WO2003009702A1

WO2003009702A1 - Animal feed with antimicrobial reactive oxygen species

Info

Publication number: WO2003009702A1
Application number: PCT/EP2002/008160
Authority: WO
Inventors: Nicolaas Charles Marie Laane; Alexander Ferdinand Arendsen
Original assignee: Dsm Ip Assets B.V.
Priority date: 2001-07-20
Filing date: 2002-07-22
Publication date: 2003-02-06

Abstract

As an alternative to an antibiotic, animal feeds contain a source of reactive oxygen species (ROS) having antimicrobial activity. Suitable ROS include hydrogen peroxide, hydroxyl radical, peroxyl radical, nitric or nitrous oxide, sulphuric acid, ozone, peroxynitrite, hypocholorus acid, persulfate, peracetic acid and percholorate. The ROS may be added to the feed or may be generated in situ, by a chemical ROS generator (perborate, persulphate, percarbonate) or enzymatic ROS generator (glucose oxidase). The ROS may be converted into a second, more potent, ROS by an ROS converter which may be an enzyme (chloroperoxidase or lactoperoxidase) or a chemical (ferrous or nitrate ions). The composition may also contain an ROS stabiliser which is an inhibitor of an enzyme which consumes or removes ROS (such as an inhibitor of catalase, superoxide dismutase or glutathione peroxidase).

Description

ANIMAL FEED WITH ANTIMICROBIAL REACTIVE OXYGEN SPECIES

Field of the Invention

The present application relates to animal feeds, and additives or premixes therefor, that contain a reactive oxygen species (ROS) or a substance that can generate or produce such a species. The species is usually inorganic, such as a compound, radical or anion and canhe capable of acting as an antimicrobial agent.

Background of the Invention

Monogastric animals such as pigs, poultry, veal calves and fish are grown intensively for the production of meat, fish and eggs. These animals are fed diets containing a variety of raw materials of animal and/or vegetable origin to supply energy and protein. Most of the feed that is consumed is produced commercially, but a significant part is produced on the farm and fed directly. The feed is often supplemented with vitamins and minerals to meet the animal's nutrient requirements. The use of industrially produced enzymes in these feeds has now almost become common practice.

The enzymes are used to promote growth and feed conversion, and to reduce the environmental pollution produced by manure from pigs, poultry and fish. However, feed costs are the most important cost factor in animal production. While antibiotics have been routinely added to animal feed, the resistance of pathogenic bacteria (to humans) against antibiotics has been increasing rapidly. This has made it more difficult to cure people from bacterial infections, and the widespread use of antibiotics in animal feed has been blamed by various experts in the acceleration of build-up resistance to various antibiotics. This has led to a ban on the use of most antibiotics as growth promoters in animal feed in the European Union. It is likely that other countries will follow this example due to pressure from consumer and healthcare organisations. The feed industry is therefore interested in alternative additives with growth promoting effects, without any therapeutic side effects in humans.

WO-A-00/21381 (DSM N.V.) teaches animal feeds which contain at least two antimicrobial enzymes and a polyunsaturated fatty acid (PUFA). One of these enzymes can be lysozyme. At that time it was not realised that a PUFA could disrupt the outer phospholipid layer of bacteria, and while it was known that the PUFA was beneficial, its mechanism was unknown, and therefore its use as a compound for disrupting the outer phospholipid layer was not disclosed in that document. Indeed this document does not contain a general disclosure of using phospholipid-disrupting agents in animal feed.

Other documents that refer to antimicrobial effects for more than one component, include a combination of:

(a) membrane disrupting peptides and lysozyme (Winans et al, Biochemistry 38:11700-11710 (1999));

(b) lysozyme, hydrogen peroxide and ascorbic acid (T.E. Miller, J. Bacteriol 98(3):949-955 (1969));

(c) lysozyme with EDTA, hydrogen peroxide or ascorbic acid (Peng et al, Am. J. Physiol. Cell Physiol. 279:C797-C805 (2000)).

Other documents of interest include WO-A-00/21381 (referring to the use of two antimicrobial enzymes and an enzyme enhancer, such as a PUFA), and WO-A-96/36244 (which refers to the application of phospholipases in animal feed), EP-A-0346909 (which refers to fermentative procedures for fodder, and the addition of carbohydrate-splitting enzymes) and WO-A-96/15682 (which refers to fodder and drinking additives that contain lysosyme and horseradish peroxidase).

Description of the Invention

The present invention provides an animal feed, and an additive or premix composition therefor, comprising a source of (e.g. antimicrobial) reactive oxygen species (ROS). This may allow the improvement of growth and feed conversion ratio of farm, monogastric and/or non- ruminant animals such as pigs, piglets, poultry, (veal) calves and aquatic animals such as fish, and can allow one to reduce the amount of, or omit, an antibiotic as a growth promoter.

A first aspect of the present invention relates to an' animal feed composition, comprising:

(a) a reactive oxygen species (ROS), namely an (e.g. antimicrobial) oxygen- containing moiety; or

(b) a substance that generates or produces such a species (a ROS generator); and

(c) optionally, a ROS converter (e.g. for converting one ROS species into another ROS). Composition of the Bacterial Cell Wall

Based on colour formation following staining, bacteria can be divided into two classes, namely Gram positive and Gram negative bacteria. These two classes differ both in the composition and the structure of their cell walls. The major component for both Gram positive and Gram negative bacteria cell walls is peptidoglycan. The peptidoglycan is a thick rigid layer consisting of an overlapping lattice of two sugars that are cross-linked by amino acid bridges. The exact molecular structure of this layer is species specific.

The two sugars are N-acetyl glucosamine and N-acetylmuramic acid, which are linked through a β-l,4-glycoside bond. Attached to N-acetylmuramic acid, which is a compound uniquely found in bacterial cell walls, is a side chain usually consisting of four amino acids. The most commonly found amino acids are L-alanine, D-alanine, D-glutamic acid, and a di-basic amino acid, usually diaminopimelic acid. The N-acetylglucosamine, N-acetylmuramic acid and the amino acid side chain forms a single peptidoglycan unit that can link with other units via covalent bonds to form a repeating polymer.

The polymer is further strengthened by crosslinks between the third amino acid (D- glutamic acid) of one unit and the fourth amino acid (diaminopimelic acid) of the next glycan tetrapeptide. The linker peptide of some bacteria contains glycine, serine and threonine. The degree of crosslinking determines the degree of rigidity. Peptidoglycan can be thought of as a strong woven mesh that holds the cell shape. It is not a barrier to solids, since the openings in the mesh are large enough for most type of molecules to pass through them.

The cell walls of Gram positive bacteria consist almost entirely of the peptidoglycan layer, which forms a heavy crosslinked woven structure that wraps around the cell. It is very thick with peptidoglycan accounting for about 50% of the weight of the cell, and 90% of the weight of the cell wall. It is about 20 to 80nm thick.

The cell walls of Gram negative bacteria contain markedly less peptidoglycan, only 15 to 20% of the cell wall being made up of peptidoglycan,- and this is only intermittently crosslinked.

Gram negative bacteria further differ from their Gram positive counterparts in that their cell wall contains an extra lipid layer. This is the phospholipid layer or outer membrane. This outer membrane, which is made of a lipopolysacchari.de layer, encloses the periplasmic space. The lipid portion of this layer contains lipid A, a toxic compound that is responsible for most of the pathogenic effects associated with harmful Gram negative bacteria. Reactive Oxygen Species (ROS)

The composition may comprise a source of the reactive oxygen species (ROS), such as the ROS itself, or a substance capable of generating or producing an ROS (herein after referred to as an ROS generator), or it may contain both. The composition may also contain a substance capable of converting one (e.g. first) ROS into another (e.g. second) ROS.

