During lactation in ruminants there is a marked increase in protein requirement.

This need is presently met by supplementing the feed with expensive protein sources.

Since only part of this protein will be used as protein source by the ruminant (a substantial part is subjected to microbial proteolysis in the rumen) milk production is reduced as a consequence.

In addition, nitrogen in manure contributes to environmental pollution. Ammonia from manure contributes to acid rain; microbial oxidation of ammonia to nitrate and nitrite causes problems in drink water supply. The reduction of ammonia in manure is therefore desirable.

Amino acids are absorbed in the small intestine in ruminants. Accordingly efforts have been made to increase the quantity of protein and amino acid delivered to the small intestine, particularly since feed is the most important cost factor in the ruminant industry. Supplementation of protein in the small intestine would bypass the fermentation in the rumen and could result in increased milk production as shown in fistulated animals (Broderick, G. A., Wallace R. J. and Orskov E. R., Control of rate and extent of protein degradation, in: Physiological aspects of Digestion and Metabolism in Ruminants [Tsuda T., Sasaki Y., and Kawashima R., Eds] London Academic Press 1991 (541). This, however, is not practical since although rumen-protected amino acids are commercially available, and are used on a large

scale, the formulation to protect them against rumen proteolysis is an expensive process.

Attempts have been made to improve the efficiency of digestion by ruminants by addition of various compounds including ionophores, oxidizing agents, surfactants, enzymes, rumen protected amino acids, and enzyme enhancers (Shelford, J. A and Kamande, G.; Canadian Patent Application 2,126,895 (1994)). However, the effects of these compounds in improving growth, feed conversion and milk production have been inconsistent. Thus in recent years the feed industry has started to use industrially produced enzymes to complement the enzymes produced in the gastrointestinal tract of the animals. Examples comprise phytases, alpha-amylases, proteases and various plant cell wall degrading enzymes.

Protein cross-linking enzymes are known. Japanese patent application 05/058605 (Ajinomoto) refers to the use of transglutaminase to produce food for domestic pets such as cats and dogs. Transglutaminase is added to the meat of domestic fowl animals and fish and/or by-products thereof and after allowing the enzyme to act the total contents are sterilized by heating. The purpose of this treatment is to improve the mouthfeel, the addition of the transglutaminase resulting in the formation of meat particles giving a better eating sensation ("bite"). The transglutaminase is not used as a feed ingredient; instead its use is only in vitro followed by heat sterilization thereby inactivating all enzyme activity. There is also no reference to a ruminant feed.

Other possible industrial applications of transglutaminase have been reviewed recently in Zhu, Y, Rinzema, A., Tramper, and Bol, J. Applied Microbiology Biotechnology 44: 277-282 (1995) but these do not include use of transglutaminase as a feed ingredient. All reported applications aim at improving taste, texture and shelf-life of human food. Transglutaminase is also suggested for in vitro use to prepare food ingredients for human consumption in Japanese Patent Application JP 04/349869 (1992). Here transglutaminase is used in vitro to deaminate casein in order to promote the absorption of minerals from the proposed food ingredients in the human intestine.

It is desirable to increase the amount of nitrogen available to a ruminant in its feed, and to do so more efficiently than the prior art.

Thus in a first aspect of the present invention there is provided a ruminant feed composition comprising an edible (to the ruminant) protein-containing feed substance where the protein has been cross-linked and/or the composition additionally comprises (at least one) protein cross-linking enzyme. The invention may thus allow one to decrease the degradation (or consumption by micro- organisms) of the edible (or digestible) protein in the rumen in order to deliver a greater proportion of the total protein to the small intestine. At its broadest the invention aims to cross-link the digestible protein to reduce protein (a nitrogen source for the ruminant) degradation either during ensilage or inside the rumen.

Protein degradation in silage can be reduced by a rapid drop in pH resulting in an inhibition of fermentation and proteolytic activity. However, this acidification is often not practical for farmers and so the use of cross-linking enzymes to inhibit enzymatic proteolytic activity during ensilage can provide a solution to the problem of increasing protein bio-availability to a ruminant.

The cross-linking enzyme can be employed as a ruminant feed ingredient or supplement and can catalyse the cross-linking of the proteins in the feed substance either before feeding to the ruminant (or even before addition of the feed substance to the feed composition) or during fermentation in the rumen. Thus the cross-linked proteins in the feed substance can result from the use of the cross-linking enzyme. Microbial proteolysis either inside the ruminant (e. g. in the rumen) or during ensilage (fermentation of the feed substance) can thus be reduced due to this cross-linking. Later on, in the small intestine, the (enzymatically) cross- linked protein is degraded by enzymes inside the animal (e. g. proteases) to amino acids which can then be absorbed and used as nitrogen source by the ruminant.

