted in a content of 0.47 μg g^"1 soluble protein in the barley diet with the transgenic malt. No β-glucanase was detected in the diet containing Golden Promise malt. Table 2. Analyses of diets and ingredients

GP, Golden Promise; TL, transgenic line 5607. *Neutral Detergent Fiber, acidic Detergent Fiber.

Weight Gain, Feed Consumption, Feed efficiency and Dry Matter of Excreta. The increase in weight of the chicken over the 21 day period is presented in FIGURE 2. Each point represents the average weight of 60 birds. An analysis of variances revealed that at day 20 the weight of broiler chicks fed barley diet with addition of malt containing heat-stable (l,3-l,4)-β-glucanase was not significantly different from the weight of broilers fed co diet. The weight gain of the birds fed the barley diet was significantly slower and that of the barley diet with the addition of malt lacking (l,3-l,4)-β-glucanase resulted in a growth curve intermediate between that of the birds fed co diet and that for the barley diet. The values are provided in Table 3.

Table 3. Weight gain, feed consumption, feed efficiency, and excreta dry matter

*Means with different letter within the day of trial are significantly different at 5% level.

Broiler chicks fed com or barley with transgenic malt consumed up to day 13 an equal amount of feed, whereas consumption was less by broilers on barley diet (Table 3). Consumption was considerably lower by the chicks on co diet than on the barley diets when measured on day 20. This resulted in a feed efficiency of 0.8 for co . Feed efficiencies of 0.7 were observed for all diets on day 13 and for barley with transgenic malt at day 20. The efficiencies of the other two barley diets were with 0.6 distinctly lower. In general feed efficiencies of 0.8 and 0.7 are considered within the normal range. The dry matter of the excreta (Table 3) increased on all diets as the broiler chicks grew with limited differences at a given day. Number of chickens with adhering sticky droppings. An analysis of the number of chickens with sticky droppings adhering to their down among the 60 on a given diet over the trial period is presented in the bar-graph of FIGURE 3. Only two chickens on com diet had sticky droppings on day 14 and 17 respectively. On the barley diet between 15 and 17 chicks per diet had sticky droppings adhering to their cloaca region throughout the trial. Floor bonitations confirmed the extensive sticky droppings by the pens feeding on barley diet. A significant reduction of the droppings adhering to the down is observed by supplementing the barley diet with barley malt. The transgenic malt addition to normal barley reduced the occurrence of the sticky dropping to a frequency of 2 to 7 among the 60 chicks on this diet at a given day. A further increase of the amount of transgenic barley added, is likely to eliminate the undesirable droppings completely.

Contents of (l,3-l,4)-β-GIucans in Diets, Malts and the Gastrointestinal Tract. The (l,3-l,4)-β-glucan content of the barley cv. Baronesse used in the feed trial amounted to -30 mg g^"1 flour. About 8 mg g^"1 grain were extractable with water at 65°C (Table 4). The diets with Golden Promise and transgenic malt contained a somewhat higher amount of soluble β-glucans. The two malts had a low content of water-extractable β-glucans. The effect of the heat-stable (l,3-l,4)-β-glucanase on the content of (1,3-1, 4)-β-glucans in different parts of the gastrointestinal tract and in the excrements of chicks raised on barley diet with added transgenic malt is compared in FIGURE 4 with the corresponding contents in the birds fed the barley diet without this addition. On barley diet, the intestine and the excrements contained 3.1±0.8 and 4.5±0.7 mg g^"1 wet weight water extractable (l,3-l,4)-β-glucans at day 20. This was reduced to 0.8±0.2 and 2.2±0.4 mg g^"1 in the chicks raised on barley diet with the transgenic malt addition. A reduction of the limited amount of soluble β-glucans by the enzyme is also seen in the glandular stomach and the caecum. The amount of insoluble β-glucans in the digesta from the glandular stomach and intestine and in the excrements of the chicks on barley diet is low (1.2, 0.9, 0.6 mg g^" '). An effect of the enzyme is only evident in the intestine. The amount of β-glucans in the caecum of the broilers on barley diet is below 1 mg g^~\ but the enzyme addition in the malt decreased both the soluble and insoluble β-glucan content.