The ROS can be thought of as an oxygen containing moiety which suitably has antimicrobial activity. Suitably the ROS is an inorganic compound, a cation or a radical. It may be neutral or negatively charged (an anion). It may be an oxidant or a reductant (i.e. an oxidising or reducing agent), and in some cases it may act as both (such as the superoxide anion). The ROS can be a radical or non-radical species. It may be organic or inorganic, but preferably it is inorganic.

ROS are known in the art, and the term "reactive oxygen species" is an art-recognised term, often used to describe oxygen-containing moieties that are toxic to cells. For example, they may damage DNA, cause membrane disruption and/or release Ca ions from or withm the cells (thus leading to activation of Ca²⁺ dependent proteases and nucleases). It is known that some of this damage may be mediated by the presence of metal ions, such as iron or copper ions. ROS are generally thought to include inorganic moieties such as hydrogen peroxide, the hydroxyl radical, formaldehyde, hypochlorous acid, nitric oxide, the peroxyl radical (or hydroperoxide), peroxynitrite anion, singlet oxygen and the superoxide anion. The term ROS in this specification includes not only these moieties, but other ones, such as peracids, for example peracetic or perbenzoic acid, persulphate, permanganate, perchromate or perborate (e.g. Na salt).

The ROS preferably has at least two atoms and/or no more than four or five atoms. It will of course be appreciated that at least one (or two) of these atoms is an oxygen atom.

The ROS can thus be an oxygen containing moiety or species with antimicrobial activity, for example due to its ability to be involved in an oxidising/reducing (redox) reaction. Preferably the ROS has a redox potential of from -1.0 to +1.5 V, preferably from 0.0 to +1.0 V (measured against NHE - normal hydrogen electrode).

The antimicrobial ROS may be bacteriostatic, growth-retarding or antipathogenic, and may result from catalysis or a reaction involving an enzyme. The ROS is suitably toxic to bacteria, such as Gram positive and/or Gram negative bacteria. The ROS generator may be a protein, e.g. an enzyme, such as a (phospho)lipase, oxidase, peroxidase, lipoxygenase, synthase and/or phosphorylase.

Preferably the ROS comprises (hydrogen) peroxide (e.g. H₂O₂), HOC1, hypothiocyanate (OSCN^" or HOSCN), and/or a hydroxyl radical (OB>). The amount or concentration of the ROS (or ROS generator) is suitably sufficient for the ROS to be antimicrobial, in other words by an antimicrobially effective amount (or the ROS generator to be in a sufficient concenfration to generate an antimicrobially effective amount of the ROS, e.g. in vivo).

Examples of reactive oxygen species

Listed below are reactive oxygen species (ROS) contemplated by the invention:

O ^* Superoxide radical/anion

H₂O₂ Hydrogen peroxide

OH* Hydroxyl radical

ROO^» Aliphatic and or aromatic peroxyl radical (or hydroperoxide, ROOH)

HOO* Peroxyl radical (or peroxide anion HOO^") O₂ Singlet oxygen

NO* Nitric oxide

N O Nitrous oxide

SO₂ Sulphur dioxide

O₃ Ozone

ONOO^" Peroxynitrite anion

CIO^", HOCl Hypochlorous acid (and other hypohalous acids, general formula: HOHalogen or

HalogenO^") OSCN^" Hypothiocyanate

S₂O₈ ^" Persulphate (and other inorganic peracids, e.g. permanganate, perchromate and perborate) C₂H₃O₃ ^" Peracetic acid

RCOOOH Peracids (organic) C₆H₅CO₃H Perbenzoic acid

ClO₄ ^" Perchlorate (and other perhalates, general formula: HalogenO^" ₄)

HCHO Formaldehyde

Detection of ROS

Short-lived free radicals like O₂ ^' and OH* can be detected by elecfron spin resonance (ESR) or electron paramagmetic resonance (EPR) spectroscopy. ESR spectroscopy depends on the interaction of an external homogeneous magnetic field with the magnetic moment of an unpaired electron within a free radical molecule. A single unpaired or free elecfron, which has a spin quantum number (M_s) of ±1/2, assumes two orientations in the magnetic field. The two orientations of the free elecfron exist at different energy levels, where the difference in energy is referred to as the Zeeman splitting. As the magnetic field (H) increases the magnitude of the Zeeman splitting also increases.

By definition, radicals are very reactive, and, as a result, can be short lived. The spin trapping method was developed to extend the limits of ESR spectroscopy so that lower concentrations of free radicals could be detected indirectly. This method involves the trapping of reactive short-lived free radicals by a diamagnetic spin trap compound via an addition reaction to produce a more stable free radical product or spin adduct. The spin adduct that is formed is paramagnetic and has an ESR spectrum with a hyperfine splitting constant and g- value characteristic of the type of reactive free radical trapped. The most commonly used spin trapping agents in biological systems are nitrone derivatives. Examples of commonly used nifrones are a-phenyl-N-tert butyl nitrone (PBN); 5, 5 -dimethyl- 1-pyrroline-N-oxide (DMPO); and a-(4-pyridyl-l-oxide)-N-tert-butyl nitrone (POBN).

Assay conditions for the detection of radicals

- Prepare a 2.5 M PBN solution in 50% ethanol

Suspend the radical-containing sample in buffer (NaAc/HAc pH 4, minimum 200 μl)

Add PBN to a final concentration of 0.05 M, mix

Incubate on ice for 30 minutes

Transfer to quartz EPR tube

Acquire EPR spectrum at room temperature on X-band spectrometer at 1.25 Gauss frequency modulation; a suitable magnetic field is from 3000 — 4000 Gauss. Microwaver power: should be set such that power saturation is avoided (e.g 20 mW)

Compare experimental spectra against spectrum of a Cu²⁺-EDTA solution of known concentration.

H₂O₂ (a ROS) may be detected for instance by using a peroxidase, for example horseradish peroxidase, in presence of a suitable chromophoric substrate. Examples of suitable chromophoric substrates include: 5-amino-2,3-dihydro-l,4-phthalazinedione; 3-amino-9- ethylcarbazole; 5-aminosalicylic acid; 2,2'-azinobis(3-ethyl)benzthiazoline-6-sulfonic acid; 4- chloro-1-naphtol; 3,3'-diaminobenzidine; o-dianisidine; guaiacol; o-phenylenediamine; pyrogallol; and 3,3',5,5'-teframethylbenzidine. Assay conditions and protocol for the detection of^O Tris-phospate buffer pH 7 (solution A) o- dianisidine (solution B): 2 gram per litre in pH 7.0

Horseradish peroxidase (solution C) : 60 U per ml in pH 7.0

Glycerol

H₂O₂ standard: 1%

Hydrochloric acid: 5 M

Preincubate all solutions at least 10 minutes at 30°C.

Add to 1 ml A, 0.1 ml B, 0.1 ml C and 0.8 ml glycerol. Mix carefully.

Add 10 to 20 microliters of a sample containing in between 0- 3 μmol H₂O₂

Incubate 30 minutes at 30°C.

Stop reaction by adding 2 ml 5M HC1. Mix.

Oxidized o-dianisidine is measured in a spectrophotometer at 525 nm.

Compare with H₂O₂ standard 10 μl

Delta extinction coefficient o-dianisidine: 1.13 X 10⁴M cm^"1.

The amount of ROS (or its generator) is preferably such that it is effective, e.g. antimicrobial, or that it can produce enough antimicrobial substance to kill microbes (e.g: bacteria).: In the case of H₂O₂, this ROS may be present at a concentration from 10 or 17 to 17,000 or 25,000, preferably from 30 or 43 or 170 to 2,500, 5,000 or 8,500, and more preferably from 85, 200 or 340 to 1,700, 3,000 or 4,300 μg per kilogram (or unit) of animal feed. Thus preferably the ROS (or generator) may be present at a concentration at, or to give, from 0.5 to 500 , preferably from 1.25 or 5 to 75 or 250, and more preferably from 2.5 or 10 to 50 or 125 μmol per kilogram (or unit) of animal feed.