The rumen has high microbial enzyme activity and bacteria are the most important organisms in the breakdown of protein. Different types of proteases, such as serine proteases and metalloproteases, as well as various types of peptidases are involved in rumen proteolysis. Due to this high proteolytic activity and the variety of different proteases and peptidases present it was considered, prior to the invention, unlikely that it would be possible to protect proteins from all types of proteolysis merely by

enzymatic cross-linking of proteins in situ. However, unexpectedly it was found that this was so and the addition of a protein cross-linking enzyme resulted in a marked protection of the protein against proteolytic breakdown in the rumen. Consequently this increased nitrogen utilization resulting in increased growth and milk production for the ruminant.

The feed substance may be derived from meat (which includes fish) such as meat, bone or fish meal. In another embodiment the substance may comprise only plant (e. g. vegetable or vegetative) matter (in the form of digestible protein for the ruminant), and so here the feed can be devoid of meat and/or fish. The feed substance will often comprise silage (fermentated fodder or grass), forage, concentrates or compound feed. It may be free of dairy products, e. g. casein.

One can thus provide one or more protein cross-linking enzymes (e. g. transglutaminase) as an ingredient for the feed composition, such as silage or ready-for-use ruminant feed. The enzyme (s) can be (e. g. naturally) derived from microorganisms, plants and animals or can be synthetic or recombinant. The enzyme will be in an active form, if present, that is to say it is capable of cross-linking protein. This excludes inactive (e. g. denatured, such as by heat) forms of the enzyme that have been described in prior art compositions.

The cross-linking enzyme (e. g. transglutaminase) is preferably recombinantly produced, such as by heterologous expression of an encoding gene or cDNA in a

suitable host organism, or, alternatively, by homologous (e. g. over expression) of a suitable endogenous gene. The enzyme may be produced by a (wild-type) strain of e. g. a Streptoverticilliacm species. Illustrative strains include Streptoverticillium griseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamonezcm IFO 12852, Streptoverticillium mobaraense IFO 13819 and other species (cf.

JP-A-64-27471).

Alternatively, the enzyme may be produced (e. g. recombinantly by expression of a heterologous gene) by a micro-organism such as a bacteria, yeast or fungus (e. g. <BR> <BR> <BR> <BR> <BR> filamentous fungi). Preferably the organism is of the genus Streptomyces, Bacillus,<BR> <BR> <BR> <BR> <BR> <BR> Escherichia, Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, Candida,<BR> <BR> <BR> <BR> <BR> <BR> <BR> Aspergillus, Trichoderma, Penicillium, Mucor, Fusarium or Humicola. Typical preferred<BR> <BR> <BR> <BR> <BR> <BR> (production) organisms are E. coli, Streptomyces e. g. Streptomyces lividans, Bacillus e. g.

Bacillus licheniformis, Saccharomyces e. g. Saccharomyes cerevisiae, Kluyveromyces lactis <BR> <BR> <BR> <BR> <BR> and Aspergillus e. g. Aspergillus niger. The enzyme can be produced by fermentation (of the organism) and then additional processing to recover the enzyme.

One suitable group of protein cross-linking enzymes are the transglutaminases.

Transglutaminase (EC 2.3.3.13; protein-glutamine gamma-glutamyltransferase) is an enzyme capable of catalysing acyl-transfer reactions introducing covalent cross-links between proteins as well as peptides and various amino acids. Large scale production of transglutaminase is now possible by cloning a (microbial) gene encoding transglutaminase and expression in a suitable host.

There are a number of enzymes other than transglutaminase that are effective in cross-linking proteins. Protein disulfide isomerase (EC 5.3.4.1), for example, catalyses the reduction of disulphide groups in proteins to reactive sulphydryl residues. Oxidation of these sulphydryl groups results in a covalent cross-link between two polypeptide chains or between two residues in the same chain.

Sulphydryl oxidase (EC 1.8.3.2) or thiol oxidase is capable of catalyzing the oxidation of sulphydryl groups in proteins to disulfide bonds. This enzyme is found in both animals and microorganisms.

Polyphenol oxidase (EC 1.14.18.1; previously classified as EC 1.10.3.1) is also known as catechol oxidase, tyrosine oxidase, tyrosinase, phenolase or phenol oxidase. This enzyme is widespread in nature and is found in many different types of animals such as e. g. mammals, insects, fish, molluscs, nematodes, plants, and microorganisms. The enzyme catalyses the oxidation of phenols to ortho-diphenols which are subsequently oxidized to ortho-diquinone by the same enzyme. The ortho-diquinones react with sulphydryl groups or with amine groups to form protein cross-links.