Analysis of heat-stable (l,3-l,4Vβ-glucanase in the gastrointestinal tract and excreta. As illustrated in FIGURE 5, the amount of enzyme activity in the intestine was 0.40 μg g^"1 soluble protein, corresponding to the amount of enzyme present in the diet with transgenic malt (0.47 μg g^"1 soluble protein). The enzyme activity in the glandular stomach was 0.11 μg g^"1 due to the limited amount of feed that was present in this stomach. The caeca, which are enlarged in broilers on barley diet compared to the size seen in the chicks on com diet, concentrate the enzyme to an activity of 5.2 μg g^"1 and also the excreta accumulate high amounts of active heat-stable (l,3-l,4)-β- glucanase. This matches with a strong reduction of the β-glucans in the caeca and excrements (FIGURE 4). The heat-stable β-glucanase was characterized by SDS- PAGE, followed by Western blotting and decoration with a specific antibody. Purified, unglycosylated enzyme expressed in E. coli, and purified, glycosylated enzyme from transgenic barley were employed as standards. Glycosylated, recombinant (l,3-l,4)-β-glucanase was present in the extracts from the intestine, excreta and caeca, but is absent in the caeca of the birds fed co , barley, or barley with Golden Promise malt. The limited amount of enzyme present in the glandular stomach was not revealed in the Western blot. The presence of the glycosylated enzyme in the caeca testifies to its origin from the transgenic barley, and excludes the possibility that the (l,3-l,4)-β-glucanase is produced by the uric acid decomposing anaerobic bacteria of the caeca.

Viscosity of digesta in the gastrointestinal tract. The measurements confirm that a barley diet leads to a higher viscosity in the glandular stomach and intestine than a com diet. The addition of barley malt or transgenic malt reduces the viscosity in these two parts of the digestive tract. Co diet resulted in a higher viscosity of the caecum contents than the barley diet and the barley diet with an addition of normal malt. Transgenic malt increased the viscosity towards and above that observed for com diet. The high viscosity in the caeca is due to accumulation of volatile fatty acids, an important nutrient for chickens. Conclusions. The results presented in this Example demonstrate that inclusion of 6.2% transgenic malt containing a protein-engineered, thermotolerant (l,3-l,4)-β-glucanase (4.6 μg g^"1 soluble protein) (SEQ ID NO:l) provides a weight gain of Hubbard High Yield Broilers indistinguishable from that of presently employed com diets. The gene encoding the enzyme (SEQ ID NO:l) is expressed with an α-amylase gene promoter in the aleurone and synthesized with a signal peptide for secretion into the endosperm of the germinating grain. The enzyme concentration in the diet was 0.47 μg g^"1 soluble protein. The excellent growth and survival of the 60 broiler chickens receiving this diet shows the transgenic malt not to be toxic. The chicks did not develop the extensive unhygienic sticky droppings characteristic for chickens fed on barley diets. Advantages in using the transgenic malt containing the thermostable (1,3- 1 ,4)-β-glucanase (SEQ ID NO:l) for chicken feed are several. The required malt corresponding in amount to the feed ingredients such as fish meal, beef tallow or dicalcium phosphate can be added to any normal barley grown in a given area and constituting the major basis of the feed. It provides an alternative to the use of grain com, which is more extensively used and needed as food for humans than barley. Co grain is also 30-50% more expensive. Only 10% of the barley harvest in the US is used as malt for beer and less than 1% for production of ingredients in human food (Washington Agricultural Statistics 1997-1998). The State of Washington produces annually 40 million broilers with imported grain com. If barley is to be used for raising this number of broiler chickens it would require 3.400 tons of presently available transgenic malt and 280,000 tons of normal barley i.e., -1/3 of the barley harvest of the State of Washington. Barley is needed in Washington agriculture for crop rotation. When the transgenic barley plants that produce larger amounts of enzyme during grain maturation become available, mature grain can be used as additive and a level corresponding to the present vitamin mix will suffice. The thermostability of the enzyme allows it to survive the heat generated by pressing barley into feed pellets and pasteurization of the feed required to prevent infection of the chicks by Salmonella typhimurium.