Chemistry of free radicals and other reactive oxygen species

A free radical (radical - Latin derivation - radix - root or fundamental) can be thought of as any species capable of independent existence that contains one or more unpaired electrons, an unpaired electron being one that is alone in an orbital.

Most biological molecules are non-radicals, containing only paired electrons. Free radicals can be cationic, anionic or have neutral characteristics. The term reactive oxygen species (ROS) is used in the art and has been introduced to describe collectively not only O₂ ^' and OH* (radicals) but also H₂O₂ (a non-radical). Hypochlorous acid (HOCl), produced by certain heme or vanadium peroxidases, is also a non-radical, having no unpaired electrons. H O₂, OH^#, HOCl are sometimes collectively called "oxidants".

Radicals can react with other molecules in a number of ways. If two radicals meet, they can combine their unpaired electrons and join to form a covalent bond (a shared pair of electrons). Radicals react with non-radicals in several ways. A radical may donate its unpaired electron to a non-radical (a reducing radical) or it might take an electron from another molecule in order to form a pair (an oxidizing radical).

A radical may also join onto a non-radical. Whichever of these three types of reaction occurs, the non-radical species becomes a radical. A feature of the reactions of free radicals with non-radicals is that they tend to proceed as chain reactions, where one radical creates another.

ROS generators (compound or enzyme) that produces an (antimicrobial)ROS

The ROS generator may be instead of, or in addition to, the ROS itself. Hence the composition of the invention may comprise one or more ROS and/or ROS generator(s).

The ROS generator may be an oxidase, in which case the ROS generator may be H₂O₂, HOCl and/or OSCN^". Suitable oxidases include glucose oxidase, galactose oxidase, hexose oxidase, methanol oxidase, xanthine oxidase, sulphydryl oxidase, NADPH oxidase, nitroalkane oxidase or a (e.g. poly) phenol oxidase (such as catechol oxidase or tyrosinase), monoamine oxidase, copper amine oxidase or cytochrome oxidase. The enzyme may be a peroxidase (such as horseradish peroxidase, chloro or bromo peroxidase, lactoperoxidase), a lipoxygenase or a nitric oxide (NO) synthase (which generates nitric oxide).

The enzyme may be naturally occurring or may be produced recombinantly. The enzyme may have been produced by expression of a heterologous gene in a microorganism, for example in Kluyveromyces lactis or Aspergillus. The enzyme may be present as an inactive pro-form that can be activated on ingestion, for example in the GI tract, suitably by a proteolytic processing.

The amount of the ROS generator (e.g. enzyme) is preferably such that is effective, e.g. antimicrobial, or that it can generate enough ROS to kill microbes (e.g. bacteria).

The ROS generator can be a substance that is capable of generating or producing an ROS. Preferably, the generator will only be active, in other words capable of generating or producing an ROS, in situ, in other words, once inside the animal or when it is contacted with water. The composition may be such so that, outside the animal, the generator is inactive, or not activated. However, once ingested, or when the generator comes into contact with, for example, water, it may then be able to generate or produce an ROS.

The ROS generator may be a (inorganic or organic) chemical or a protein (e.g. enzyme). Enzvmic ROS Generator

The ROS generator may comprise a (phospho)lipase, an oxidase (for example to produce H₂O₂), lipoxygenase (to produce a peroxyl radical or hydroperoxide), (e.g. NO) synthetase (e.g. for NO), a reductase (for NO), a peroxidase (for HOHalogen), a phosphorylase and/or a lactoperoxidase (for OSCN^").

The ROS generator may be an oxidase, in which case the toxic compound may be H₂O₂, HOCl and/or OSCN^". Suitable oxidases include glucose oxidase, galactose oxidase, hexose oxidase, methanol oxidase, xanthine oxidase, sulphydryl oxidase, NADPH oxidase, nitroalkane oxidase or a (e.g. poly) phenyl oxidase (such as catechol oxidase or tyrosinase), monoamine oxidase, copper amine oxidase or cytochrome oxidase. Co-substrates (e.g. glucose or Cl^") may be added to the composition or feed, or they may be naturally present. Examples of ROS generating enzymes include:

Lactoperoxidase

Peroxidase-like enzymes (V-phytase)

Xanthine oxidase

Haloperoxidase (e.g. bromo or chloro peroxidase) Micro)peroxidase

Lipoxygenase

Glucose oxidase

Hexose oxidase

Sulphydryl oxidase

Xanthine oxidase

Galactose oxidase

Amino acid oxidase

Amine oxidase

Methanol oxidase - NAD(P)H oxidase

The enzyme may be naturally occurring or may be produced recombinantly. The enzyme may have been produced by expression of a heterologous gene in a microorganism, for example in Kluyveromyces lactis or Aspergillus. The enzyme may be present as an inactive pro-form that can be activated on ingestion, for example in the GI tract, suitably by a proteolytic processing. Oxidases may be detected on the basis of the production of H₂O₂ in the presence of a suitable substrate and a peroxidase and a chromophoric substrate (see page 6, assay conditions for radical detection).

A preferred oxidase is a sugar or hexose oxidase, such as glucose oxidase. Glucose oxidase can be assayed in an assay mixture containing glucose, horse radish peroxidase, and o- dianisidine as elecfron acceptor. The oxidized form of o-dianisidine turns brown and then reveals the glucose oxidase activity. The absorbance at 525 nm can be monitored with a spectrophotometer.

Assay conditions and protocol for the detection of glucose oxidase

Glucose oxidase in Tris-phospate buffer pH 7 (solution A) (max 10 IU / ml) o- dianisidine (solution B): 2 gram per liter in pH 7.0

Horseradish peroxidase (solution C): 60 U per ml in pH 7.0

Glycerol

H₂O₂ standard: 1%

Hydrochloric acid: 5 M

Preincubate all solutions at least 10 minutes at 30°C.

Add to 1 ml A, 0,1 ml B, 0,1 ml C and 0,8 ml glycerol. Mix carefully.

Add 10 to 20 microliters of a sample containing in between 0- 10 IU glucose oxidase per ml

Incubate 30 minutes at 30°C.

Stop reaction by adding 2 ml 5M HC1. Mix.

Oxidized o-dianisidine is measured in a spectrophotometer at 525 nm.

Delta extinction coefficient o-dianisidine: 1.13 X 10 M^"1 cm^"1.

Compare with internal standard 0 to 10 IU per ml.

The amount of the enzyme is preferably such that it is effective, e.g. antimicrobial, or that it can produce enough antimicrobial substance to kill microbes (e.g. bacteria). The enzyme may be present at a concentration to give from 10 to 10,000 , preferably from 25 or 100 to 1,500 or 5,000, and more preferably from 50 or 200 to 1,000 or 2,500 International Units (IU) per kilogram (or unit) of animal feed (such as for oxidases e.g. glucose oxidase). An LU is defined as the conversion of 1 μmol per minute.. Chemical ROS generator

Alternatively, the ROS generator may be an inorganic compound or a precursor for the ROS. The precursor may produce the ROS when in contact with water, for example it may dissolve in water to generate the ROS. Thus, if the ROS generator is a chemical, it may comprise a peroxide (such as hydrogen peroxide) for OH* or ROO^" or SCN (for OSCN^"). Peracids, such as perborate, persulphate, perbenzoic acid, and peracetic acid, and (auto)oxidizable sugars are examples of chemical ROS generators.

Generation of ROS

O₂ ^' (superoxide radical) is a species that is generated by adding one electron onto molecular oxygen. O₂ ^" formed in vivo, whether functionally or accidentally, can be disposed of by superoxide dismutase (SOD). Simple sugars can autooxidize under physiological conditions generating O₂ ^' . Reduced semiquinones can bind and interchelate with DNA generating O₂ ^' in situ. O₂ ^" can be formed by spontaneously by irradiation, or enzymatically, e.g. by xanthine oxidase and by NAD(P)H oxidases.