Lysyl oxidase (EC 1.4.3.13) or lysyl-protein-6-oxidase is an important enzyme in the formation of protein cross-linking in collagen and elastin. Lysyl oxidase catalyses the oxidative deamination of lysine to an alpha-amino adipic-delta-semialdehyde or its hydroxide. These reactive molecules react with other amino acids to form

cross-links.

Peroxidase (EC 1.11.1.7) is capable of catalysing a large number of reactions in proteins in the absence or presence of hydrogen peroxide. Hydroxylation and peroxidation activities of peroxidase cause protein cross-linking. However cross- linking enzymes other than peroxidase (s) can be used depending on the circumstances.

Lipoxygenase (EC 1.13.11.12, also lipoxydase or linolate: oxygen oxidoreductase) catalyses the oxidation of unsaturated fatty acids to fatty acid peroxo radicals. These reactive molecules cause protein cross-linking.

Many kinds of oxidases produce hydrogen peroxide and cause protein cross-linking in an indirect way. Glucose oxidase (EC 1.1.3.4) for example, catalyses the oxidation of glucose to gluconolactone and hydrogen peroxide which in its turn oxidises proteins, and can be employed although it can be omitted in some embodiments (e. g. the feed substance comprises silage).

The protein cross-linking enzyme (s) used in the invention will usually be either one which is not usually a natural constituent of the ruminant feed substance (s) or is present in the feed at a concentration different from its natural concentration. The enzyme can be added to the feed composition separately from the feed substance (s), alone or in combination with other feed additives. Alternatively or in addition the

enzyme can be an integral part of one of the feed substances. A suitable enzyme in the form of a supplement may be added to the feed substance, e. g. silage, or directly to the feed composition such as after other feed substance (s) have been added or admixed.

A particularly preferred method for the (exogenous) addition of the cross-linking enzyme additive to ruminant feed is to add the enzyme as or in transgenic plant material e. g. (transgenic) seed. The enzyme may thus have been synthesized through (heterologous) gene expression in the (part of) the plant. The gene encoding the desired enzyme is cloned in a plant expression vector, under control of the appropriate plant expression signals, e. g. a tissue specific promoter, such as a seed specific promoter. The expression vector containing the gene encoding the enzyme is subsequently transformed into plant cells and transformed cells are selected for regeneration into whole plants. The thus obtained transgenic plants can be grown and harvested, and those parts of the plants containing the (heterologous) enzyme can be included in the feed, either as such or after further processing. The enzyme may be present in a seed of the transgenic plant or it may be present in other plant parts such as roots, stems, leaves, wood, flowers, bark and/or fruit.

For expression of transglutaminase or other cross-linking enzymes in transgenic plant materials, preferably in seed, reference is made to WO-A-91/14772 which discloses general methods for the (heterologous) expression of enzymes in plants, including methods for seed-specific expression of enzymes.

The addition of the enzyme in the form of transgenic plant material, e. g. transgenic seed containing the e. g. transglutaminase, may require the processing of the plant material so as to make the enzyme available, or at least improve its availability. Such processing techniques may include various milling and grinding techniques or thermomechanical treatments such as extrusion or expansion.

The protein cross-linking enzyme such as transglutaminase may be added to the feed composition (which includes silage) at concentration which varies as a function of diet composition and target animal species. Generally between 1 and 1,000,000 units of enzyme per kg protein in the feed composition is suitable. Preferably 10-10,000 U are added per kg of protein in feed. A unit of transglutaminase activity is described in Example 1. Usually the amount of protein in the feed composition is from 15-25% (w/w). Note that these amounts are based on ability to cross-link proteins and so excludes enzymes denatured by heating.

Suitably the ruminant (order Ruminantia) is a cow, sheep, deer or goat. The ruminant is preferably a lactating animal (or at least capable of lactating).

The present invention will now be described by way of example with reference to the following Examples which are provided by way of illustration and are not intended to limit its scope.

COMPARATIVE EXAMPLE 1 Generation of transglutaminase enzvme units Transglutaminase from Streptoverticillium sp. was cloned and (over) expressed in Streptomyces lividans as described in US patent no. 5,420,025. Transformants of <BR> Streptomyces lividans expressing the Streptoverticillium transglutaminase gene were grown in a complex medium as described in this patent. The fermentation broth had transglutaminase activities ranging from 1-10 U/ml. The microorganism was separated from the broth by means of centrifugation for 10 min. at 12,000 g at 4°C.