The barley feed used in this study contained 8 mg g^"1 water-soluble and 22 mg g^"1 insoluble (l,3-l,4)-β-glucan (Table 4). The barley diet including the transgenic malt had a somewhat higher soluble (12 mg g^"1) and a lower insoluble (14 mg g^"1) (l,3-l,4)-β-glucan content. Table 4. Water-extractable and total β-glucan content in diets and malts

In the glandular stomach, the intestine and the excrements increasing amounts of water-soluble (l,3-l,4)-β-glucans have been determined for the birds fed barley diet without transgenic malt, whereas the insoluble content stayed more or less constant (FIGURE 4). For the interpretation of these values one has to consider the time solid phase food spends in various parts of the digestive tract (mean retention time). For 1800 g broilers on com-canola or corn-soybean diet this is 39 min for the gizzard and glandular stomach, 191 min for the intestine, 119 min for the caeca and 56 min for the rectum (Shires, A., Thompson, J.R., Turner, B.V., Kennedy, P.M. & Goh, Y.K. (1987) Poultry Science 66:289-298). In the gizzard and glandular stomach the feed is retained a relatively short time.

The concentration of the insoluble and soluble (l,3-l,4)-β-glucans in the glandular stomach was reduced to 5 and 25% of that in the diet, respectively. A reduction to 13% was also registered in the chickens fed the diet with transgenic malt. This reduction is possibly effected by the HC1 secreted with 93 mM l^"1 in the stomach together with pepsinogen. (Denbow, M. (2000) in Sturkie 's Avian Physiology, ed. Whittow, G.C. (Acad. Press, New York) 5^th Ed. pp. 299-325; Long, J.F. (1967) Am. J. Physiol. 212:1303-1307.) The pH of the gastric secretions in the gizzard and glandular stomach is 2 to 3, although the contents of the stomach has usually a higher pH due to the presence of ingesta (Denbow, M. (2000) in Sturkie' s Avian Physiology, ed. Whittow, G.C. (Acad.Press, New York) 5^th ed. pp. 299-325). During the approximately 3 h retention of the feed in the intestine the data reported herein indicate a strong increase in the concentration of soluble β-glucans (FIGURE 4). This results from an ongoing conversion of insoluble to soluble (1,3- l,4)-β-glucans and accumulation of the soluble part. The soluble (l,3-l,4)-β-glucan in the intestine of the chicks on barley diet was measured to be 3.1±0.8 mg g^"1 (n=6 pens). This value was reduced to 0.83±0.2 mg g^_1 (n=6 pens) in the chicks on barley diet containing 6.2% transgenic malt (FIGURE 4). This depolymerization of the soluble (l,3-l,4)-β-glucan was carried out by the heat-stable (l,3-l,4)-β-glucanase present in the intestine with an activity corresponding to that in the diet. Collection of excrements over a 48 h period yielded a content of 4.6 mg g^"1 soluble (l,3-l,4)-β- glucans (FIGURE 4). The stickiness of the droppings is due to the accumulation of this large amount of soluble β-glucans. The activity of the heat-stable β-glucanase effects the reduction of the soluble β-glucans in the excreta of chicks on barley diet with the transgenic malt. The reduction was from 4.6 mg g^"1 (n=6 pens) to 2.3 mg g^"1 (n=6 pens). Active heat-stable enzyme had accumulated in the excreta to a 7.5 fold higher concentration than in the feed (FIGURE 5).