H₂O itself is of course not a free radical. It can cross biological membranes. Intracellularly it reacts with phospholipids, carbohydrates, metalloproteins and DNA and so causes damage. H₂O₂ can also be converted to hydroxyl radical (OH*) by metal ions, such as ferrous ion (the so-called Fenton reaction). Sugars (e.g. glucose, mannose and deoxy sugars) can autooxidize to produce H₂O . Semiquinones can bind and interchelate with DNA generating H₂O in situ. H₂O₂ can be formed enzymatically by oxidases, such as carbohydrate oxidases (e.g. glucose oxidase), amine oxidases, amino acid oxidases, and aromatic ring oxidases (e.g. (poly)phenol oxidases).

HO- is the most reactive radical known to chemistry. It can attack and damage almost every molecule found in living cells at a diffusion-controlled rate, i.e. it reacts as soon as it comes into contact with another molecule in solution. Since it is so reactive, HO- generated in vivo barely persists for a microsecond and rapidly combines with molecules in its immediate vicinity. Reactions of HO- with biological molecules, most of which are non-radicals, can set off chain reactions. Reactions of HO- include its ability to interact with the purine and pyrimidine bases of DNA, leading to radicals that have a number of possible chemical fates. HO- can also abstract hydrogen atoms from many biological molecules. OH* can be formed chemically from H O₂ by metal ions, such as the ferrous ion (Fe²⁺, the Fenton reaction).

Peroxyl^'radicals (ROO^" can be generated enzymatically by lipoxygenases, or chemically by peroxides. Singlet oxygen ^JO can be generated e.g. by ultraviolet light (or by cobalt-, lanthanide- or molybdenum compounds, e.g. salts, for example if they contact H₂O₂).

Nitric oxide (NO*) can be generated chemically, but also enzymatically by NO synthase (in a reaction which involves arginine, NADPH, and O₂). Also, NO is an intermediate in biological nitrate reduction. (Heme or copper contaimng) nitrite reductase can convert nitrite to nitric oxide.

Nitrous oxide (N₂O) can be synthesized chemically, and enzymatically by (e.g. copper- containing nitrite) reductases or by (e.g iron-containing) NO reductases, which participate in biological denitrification.

Sulphur dioxide (SO₂), ozone, and peroxynitrite (ONOO^~) can all be generated chemically.

Ozone (O₃) can be generated chemically or by the action of ultraviolet light. It is a strong oxidant, and can generate radicals such as hydroperoxyl radicals.

HOCl and other hypohalous acids can be generated enzymatically by (e.g. chloro- and bromo)peroxidases, such as in the presence of halides and H₂O , and by (e.g myelo)peroxidases (MPO) in neutrophils. HOCl is a strong oxidant, and can act as a bacteriocidal agent.

Hypothiocyanate (SCNO^~) can be generated enzymatically, for example by lactoperoxidase (also called LP system), such as in a reaction involving H O₂ and SCN.

Peracids (persulfate, permanganate, perchromate, perborate or perbenzoic acid) are bacteriocidal. Peracetic acid can be synthesized by conversion of H₂O₂ by TAED.

Damage caused by ROS to microbes

Oxygen containing free radicals are highly reactive species. They can cause severe damage, e.g. by hydrogen absfraction, bond scission and polymerization reaction. All membrane lipids contain polyunsaturated fatty acids (PUFAs) and these are susceptible to free radical damage by lipid peroxidation.

Lipid peroxidation is a free radical process, where a primary reactive free radical interacts with a polyunsaturated fatty acid (PUFA) to initiate a series of reactions, resulting in peroxide products (called diene conjugates).

Free radicals can also oxidize amino acids such as cysteine, methionine, tyrosine etc. Furthermore, free radicals can damage or affect the structure of DNA. OH* attacks the nucleotide bases to cause opening of the purine ring to form derivatives, such as 8- hydroxyguanine. When the ring is broken, DNA replication is effectively stopped. Other free- radical damage occurs to the deoxyribose-phosphodiester backbone of the DNA. Also, semiquinones can bind and interchelate with DNA generating O₂ ^' and H₂O₂ in situ.

ROS converters (one ROS into another ROS)

The composition of the invention may, alternatively or in addition to the ROS generator, comprise an ROS converter. Such a converter may be able to convert, activate (or even consume) one ROS into another ROS. Thus there may be present a first ROS, and this may be converted into a second ROS by the ROS converter. The second ROS may be more active than the first, for example, it may have a higher redox potential than the first (that is to say the value may be more positive or more negative than the first ROS). The first ROS maybe produced by a ROS generator.

The first ROS may therefore be hydrogen peroxide, the superoxide anion, a peroxyl radical, nitric oxide or singlet oxygen. This may be converted into a second ROS, which may be a hydroperoxide hypohalous acid (or hypohalide anion), hypothiocyanate, hydroxyl, peroxynitrate anion and/or a peracid. The ROS converter, as with the ROS generator, may be a chemical (such as ferrous irons, nitrate, TAED (tefracetylethylenediamine), Mn-TACN (manganese- 1,4,7-trimethyl-l, 4, 7-triazacyclononane), a cobalt, lanthanide or molybdenum salt, (e.g. Fe) porphyrin) or an enzyme, such as a peroxidase (e.g. lactoperoxidase, soybean peroxidase, horseradish peroxidase, haloperoxidase) or a peroxidase-type enzyme (e.g. V- phytase).

Enzymatic ROS converters

The second ROS may be generated by an enzymatic ROS converter. This may be one or more ROS converting or "activating" enzymes (such as a peroxidase, for example chloroperoxidase). A ROS activating enzyme may propagate or convert one ROS into another (for example HOCl from H₂O₂ and Cl^" in the case of chloroperoxidase). Substrates (e.g. glucose) or co-substrates (e.g. Cl^") may be added to the composition or feed, or they may be naturally present.

The enzyme may be naturally occurring or may be produced recombinantly. The enzyme may have been produced by expression of a heterologous gene in a microorganism, for example in Kluyveromyces lactis or Aspergillus. The enzyme may be present as an inactive pro-form that can be activated on ingestion, for example in the GI tract, suitably by a proteolytic processing. Peroxidases may be detected on the basis of the oxidation of a suitable chromaphoric substrate (see page 6, assay conditions for radical detection) and H₂O₂. Hence peroxidases (soybean, horseradish and microperoxidase) are preferred.

A preferred peroxidase is horseradish peroxidase. Horseradish peroxidase can be assayed in an assay mixture containing o-dianisidine and H O₂. The oxidized form of o-dianisidine turns brown and then reveals the peroxidase activity. The absorbance at 525 run can be monitored with a spectrophotometer.

Assay conditions and protocol for the detection of peroxidase

Tris-phospate buffer pH 7 (solution A) (max 10 IU / ml) o- dianisidine (solution B): 2 gram per litre in pH 7,0

Glycerol

H₂O₂ standard: 1%

Hydrochloric acid: 5 M

Preincubate all solutions at least 10 minutes at 30°C.

Add to 1.1 ml A, 0.1 ml B, and 0.8 ml glycerol. Mix carefully.

Add 10 to 20 microlitres of a sample containing in between 0- 10 IU peroxidase per ml.

Incubate 30 minutes at 30°C.

Stop reaction by adding 2 ml 5M HC1. Mix.

Oxidized o-dianisidine is measured in a spectrophotometer at 525 nm.

Delta extinction coefficient o-dianisidine: 1.13 X 10⁴M^"1 cm^"1.