The liquid containing the enzyme was concentrated by means of ultrafiltration (cut off was 1,000 D) to a final enzyme concentration of 25 U/ml.

Enzyme analysis was then carried out as described in J. Biol. Chem. 241: 5518 (1966) using benzyloxycarbonyl-L-glutamylglycine and hydroxylamine as substrates. One unit of enzyme activity is defined as the amount of enzyme catalysing the formation of one 1 micromole of hydroxamic acid per minute at 37°C.

COMPARATIVE EXAMPLES 2.3 and EXAMPLES 4 to 7 Inhibition of proteolytic activity in the rumen with a transglutaminase Rumen fluid collection and preparation of rumen protease was as follows.

A rumen-fistulated, lactating cow of 700 kg weight was fed on a mixture of maize silage and dairy concentrate twice per day. Water was available ad libitum.

Rumen fluid was collected 3 hours after morning feeding and sieved through a 1 mm <BR> <BR> <BR> <BR> <BR> strain. The sieved fluid was centrifuged for 5 min at 1000 g at 4°C to remove solids and then centrifuged for 20 min at 25, C00 g at 4°C to concentrate rumen microorganisms. The supernatant liquid is referred to as rumen protein. The liquid was lyophilized and stored at 4°C until required.

Protase activity present in the concentrated rumen microorganisms was determined by a known method such as that described by Manadeval, S., Sauer, F. D. and Erfle, J. D. (1987) in Can. J. Anim. Sci. 67: 55. Final extracts were concentrated by means of ultrafiltration under anaerobic conditions and lyophilized. Lyophilized protease extracts from the concentrated rumen microorganisms are referred to as crude rumen protease.

Determination of rumen protease activity Rumen protein was analysed for crude rumen protease activity as follows. Rumen protein was dissolved in 0.1 M phosphate buffer (pH 6.5) to give a final rumen protein concentration of 5 g/L. Crude rumen protease was added to this mixture and then incubated with stirring at 37°C; various amounts of crude rumen protease were added in order to get linear enzyme activity over a period of 30 min. After 10,20 and 30 min. of incubation the reaction was terminated by the addition of 15% trichloroacetic acid to a final concentration of 0.3% (w/v) TCA at 4°C. Precipitated protein was removed by means of centrifugation for 10 min at 10,000 g. Free amino acids in the supernatant were estimated using the known ninhydrin method (Rosen, H., 1957, Arch. Biochem. Biophys. 67: 10).

Inhibition of proteolytic activity bv transglutaminase Rumen protease activity was determined as described supra. Prior to the addition of crude rumen protease to rumen protein various amounts of transglutaminase units were added and allowed to act for 15 minutes (Examples 6 and 7). Simultaneous addition of transglutaminase and crude rumen protease was tested as well (Examples 4 and 5). Activities of transglutaminase tested varied between 50 and C. 05 IU per experiment equivalent to respectively 10,000 and 10 U/kg of substrate protein. The addition of transglutaminase resulted in a marked inhibition of rumen protease activity (Table 1). A pre-incubation period of substrate with transglutaminase lead to a more pronounced inhibition of proteolytic activity.

TABLE 1

Example Pre-incubation Proteolytic activity period (min) with (mg amino acids/60 min) transglutaminase 2. Negative control (no n. a. 0.3 crude rumen protease, no transglutaminase) 3. Positive control (crude n. a. 5.1 rumen protease, no transglutaminase) 4. As2+50U 0 2.4 transglutaminase 5. As 2 + 0.05 U 0 3.3 transglutaminase 6. +50U2 15 1.1 transglutaminase 7. As2+0. 05U 15 2.5 transglutaminase COMPARATIVE EXAMPLES 8. 9 and EXAMPLES 10 to 13 Inhibition of proteolytic activity bv glucose oxidase Glucose oxidase, as a typical example of a protein cross-linking oxidase, was tested

for its effects on the inhibition of proteolysis in the rumen. Glucose oxidase was obtained as the commercial product sold under the trade mark FERMIZYME GO from Royal Gist-brocades, Bakery Ingredients Division, PO BOX 1,2600 MA DELFT, The Netherlands. This enzyme preparation exhibits an activity of 500 Sarett Units per gram. One Sarett unit is the amount of enzyme that will cause an uptake of 10mm3 of oxygen per minute in a Warburg manometer at 30°C in the presence of excess oxygen and 3.3% glucose monohydrate in a phosphate (buffer pH 5.9).

Rumen protease activity was determined as described in Examples 2 to 7. Prior to the addition of crude rumen protease to rumen protein various amounts of glucose oxidase were added and allowed to act for 15 minutes (Examples 12 and 13). Activities of glucose oxidase tested varied between 50 and 0.25 Sarett Unit per experiment equivalent to respectively 10,000 and 50 Sarett Units/kg of substate protein.