Development of longer caeca is observed in birds on high fiber diets (McLelland, J. (1989) J. Exp. Zool. Suppl. 3:2-9). In agreement therewith a larger size of the caeca was observed in the chickens on barley diets with the high fiber content than in the birds on co diet with the lower fiber content. The main function of caeca in birds is nutritional. They take part in the digestion of fine particulate matter, food fiber, and in the production of volatile fatty acids, mainly acetate, propionate and butyrate (Braun, E.J. & Duke (1989) "Function of the Avian Cecum," J. Exp. Zool. Suppl. 3:1-130; Goldstein, D.L. & Skadhauge, E. (2000) in Sturkie' s Avian Physiology, ed. Whittow, G.C. (Acad. Press, New York) 5^th ed. pp. 265-297). Large amounts of uric acid, intestinal and urethral water are moved by peristalsis and antiperistalsis into the caeca (Skadhauge, E. (1981) Osmoregulation in birds (Springer Verlag, New York); Skadhauge, E. (1993) Adv. Comp. Environ. Physiol. 16:67-93; Braun, E. (1993) in New Insights in Vertebrate Kidney Function, eds. Brown, J.A., Balment, R.J., Rankin, J.C. (Cambridge Univ. Press, Cambridge) pp. 167-188). Volatile fatty acids, which can reach a concentration of 125 mM, water and ions are reabsorbed through the caecal wall into the blood stream. A large number of different anaerobic bacteria, including Clostridium species, are present in the caeca at titers of 10⁸-10¹⁰ g^"1 caecal material (Barnes, E.M. & Impey, C.S. (1974) /. Appl .Bact. 37:393-409). They decompose uric acid to ammonium and ferment polysaccharides to acetate, propionate and butyrate. The production of volatile fatty acids in the caeca can meet 11-18 % of the energy needs for the birds' basal metabolism (Annison, E.F., Hill, K.J. & Kenworthy, R. (1968) Br. J. Nutr. 22:207- 216). Caecectomy significantly reduces the metabolization of feed, volatile fatty acid absorption and digestibility of polysaccharides (crude fiber), if the birds are preconditioned to a high fiber diet (Chaplin, S.B. (1989) J. Exp. Zool. Suppl. 3:81- 86). The amount of soluble and insoluble (l,3-l,4)-β-glucan in the caecum of chickens on barley diet is low, but significantly lowered by addition of transgenic malt. The 11-fold concentration of the heat-stable (l,3-l,4)-β-glucanase in the caecum (FIGURE 5) is remarkable. It shows that potentially other recombinant enzymes in chicken feed can be concentrated in the caecum and used to probe enhancement of utilization of other fibers than (l,3-l,4)-β-glucans. EXAMPLE 4 This example sets forth two protocols for genetically transforming barley plants.

A biolistic method for genetically transforming barley plants. CIM medium (pH 5.8) contains Murashige and Skoog medium (Murashige and Skoog, Physiol. Plant 15:473-497 (1962)) supplemented with 30 g/L"¹ maltose, 1.0 mg/L thiamine- HCL, 0.25 g/L myø-inositol, 1.0 g/L casein hydrolysate, 0.69 g/L L-proline, and 2.5 mg/L"¹ dicamba, solidified by 3.5 g/L phytagel. SGM medium (pH 5.6) consists of Murashige and Skoog medium with the ammonium nitrate concentration changed to 165 mg/L supplemented with 62 g/L maltose, 0.4 mg/L thiamine-HCL, 0.1 g/L myø-inositol, 1.0 g/L casein hydrolysate, 0.75 g/L glutamine, and 1 mg/L 6-benzyl- amino purine, solidified with 3.5 g/L phytagel. RGM medium is CIM medium without any dicamba added. Immature zygotic embryos (1.5 - 2.5 mm) are excised from barley, such as barley variety Golden Promise, and bisected longitudinally. The cut embryos are placed, scutellum-side down, onto CIM medium without bialaphos and incubated at 24°C in the dark for 12 to 24 hours. The immature embryos are then transferred to CIM medium without bialaphos, but which includes 0.4 M mannitol for 4 to 6 hours, then bombarded with gold particles bearing linearized plasmid DNA. One day after bombardment, the embryos are transferred to CIM medium containing 5 mg/L bialaphos.