The amount of the enzyme is preferably such that it is effective, e.g. antimicrobial, or that it can produce enough antimicrobial substance to kill microbes (e.g. bacteria). The enzyme may be present at a concentration to give from 10 to 10,000 , preferably from 25 or 100 to 1,500 or 5,000, and more preferably from 50 or 200 to 1,000 or 2,500 International Units (IU) per kilogram (or unit) of animal feed, for example for a peroxidase. An IU is defined as the conversion of 1 μmol per minute.

Chemical ROS convertor

The composition of the invention may comprise one or more chemical ROS converters. Examples of chemical ROS convertors include: cobalt, lanthanide, and molybdenum salts (generating singlet oxygen from H₂O₂) Ferrous iron (generating OH^»)

Manganese-TACN (an activated μ-(per)oxo Mn complex) TAED (generating peracetic acid) nitrate (generating ONOO^" in the presence of H₂O₂)

Iron porphyrins ("heme") may form iron (IV)-oxo (ferryl) compounds (conversion of H₂O₂ to hydroperoxide or oxygen radicals)

Enzymatic ROS generator plus enzymatic ROS convertor

ROS generation in the GI tract can also be effected by adding a combination of two or more enzymes to the feed, namely one or more ROS generating enzymes (such as an oxidase, for example glucose oxidase) plus one or more ROS converting enzymes (such as a peroxidase, for example lactoperoxidase). The second enzyme (the ROS converting enzyme) can convert a first ROS (generated by the first substance, ROS generator e.g. an enzyme) to form another, second, and perhaps more potent ROS.

Co-substrates may be added, or they may be naturally present. Examples of suitable co- substrates for the ROS activating enzymes include thiocyanate (SCN^"), chloride (Cl^"), bromide

94-

(B ), ferrous iron (Fe ), and polyunsaturated fatty acids (PUFAs) such as arachidonic acid (ARA). Upon conversion oxidation by a ROS converting enzyme (e.g. a peroxidase) with H₂O₂ as an oxidant these co-substrates can be converted into hypothiocyanate (OSCN^"), hypochloride (CIO^"), and hypobromide (BrO^").

Chemical ROS generator plus enzymatic ROS convertor

The composition of the invention may also comprise one or more chemical ROS generators plus one or more enzymatic ROS convertors. For example, the composition may contain sodium perborate in combination with a peroxidase (such as chloroperoxidase). Upon dissolving in water sodium perborate generates H₂O₂, which can be converted by chloroperoxidase to HOCl in the presence of Cl^" (either present in the feed or added to it).

Enzymatic ROS generator plus chemical ROS convertor

The composition of the invention may also comprise one or more enzymatic ROS generators plus one or more chemical ROS convertors. ROS may be generated by one or more enzymatic ROS generators (e.g. an oxidase, for example glucose oxidase) in the presence of one or more ROS activating chemicals. For example, H₂O can be (reductively) converted to a second, (more potent) ROS, OH^«, in the so-called Fenton reaction by metal ions such as Fe²⁺. Substrates (e.g. glucose) may be added, or they may be naturally present. For example, the composition may contain an oxidase (such as glucose oxidase) in combination with ferrous iron. In the presence of glucose (either present in the feed or added to it) and oxygen glucose oxidase produces H₂O₂, which is converted to OH^» by concomitant oxidation of ferous iron (either present in the feed or added to it).

Chemical ROS generator plus chemical ROS convertor

The composition of the invention may also comprise one or more chemical ROS generators plus one or more chemical ROS convertors. For example, the composition may contain sodium perborate in combination with Fe²⁺ ions). Upon dissolving in water sodium perborate generates H₂O₂, which is converted to OH* by concomitant oxidation of the ferrous iron (either present in the feed or added to it).

The ROS may also be generated chemically. For example, peracids such as perborates or persulphates will form H₂O₂ (a ROS) upon dissolving. Other chemicals such as nitrate activate one ROS (namely H₂O₂) to form a second ROS, (in this case ONOO^"). In another example, a ROS generated by a ROS generating chemical (such as perborate generating H₂O ) is converted to a second ROS by a ROS activating chemical (such as nitrate forming ONOO^").

Combinations of ROS generators and converters

The following indicates the use of both chemical and enzyme ROS generators and (optionally) ROS converters.

ROS generator ROS converter

enzyme - glucose oxidase enzyme - (chloro)peroxidase (CIO)^"

- (lacto)peroxidase (OSCN^", Halogen O)

OR ROS OR

chemical - perborate chemical - Fe²⁺ (converts H₂O₂ to Fe ,3^J+⁺ +_. OH^» + OH^")

- nitrate (produces ONOO^") The invention thus relates to compositions having a ROS generator and a ROS converter. Suitable generators (and the ROS they generate) and ROS converters (if present) are as shown below.

ROS generator (1st) ROS ROS converter (2nd) ROS

Enzymes (e.g. glucose) oxidase H₂0₂ (chloro) peroxidase CIO^' (HOCl) (lacto) peroxidase OSCN^", HalogenO^"

(xanthine) oxidase H₂O₂ and ₂ ^'

(NADPH) oxidase o₂'^~

(lipo)oxygenase ROO^"

NO synthase NO

(nitrate) reductase N₂O

Chemical Perborate H₂O₂ Fe²⁺ OH^"

Nitrate ONOO^"

TAED Peracetic acid Oxo-species

TACN ^JO₂

Co/La/Mo Salt

Concentrations of ROS converters and generators

The following table gives preferred, more preferred and optimal ranges for the ROS itself, ROS generators (chemical and enzymatic) and ROS converters (chemical and enzymatic).

A preferred ROS generator or converter is a sugar or hexose oxidase, such as glucose oxidase. This enzyme may be present at a concentration to give from 10 to 10,000 , preferably from 25 or 100 to 1,500 or 5,000, and more preferably from 50 or 200 to 1,000 or 2,500 Sarrett U per kilogram (or unit) of animal feed. Thus preferably the enzyme may be present at an amount, by weight, to give a final concentration in the animal feed of from 0.05 to 50 milligrams of protein per kg of feed, preferably from 0.08 or 0.13 to 7.5 or 25 milligrams of protein per kg of feed, and more preferably from 0.25 or 0.5 to 5.0 or 10 milligrams of protein per kg of feed, for example for A. niger-dsάved glucose oxidase.

ROS stabilisers

The composition of the invention may also comprise one or more ROS stabilisers, such as a compound that can prolong the lifetime of the ROS (suitably in situ). The stabiliser may be an inhibitor of an enzyme that removes, reacts with or consumes an ROS. Such an enzyme may be a catalase, a superoxide dismutase or a glutathione peroxidase. Thus, the stabiliser may comprise, or be a source of, fluoride or sulphide ions, nitric oxide, sulphite ions or sodium azide (all inhibitors of catalase) or aminotriazole (3-amino-l, 2, 4-triazole), a superoxide dismutase inhibitor.

Further examples of the types of compounds used in the invention are presented below.