Simultaneous addition of glucose oxidase and crude rumen protease was tested as well (Examples 10 and 11). The addition of glucose oxidase resulted in a marked inhibition of rumen protease activity (Table 2). The preincubation of substrate with glucose oxidase lead to more pronounced inhibition of proteolytic activity.

TABLE2

Example Preincubation period Proteolytic activity (mg (min) with glucose amino acids/60 min) oxidase 8. Negative control (no n. a. 0.3 crude rumen protease, no glucose oxidase) 9. Positive control n. a. 5.3 (crude rumen protease, no glucose oxidase 10. As 9 + 50 Sarett 0 2.9 | Units glucose oxidase 11. As 9 + 0.25 Sarett 0 3.8 Unit glucose oxidase 12. As 9 + 50 Sarett 15 2.2 Units glucose oxidase 13. As 9 + 0.25 Sarett 15 3.2 Unit glucose oxidase COMPARATIVE EXAMPLE 14 and EXAMPLES 15 and 16 Effects of transglutaminase on silage qualitv Forage grass was harvested with a self-propelled metered chopper equipped with grinding plates to damage the kernels. The crop contained 32% dry matter and 24% of this dry matter consisted of crude protein.

Transglutaminase was added to the silage at 10 and 10,000 U/kg silage protein by spraying the liquid enzyme on to the crop after wilting while mixing in a concrete mixer. Controls received equal amounts of water.

The crop was ensiled in 1-litre preserving jars. Duplicate samples were taken and analysed (Spoelstra, S. F. 1983, Neth. J. Agric. Sci. 31: 89-92) after 90 days of storage at ambient temperature. The results are shown in Table 3.

TABLE 3

Example Dry pH Crude protein NH3 Matter g/kg g/kg g/kg dry matter drv matter 14. No 303 3.9 21.6 2.9 transglutaminase 15.10U 309 3.8 22.7 2.1 transglutaminase per kg protein 16.10,000 U 311 3.7 23.4 1.5 transglutaminase per kg protein The results clearly indicate the protective effect of the addition of transglutaminase on proteolysis in silage. The nutritional value was increased due to addition of the transglutaminase since the valuable protein was degraded to a lesser extent by the microorganisms. The degree of remaining proteolysis is still good enough to allow for a rapid fermentation process as demonstrated by the drop in pH.

EXAMPLES 17 and 18 Effects of transglutaminase on zootechnical results of dairv cows fed on maize forage and dairv concentrates Grass forage was supplemented by being sprayed with liquid transglutaminase produced by the method of Example 1 at two dosages, 10 and 10,000 U/kg of protein. Total rations consisted of 80% grass forage and 20% dairy concentrate.

Experiments were performed with 5 Friesian dairy cows for each enzyme concentration.

Performance data (Table 4) obtained clearly demonstrated the favourable effects of transglutaminase on milk yield and feed conversion ratio (milk/dry matter intake).

Feed conversion ratio was improved by 5% at the higher enzyme dose.

TABLE 4

Parameter Untreated 10 U/kg 10,000 U/kg control protein protein Milk 25.3(kg/day) 26.2 26.9 Protein (g/day) 793 845 805809Fat(g/day) 801 Intake 15.1 15.3 15.4 (kg dry matter/da Feed conversion ration (milk/dry 1.67 1.71 1.75 matter intake)

CLAIMS 1. A ruminant feed composition comprising an edible protein-containing feed substance where the protein has been cross-linked and/or the composition additionally comprises a protein cross-linking enzyme.

2. A feed composition according to claim 1 in which the protein cross-linking enzyme comprises protein disulphide isomerase, protein disulphide reductase, sulphydryl oxidase, polyphenol oxidase, lysyl oxidase, peroxidase, glucose oxidase or a transglutaminase.

3. A feed composition according to claim 1 or 2 wherein the protein cross-linking enzyme is derived from an animal, a plant or a microorganism.

4. A feed composition according to any preceding claim wherein the protein cross-linking enzyme is of microbial origin and/or is a recombinant protein.

5. A feed composition according to any preceding claim wherein the protein cross-linking enzyme comprises a transglutaminase.

6. A feed composition according to claim 5 wherein the enzyme is derived from, produced by or present in a microorganism which is a bacterium, yeast or filamentous fungus.

7. A feed composition according to claim 6 in which the microorganism is of the genus Streptomyces, Bacillus, Escherichia, Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, Candida, Aspergillus, Trichoderma, Penicillium, Mucor, Fusarium or H1lmicola.