During the first round of selection, calli are kept on CIM medium including bialaphos for two weeks at 24°C in the dark. During the second round of selection, the calli are transferred to fresh CIM medium containing bialaphos and incubated at 24°C in the dark for two weeks. During the third round of selection, calli are transferred to fresh CIM medium containing bialaphos and incubated for two weeks at 24°C in the dark. During the fourth round of selection, calli are transferred to fresh CIM medium containing bialaphos and incubated for two weeks at 24°C in the dark.

After the fourth round of selection, calli are transferred to shoot generation medium (SGM medium) containing 1 mg/L bialaphos, and incubated at 24°C (16 hours light/8 hours dark) for 4 weeks. The resulting plantlets are transferred to root generation medium (RGM medium) containing 1 mg/L bialaphos, and incubated at 24°C (16 hours light/8 hours dark) for 2 weeks. The resulting plants are transferred to soil and grow into maturity under a light regime of 16 hours light (16°C) and 8 hours dark (12°C). Mature seed can be harvested approximately three to four months later.

A representative method for genetically transforming barley plants using Asrobacterium. All media used in this protocol contain 5 μm copper sulfate. Callus induction medium (CIM, pH 5.8, contains Murashige and Skoog medium supplemented with 30 g/L^-1 maltose, 1 mg/L thiamine-HCL, 0.25 g/L myσ-inositol, 1.0 g/L casein hydrolysate, 0.69 g/L L-proline, and 2.5 mg/L dicamba, solidified by 3.5 g/L phytagel. Shoot generation medium (SGM), pH 5.6, consists of Murashige and Skoog medium with the ammonium nitrate concentration changed to 165 mg/L supplemented with 62 g/L maltose, 0.4 mg/L thiamine-HCL, 0.1 g/L myø-inositol, 1 g/L casein hydrolysate, 0.75 g/L glutamine, and 1 mg/L 6-benzyl-amino purine, solidified with 3.5 g/L phytagel. Root generation medium (RGM) is CIM without any dicamba added. Immature zygotic embryos (1.5 - 2.5 mm) from a barley variety, such as Golden Promise, are excised and bisected longitudinally. The cut embryos are placed on CIM medium without bialaphos, and incubated at 24°C for two days in the dark. A culture of Agrobacterium, containing the nucleic acid molecule to be transferred into the barley genome, is added dropwise to the zygotic embryos and cocultivated at 24°C in the dark for 48 hours. The Agrobacterium cells are then washed off with LB medium until no more bacteria are visible, then the embryos are washed once more with LB medium containing 200 mg/L timentin and excess liquid is allowed to drain off onto sterile filter paper. Individual embryos are transferred to CIM medium containing 4 mg/L bialaphos and 200 mg/L timentin.

During the first round of selection, calli are kept on CIM medium containing bialaphos and timentin for two weeks at 24°C in the dark. During the second round of selection, the calli are transferred to fresh CIM medium containing bialaphos and timentin and incubated at 24°C for two weeks in the dark. During the third round of selection, calli are transferred to fresh CIM medium containing bialaphos and timentin and incubated at 24°C for two weeks in the dark.

Calli are then transferred to shoot generation medium (SGM) containing timentin and 3 mg/L bialaphos, and incubated at 24°C (16 hours light/8 hours dark) for four weeks. The resulting plantlets are transferred to RGM medium containing timentin and 3 mg/L bialaphos, and incubated at 24°C (16 hours light/8 hours dark) for four weeks. The resulting plants are transferred to soil and grown to maturity under a light regime of 16 hours light (16°C) and 8 hours dark (12°C). Approximately three to four months later, mature seeds can be harvested. While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of utilizing barley grains as a foodstuff for an animal, the method comprising feeding to an animal a foodstuff comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β-glucanase portion.

2. The method of Claim 1 wherein the animal is a chicken.

3. The method of Claim 1, wherein the barley feed comprises barley grains.

4. The method of Claim 1, wherein the barley feed comprises barley meal.

The method of Claim 1, wherein the barley feed consists of barley meal.