ROS generators

Enzymatic ROS generated

Oxidases H₂O₂, HOCl, OSCN^"

Glucose oxidase }

Hexose oxidase }

Nitroalkane oxidase }

Poly(phenol) oxidase } H₂O₂

Monoamine oxidase }

Copper amine oxidase }

Cytochrome oxidase }

Sulphydryl oxidase }

Xanthine oxidase H₂O₂, O₂.^" (superoxide)

Galactose oxidase }

Amino acid oxidase }

Amine oxidase } H₂O^* ₂

Methanol oxidase }

NAD(P)H oxidase }

Lipoxygenase ROOH (hydroperoxide), ROO

(Phospho)lipase Peroxyl anion

Peroxidases HOHalogen

Nitric oxide synthase NO

Lactoperoxidase, phosphorylase • OSCN^"

Nitrate reductase N₂O

Chemical ROS generated

Peroxide OH', ROO^"

ROO7SCN OSCN^"

Perborate (e.g. Na) H₂O₂

Persulphate H₂O₂

Perbenzoic acid, peracetic acid Peracid oxidisable sugars (glucose, mannose deoxy sugars) H₂O₂

Percarbonates

ROS convertors 1st ROS 2nd ROS

Enzymatic

Lactoperoxidase H₂O₂ OSCN^"

Soybean peroxidase H₂O₂

Peroxidase-like enzymes

(Vanadate-phytase) H₂O₂ Hydroperoxide

Horseradish peroxidase H₂O₂ Haloperoxidase H₂O₂ HOHal Microperoxidase H₂O₂ Hydroperoxide

Chemical Fe²⁺ H₂O₂ OH* Nitrate H₂O₂ Peroxynitrite (ONOO^") TAED H₂O₂ Peracid, e.g. peracetic acid) Mn -TACN H₂O₂ Hydroperoxide Cobalt, lanthanide, and molybdenum salts (generating singlet oxygen) H₂O O^] ₂

Manganese-TACN (an activated μ- (per)oxo Mn complex) Iron porphyrins ("heme") may form iron (rV)-oxo (ferryl) compounds H₂O hydroperoxide oxygen radicals

Examples of co-substrates for ROS (e.g. H₂O₂) convertor enzymes SCN^" Cl^" Br^" Fe²⁺ Polyunsaturated fatty acids (PUFAs, e.g. arachidonic acid)

Of the enzymatic ROS generators (or converters), oxidases are preferred, but in certain applications oxidases are not suitable, and may therefore be excluded. The same is true of phospholipases and/or peroxidases, such as lactoperoxidase. For some uses glucose oxidase may be absent.

Antibacterial effect of free radicals and other reactive oxygen

Glucose oxidase has been used in contact lens cleaners and toothpaste. Its functional effect is brought about by the generation of H₂O₂ via oxidation of glucose by molecular oxygen. Being a powerful oxidizer, hydrogen peroxide can kill bacteria, viruses and fungi. H₂O₂ itself is not very reactive, but it can cross biological membranes, and can react with phospholipids, carbohydrates, metalloproteins and DNA. Moreover, in the presence of metal ions such, as ferrous ions, H₂O results in the formation of the hydroxyl radical (OH^») which is the most reactive radical known. Another free radical, O₂ ^" , is usually associated with oxidative stress, but it also has a functional role: activated phagocytic cells generate O₂ ^" , which kills bacteria that have invaded the mammalian body. Free radicals and other reactive oxygen species as antibacterial compounds in animals

Free radicals and the other ROS can act as antibacterial agents, such as in the gastrointestinal tract of animals. Thus, they can be used as replacements for antibiotics. However, some of the ROS radicals are extremely reactive. Hence, they may not be added to the feed, but instead they can be generated in situ (i.e. inside the animal, such as in the stomach or in the gut). A preferred way to generate ROS in the gastro-intestinal tract (GI) is by using a ROS generator, such as a precursor of a ROS or a ROS-generating enzyme or chemical. In dry formulations the enzyme(s) may not be active. However, upon ingestion by the animal the enzyme(s) will dissolve, and hence become active, whereupon ROS will be generated and/or- activated. This can result in killing of bacteria in vivo. Likewise, precursors or activators of ROS can be stable in a dry formulation. They may generate or activate ROS upon entering the GI tract of the animal.

Bacteria

As mentioned before, the compositions of (or, more precisely, the ROS used in) the present invention are active against bacteria, in particular Gram negative bacteria. A list of Gram negative bacteria which the ROS can be active against in the present invention is provided below.

Class Genera Species Acetobacteriaceae Acetobacter, Gluconobacter, Frateuria A. aceti

Alcaligenaceae, Alcaligenes, Deleya, Achromobacter A. faecalis

BacteroidaceaeBacteroides, Poφhyrornonas, Fusobacterium,

Leptotrichia and Selenomonas S. rumantium

Chromatiaceae Ameobobacter, Chromatium, Lamprobacter C. okenii

Lamprocystis, Thiocapsa, Thyocystis

Thiodictyon, Thiopedia, Thiospirillum

Enterobacteriaceae Escherichia, Salmonella, Shigella, Erwinia, E. coli

Enterobacter, Serratia

Legionellaceae Legionella L. pneumophila Neisseriaceae Neisseria, Kingella, Eikenella, Simonsiella N. gonorrheae

Alysiella

Nitrobacteriaceae Nitrobacter, Nitrospena, Nitrococus, Nitrosipra N. winogradskyi Pseudomonadaceae Pseudomonas, Xanthomonas, Zoogloea, Fraturia P. aeruginosa Rhizobiaceae Rhizobium, Bradyrhizobium, Azdrhizobium, R. lagumϊnosarum

Sinorhizobiurn

Rickettsiaceae Rickettsia, Rochalimae, Ehrlichia, Cowdria

Neorickettsia

Spirochaetaceae Triponema, Borrelia T. pallidum Vibrionaceae Vibrio, Aeromonas, Plesiomonas and V. cholerae

Photobacterium

Of these Gram negative bacteria, preferred are those of the class Vibrio, Neisseria, or Salmonella.

Although particularly effective against Gram negative bacteria, the ROS can also be active against Gram positive bacteria. Suitable Gram positive bacteria are listed below.

Class Genera Species Bacillaceae Bacillus, Sporolactobacillus B. botulinum

Sporocarcina, Filibacter, Cayophanum B. cereus,

Clostridium B. coagulans B. mycroides B. pumilis B. subtilis B. thuringiensis

Micrococcaceae Arthrobacter and Micrococcus M. luteus, M. roseus, M. lysoeiktus, M. radiochrans

Peptococcaceae Peptococcus, Peptosfreptococcus P. niger Ruminococcuss, Sarcina, Coprococcus

Preferably the Gram positive bacteria are of the group Cornynebacterium, Propionibacterium, Clostridium, Lactobacillus and/or Bifidobacterium.

Animal Feed Compositions

The substance(s), if enzymes, can be produced on industrial scale and/or may be recombinant. The enzyme may be naturally occurring or may be a (e.g. recombinant) variant or mutant thereof.

The substance, if an enzyme, is preferably recombinantly produced such as by expression of a heterologous gene or cDNA in a suitable organism, or alternatively by homologous (over)expression of a suitable endogenous gene. The glucose oxidase gene, for example, has been overexpressed in recombinant systems (WO- A- 89/12675, Chiron). Enzymes can be recombinantly expressed by expression of the gene in Aspergillus niger (Archer, D.B. et al, Bio/Technology 8: 741-745 (1990)). An enzyme mutant (produced by protein engineering) can also be used which may have better heat stability and/or stronger antimicrobial action.

A second aspect of the invention relates to a premix or additive composition to be added to one or more edible feed substance(s) or ingredient(s), for example to prepare (or for supplementation of) a feed composition (of the first aspect). This can comprise the ROS or ROS generator/producer. Preferably the additive or premix comprises from 10 to 1000, such as from 25 or 50 to 600 or 750, preferably from 75 or 100 to 250 or 500, times as much of the ROS, ROS generator, ROS converter and/or ROS stabiliser as the (animal) feed. This is because the premix can be "diluted" by a factor of 10 to 1,000 (so that the premix constitutes 10% to 0.1% of final feed) when making the animal feed. This premix may be in the form of granules or pellets.

A third aspect of the invention relates to a process for the preparation of an animal feed composition, the process comprising adding to (or supplementing) an animal feed, or to one or more edible feed substance(s) or ingredient(s), the source of the ROS (e.g. ROS or ROS generator).

The or each ROS or ROS generator can be added to the animal feed composition separately from any feed substance(s) or ingredient(s), individually or in combination with other feed additives. Alternatively or in addition the ROS or its generator can be in integral part of one of the food substances. The invention includes both preparing a feed composition with the two compounds or supplementing an existing feed composition with the ROS or its generator.