6. The method of Claim 1, wherein the recombinant carbohydrate- degrading enzyme consists of a (l,3-l,4)-β-glucanase enzyme that is at least 95% identical to a (l,3-l,4)-β-glucanase enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1.

7. The method of Claim 1, wherein the recombinant carbohydrate- degrading enzyme consists of a (l,3-l,4)-β-glucanase enzyme that is at least 99% identical to a (l,3-l,4)-β-glucanase enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1.

8. The method of Claim 1, wherein the recombinant carbohydrate- degrading enzyme consists of a (l,3-l,4)-β-glucanase enzyme that consists of the amino acid sequence set forth in SEQ ID NO: 1.

9. The method of Claim 1, wherein the ratio by weight of barley feed to barley malt is less than or equal to 9:1.

10. The method of Claim 1, wherein the ratio by weight of barley feed to barley malt is less than or equal to 5: 1.

11. The method of Claim 1, wherein the concentration of the recombinant carbohydrate-degrading enzyme in the foodstuff is from 0.5 μg/g to 2.0 μg/g.

12. The method of Claim 1, wherein the concentration of the recombinant carbohydrate-degrading enzyme in the foodstuff is from 0.75 μg/g to 1.0 μg/g.

13. The method of Claim 1, wherein the barley feed and barley malt together constitute greater than 65% by weight of the foodstuff.

14. A foodstuff comprising barley feed and transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β-glucanase portion.

15. The foodstuff of Claim 14, wherein the barley feed comprises barley grains.

16. The foodstuff of Claim 14, wherein the barley feed comprises barley meal.

17. The foodstuff of Claim 14, wherein the barley feed consists of barley meal.

18. The foodstuff of Claim 14, wherein the recombinant carbohydrate- degrading enzyme consists of a (l,3-l,4)-β-glucanase enzyme that is at least 95% identical to a (l,3-l,4)-β-glucanase enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1.

19. The foodstuff of Claim 14, wherein the recombinant carbohydrate- degrading enzyme consists of a (l,3-l,4)-β-glucanase enzyme that is at least 99% identical to a (l,3-l,4)-β-glucanase enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 1.

20. The foodstuff of Claim 14, wherein the recombinant carbohydrate- degrading enzyme consists of a (l,3-l,4)-β-glucanase enzyme that consists of the amino acid sequence set forth in SEQ ID NO: 1.

21. The foodstuff of Claim 14, wherein the ratio by weight of barley feed to barley malt is less than or equal to 9:1.

22. The foodstuff of Claim 14, wherein the ratio by weight of barley feed to barley malt is less than or equal to 5:1.

23. The foodstuff of Claim 14, wherein the concentration of the recombinant carbohydrate-degrading enzyme in the foodstuff is from 0.5 μg/g to 2.0 μg/g.

24. The foodstuff of Claim 14, wherein the concentration of the recombinant carbohydrate-degrading enzyme in the foodstuff is from 0.75 μg/g to 1.0 μg/g.

25. The foodstuff of Claim 14, wherein the barley feed and barley malt together constitute greater than 65% by weight of the foodstuff.

26. A method of making a foodstuff, the method comprising the step of mixing barley feed with transgenic barley malt, wherein the transgenic barley malt comprises a recombinant carbohydrate-degrading enzyme comprising a (l,3-l,4)-β- glucanase portion.

27. A barley cell comprising a vector comprising a nucleic acid molecule that encodes a (l,3-l,4)-β-glucanase comprising the amino acid sequence set forth in SEQ ID NO:l, wherein the nucleic acid molecule is operably linked to a promoter comprising the nucleic acid sequence set forth in SEQ ID NO:2.

28. A transgenic barley plant comprising a vector comprising a nucleic acid molecule that encodes a (l,3-l,4)-β-glucanase comprising the amino acid sequence set forth in SEQ ID NO:l, wherein the nucleic acid molecule is operably linked to a promoter comprising the nucleic acid sequence set forth in SEQ ID NO:2.