A preferred method for the addition of the compound to the animal feed is to add the substance as transgenic plant material and/or (e.g. transgenic) seed. This is particularly suitable if the substance is a protein, such as an enzyme. The compound may be synthesised through heterologous gene expression, for example the gene encoding the desired enzyme may be cloned into a plant expression vector, under control of the appropriate plant expression signals, for example a tissue specific promoter, such as a seed specific promoter. The expression vector containing the gene including the enzyme can be subsequently transformed into plant cells. Transformed cells can be selected for regeneration into plants. The thus obtained transgenic plant can be grown and harvested.

Those parts of the plants containing the heterologous (plant) protein can be included in one of the compositions of the invention, either as such or after further processing. Reference here is made to WO-A-91/14772, which discloses general methods for the (heterologous) expression of enzymes in (transgenic) plants. This includes methods for seed-specific expression of enzymes. The compound, such as the protein, may be contained in the seed of the transgenic plant. It may also however be contained in other plant parts such as roots, stems, leaves, wood, flowers, bark and/or fruit.

The addition of the substance in the form of a transgenic plant material, such as transgenic seed, may require the processing of the plant materials such as to make the compound available, or at least to improve the compounds availability. Such processing techniques may include various mechanical techniques, such as milling and/or grinding, or thermomechanical treatments, such as extrusion or expansion.

Exclusions

Preferably the animal feed of the invention does not contain any antibiotics. It may be free of a (supplementary or added) mineral component (such as zinc and/or iodine) and/or an immunomodulating agent (such as ascorbic acid). The composition may not include a combination of: lysozyme, glucose oxidase and (optionally also) arachidonic acid; or lysozyme, glucose, glucose oxidase and ascorbic acid. Other excluded compositions may include those comprising a phospholipase or a combination of PLA₂ and lysostaphin; ascorbic acid and lysozyme.

Suitably the compositions of the invention do not comprise two (e.g. antimicrobial) enzymes, for example an enzyme that disrupts the cell wall of bacteria in combination with an enzyme that generates a compound toxic to bacteria. The compositions may additionally exclude those that have these two enzymes, and an enzyme enhancer, such as a PUFA. Other excluded compositions may be ones that comprise a lactoperoxidase, myloperoxidase, lipase, phospholipase and/or xylitolphosphorylase.

Other compositions that may be outside the invention include those that comprise a carbohydrate-splitting enzyme and/or an oxygen removing enzyme, for example lactoperoxidase and/or glucose oxidase. The invention may also exclude compositions that comprise (e.g. horseradish) peroxidase and/or lysosyme. Production of substance(s) by microorganisms

Although one or more of the substances can be produced by a microorganism, for many situations (the producing) micro-organisms will not be added to or present in the feed, or at ' least live (or viable) organisms, such as bacteria, are not present in the feed. Hence in this case the composition is free from any microorganisms that produced one or more of these compounds (or micro-organisms from Streptomyces). Furthermore, the composition may be devoid of micro-organisms that produce lactic acid inside the animal (e.g. those of the genus Lactobacillus or Enter ococc s). Typically, before addition of the compounds, the feed composition will be heated to kill, or reduce the number of, any bacteria present in the feed.

Uses of animal feed

A fourth aspect of the invention relates to a process for promoting growth, feed conversion or antibacterial activity, in a monogastric or non-ruminant animal, the process feeding the animal a ROS or a ROS generator. The animal can be fed the animal feed of the first aspect or feed preparable by the third aspect.

Suitable animals include farm, monogastric and/or non-ruminant animals such as pigs (or piglets), poultry (such as chickens and turkeys), calves, veal calves or aquatic (e.g. marine) animals, for example fish.

A further aspect relates to the use of a composition of the second aspect as an additive for a monogastric or non-ruminant animal feed composition.

The compositions of the invention may be active in vivo (e.g. not in vitro), or only once ingested or inside the animal. The ROS or ROS generator may thus not be effective since the compositions may be too dry, e.g. they have a water content of no more than 10, 20, 30, 40 or 50%. There may thus not be enough water in the composition for the ROS or its generator to be (chemically or antimicrobally active). Once ingested and inside the animal (e.g. in the stomach of rumen) there may be sufficient liquid (or water) for the ROS or ROS generator to become active or effective, or be activated (e.g. antimicrobial).

Animal Feed Components

The compositions of the invention, in particular additive or premix compositions, can be either in liquid or solid form. If a solid, then this may be a powder, a granulate, extrudate or it may be pellets. For a solid form, the amount of water present may be below 20, 15 or even 10%, such as from 2 to 10%, 3 to 8% or 4 to 7%. The or each ROS or ROS generator (e.g. enzyme) may be present at from 1 to 30%, such as 2 to 20%, for example 3 to 15%, and optimally at from 4 to 14% (on a dry matter basis).

The remainder may comprise carbohydrates and/or carbohydrate polymers (such as starch and/or modified starch), for example at least 70, 80, 90 or 95%, such as from 75 to 90%. The composition may have a coating, for example if it is in a pellet, granulate, or extrudate form. There may thus be one or more coats on the outside of the composition, comprising one or more coating materials. If present, the coating (or coating materials) may be present at from 1 to 10%, such as from 2 to 6%, optimally at from 3 to 5%. The composition may have one or more stabilisers (such as glycerol and/or sorbitol) and/or one or more preservatives (such as sorbate and/or benzoate).

If the composition is a liquid, then the water (or moisture) content will be higher. The water content may be up to 40, 50 or 60%, for example from 25 to 65%, optimally from 35 to 55%>. If a stabiliser is present, this may be at an amount of from 45 to 65%, such as from 50 to 60%, optimally from 52 to 58%. The stabiliser is preferably sorbitol and/or glycerol.

A description of the preparation of pellets and granules, in particular carbohydrate based enzyme granulates, is described in WO-A-98/54980 (International Application No. PCT/EP98/03327). This and all other documents mentioned have their contents incorporated herein by reference.

The composition may comprise a carrier which may comprise at least 15% of an edible carbohydrate polymer. The carrier may be in particulate or powder form. However, if the composition is a liquid, it may be in the form of a solution or a slurry. The polymer preferably comprises glucose, or glucose-containing units, although it can contain glucopyranose units, amylose and/or amylopeptin. In addition, or instead of starch, a glucan, peptin or glycogen can be used. Preferably at least 15%, such as at least 30%, at least 40%, for example at least 60%, optimally at least 80% of the composition (or the solid carrier) comprises the carbohydrate polymer.

Additional details of enzyme-containing compositions for animal feed can be found in WO-A-98/55599 (International Application No. PCT/EP98/03328). Although this document primarily deals with phytases, its teachings are equally applicable to other proteins, in particular enzymes.

Animal feed compositions of the first aspect will usually contain one or more feed ingredients or substances. These are ingredients and substances intended for consumption by an animal, and is therefore in a form suitable for ingestion and nutrition for an animal. This will therefore usually exclude human foodstuffs, or food substances or ingredients intended or destined for consumption by humans. Preferably the feed composition is both edible and digestible by the animal.

Suitably the ingredients and/or substances have a dry matter content of at least 80, 85, 90 or 95%. The protein content of the composition (or the substances and/or ingredients) may vary considerably, but may be from 5 to 20%, such as 10 to 15%, for example vegetable and/or plant products or parts thereof, such as buckwheat, rice, wheat, barley or corn. Substances or ingredients with higher protein contents, such as from 45 to 95%, e.g. 50 to 80%, may be provided, for example peanuts, poultry feathers, soy bean (or products thereof), sunflower (e.g. seeds) or casein. Prefened animal feed compositions may therefore comprise one or more of oats, pea (seeds), peanuts, soy beans, sunflower, canola, casein, coconut, corn, meat, millet, potato, rice, safflower and/or wheat. Preferably the composition (and substances or ingredients) have a crude fibre content below 30%, 25%, 20%, 15% or even below 10%. Similarly, the calcium content may be below 2%, such as 1%, below 0.5% and preferably less than 0.2%. The total phosphorous content of the (animal feed composition) is preferably from 2 to 0.01%, such as from 1 to 0.1%, optimally less than 0.5%.

The precise substances and ingredients can vary depending on the animal to be fed. An alternative composition may comprise one or more of bakery waste, sugar beet, brewers grain, canola, cassava, corn, fababean, fish (such as anchovy or herring meal), lentils, meat and/or millet.

Preferred features and characteristics of one aspect of the present invention are applicable ,to another aspect mutatis mutandis.

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.

EXAMPLES

Characterization of antimicrobial compounds

Glucose oxidase (EC 1.1.3.4), an oxidase capable of generating hydrogen peroxide, is easily available, and in this case was obtained as a commercial product under the trade mark FERMIZYME GO™ 1500 from DSM Food Specialties, PO Box 1, 2600 MA DELFT, The Netherlands. This enzyme preparation exhibits an activity of 1500 Sarrett Units per gram. One Sarrett 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 with a pH of 5.9. The enzyme was produced by the fungus Aspergillus.

Chloroperoxidase (E.C. 1.11.1.10) from Caldariomyces fumago was obtained from Sigma- Aldrich. The enzyme was diluted in water to obtain the appropriate concenfration. The unit activity of this enzyme is expressed as the conversion of 1 micromol of monochlorodimedon to dichlorodimedon per minute at pH 2.75 at 25°C in the presence of potassium chloride and H₂O .

Peroxidase (E.C. 1.11.1.7) from horse radish was obtained from Sigma- Aldrich. The unit activity is the amount of enzyme which forms 1 milligram of purpurogallin from pyrogallol in 20 seconds at pH 6 and 20°C.

Lipoxygenase (E.C. 1.13.11.12) from soybean was obtained from Sigma-Aldrich . The unit activity is the amount of enzyme which oxidizes 0.12 nanomol of linoleic acid per minute at pH 9 and 25°C.

Peroxidase (E.C. 1.11.1.7) from bovine milk (lactoperoxidase) was obtained from Sigma-Aldrich. The unit activity is the amount of enzyme which forms 1 milligram of purpurogallin from pyrogallol in 20 seconds at pH 6 and 20°C.

Vanadium phytase (V-phytase) was prepared using phytase from Aspergillus niger. This phytase is available as a commercial product, Natuphos™, from DSM Food Specialties, PO Box 1 , 2600 MA DELFT, The Netherlands. One FTU unit of phytase is defined as the amount of enzyme that releases 1 μmol of phosphate from yo-inositol hexa[dihydrogen phosphate] (6 mM) at pH 5.5 and 37°C . Equal volumes of phytase (952 FTU/ml) and 20 μM sodium orthovanadate (Na₃VO₄, from Sigma-Aldrich) were mixed and stirred.

Ferrous ammonium sulphate and sodium persulphate were both obtained from Sigma- Aldrich.

Enrofloxacin (48,000 ppb) was obtained from Bayer.

[All other compounds were obtained from suitable sources]

Comparative Examples 1 to 5 and Examples 6 to 25: In vitro tests

In disk diffusion (also called Bauer-Kirby) susceptibility tests, small paper disks (6 mm), impregnated with known amounts of anti-bacterial ROS generator or converter were placed on the surface of BHI media plates that were inoculated confluently with a standardized suspension of Streptococcus faecalis and Escherichia coli. The anti-bacterial generator or converter, diffuse into the plate media, caused a zone of inhibition of growth of S. faecalis and E. coli around the disk.

Oxoid Iso-sensitest agar (approximately 30 g/L) was dissolved in water under boiling, and autoclaved for 15 minutes at 121°C. After the agar had cooled down to 50°C aliquots of suspensions of S. faecalis (from a suspension with an optical density of 0.963) and E. coli (from a suspension with an optical density of 1.064) were added to the agar. The agar suspension was mixed, and subsequenty poured out onto the culture disk. After solidification of the agar, disks containing the ROS generator or ROS converter or, for comparison, the known antibacterial agent enrofloxacin, were applied onto the plates, and the plates were incubated at 37°C overnight. Following overnight incubation, the diameter of the zone of growth, used as a measure of susceptibility, was measured around each disk to the nearest tenth of a mm.

The results show that adding a ROS generator (e.g. glucose oxidase or Na- persulphate) to the agar media resulted in inhibition of the growth of S. faecalis (a Gram positive bacterium) and E. coli (a Gram negative bacterium). Adding a ROS convertor to the agar media resulted a significant reduction of growth around the disk. However, adding a ROS generator plus a ROS convertor results in a significant increase of growth inhibition for both strains, corroborating the concept that the combination of a ROS generator with a ROS convertor exhibits a synergistic antibacterial effect.

Claims

CLALMS

1. An animal feed composition comprising' a reactive oxygen species (ROS) or substance capable of producing or generating a ROS (a ROS generator).

2. An animal feed additive or premix composition comprising a reactive oxygen species (ROS) or substance capable of producing or generating a ROS (a ROS generator).

3. A composition according to any one of the preceding claims wherein:

(a) the ROS has a redox potential of from -1.0 to +1.5 V;

(b) the ROS comprises hydrogen peroxide (H₂O₂), HOCl, hypothiocyanate (OSCN^"), superoxide anion, hydroxyl radical, peroxyl radical, singlet oxygen, nitrous oxide,^" sulphurous acid, ozone, peroxynitrite anion, hypohalous acid, persulphate or other inorganic peracid such as perchlorate, organic peracids such as peracetic and perbenzoic acid, and/or nitric oxide (NO); and/of'

(c) the ROS generator is an enzyme (such as an oxidase, an oxygenase or synthetase) or is a chemical (such as perborate, persulphate, or percarbonate).

4. A composition according to any preceding claim which additionally comprises a ROS converter, optionally an enzyme (such as a peroxidase) or a chemical (such as ferrous ions, nitrate, TAED and/or catalyst such as a Co/La/Mo-salt or Mn-TACN), which is capable of converting one ROS into another ROS.

5. A composition according to any preceding claim which additionally comprises a ROS stabiliser, which is suitably an inhibitor of an enzyme that consumes, removes or converts (to a non ROS) an ROS, and optionally the stabiliser is an inhibitor of a catalase (such as flouride, sulphide, nitric oxide, sulphite or sodium azide), an inhibtor of superoxide dismutase (aminotriozole) or an inhibitor of glutathione peroxidase.

6. A composition according to any of claims 4 or 5 wherein:

(i) the ROS generator comprises glucose oxidase and optionally is at a concentration of from 10 to 10,000 Sarrett units per kg of animal feed; or

(ii) the ROS converter comprises peroxidase and is optionally at a concenfration of from 10 to 10,000 IU per kg of animal feed.

7. A composition according to any of (enzymatic) claims 4 to 6 wherein the ROS generator and/or ROS converter is of animal, plant, algal or microbial origin and/or is recombinant.

8. A process for the preparation of a feed composition, suitable for a monogastric or non-ruminant animal, the process comprising adding a ROS or ROS generator to an animal feed, or mixing a feed additive or premix composition according to any of claims 2 to 7, with one or more edible feed substance(s) or ingredient(s).

9. An animal feed composition comprising an additive or premix composition according to claim 2 and one or more edible feed substance(s) or ingredient(s).

10. A process for promoting growth and/or feed conversion in a monogastric or non- rurninant animal, the process comprising feeding the animal a ROS or ROS generator as defined in any of claims 1 to 7, and optionally where the animal is a pig, piglet, poultry, veal calf or aquatic animal.