The present invention relates to the use of natural essential oil actives in combination with at least one polypeptide having lysozyme activity in the manufacture of feed for aquatic animals including fish and shrimp, especially for cold water fish as for example salmon, bream, bass and for warm water fish as for example carp, tilapia, catfish.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

SEQ ID NO: 1 is the mature amino acid sequence of a wild type GH25 lysozyme from Acremonium alcalophilum as described in WO 2013/076253.

SEQ ID NO: 2 is the gene sequence of the GH24 lysozyme as isolated from Tri- chophaea saccata. SEQ ID NO: 3 is the amino acid sequence as deduced from SEQ ID NO: 2.

SEQ ID NO: 4 is the mature amino acid sequence of a wild type GH24 lysozyme from Trichophaea saccata.

SEQ ID NO: 5 is the mature amino acid sequence of a wild type GH22 lysozyme from Gallus gallus (hen egg white lysozyme). SEQ ID NO: 6 is primer F-80470.

SEQ ID NO: 7 is primer R-80470.

SEQ ID NO: 8 is primer 8643.

SEQ ID NO: 9 is primer 8654.

INTRODUCTION The present invention relates to the use of natural essential oil actives selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, di- hydroeugenol, eugenol, meta-cresol and terpinolene in the manufacture of feed for aquatic animals including fish and shrimp, especially for cold water fish as for example salmon, bream, bass and for warm water fish as for example carp, tilapia, catfish. More particular, this invention relates to the use of at least one, preferably at least two essential oil actives as defined above in combination with a polypeptide having lysozyme activity for the improvement of the feed conversion ratio and/or daily weight gain in fish, for reducing mortality by regulating the micro flora of the gut and/or by protecting the animal against infections caused by pathogenic microor- ganisms.

Lysozyme is an O-glycosyl hydrolase produced as a defensive mechanism against bacteria by many organisms. The enzyme causes the hydrolysis of bacterial cell walls by cleaving the glycosidic bonds of peptidoglycan; an important structural molecule in bacteria. After having their cell walls weakened by lysozyme action, bacterial cells lyse resulting from osmotic pressure.

Lysozyme occurs in many organisms such as viruses, plants, insects, birds, reptiles and mammals. In mammals, Lysozyme has been isolated from nasal secretions, saliva, tears, intestines, urine and milk. The enzyme cleaves the glycosidic bond between carbon number 1 of /V-acetylmuramic acid and carbon number 4 of /V-acetyl-D-glucosamine. In vivo, these two carbohydrates are polymerized to form the cell wall polysaccharide.

Lysozyme has been classified into five different glycoside hydrolase (GH) families (CAZy, www.cazy.org): hen egg-white lysozyme (GH22), goose egg-white lysozyme (GH23), bacteriophage T4 lysozyme (GH24), Sphingomonas flagellar pro- tein (GH73) and Chalaropsis lysozymes (GH25). Lysozymes from the families GH23 and GH24 are primarily known from bacteriophages and have only recently been identified in fungi. The lysozyme family GH25 has been found to be structurally unrelated to the other lysozyme families.

Hen egg white lysozyme is the primary product available on the commercial mar- ket, but does not cleave A/,6-0-diacetylmuramic acid in e.g. Staphylococcus aureus cell walls and is thus unable to lyse this important human pathogen among others (Masschalck B, Deckers D, Michiels CW (2002), "Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure", J Food Prot. 65(12):1916-23). Furthermore, the present invention relates to a novel fish feed composition comprising as active ingredients at least one, preferably at least two or three active compound(s) selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in combi- nation with at least one microbial polypeptide having lysozyme activity.

One important factor in aquaculture is the turnover rate. Turnover rate is determined by how fast the fish grow to a harvestable size. As an example, it takes from 12 to 18 months to raise Atlantic salmon from smolt (the physiological stage when the Atlantic salmon can first be transferred from fresh water to sea water) to harvestable size. A fast turnover has several positive results. First, it helps cash flow. Second, it improves risk management. Especially, a high mortality rate is a substantial risk for fish farmers.

It is generally known that mortality rate increases by an unbalanced microflora and/or by infections caused by pathogenic microbes. Fish diseases are common, and the likelihood of an outbreak is higher over a long growing period. There is also a risk that fish will escape due to accidents, e.g. when shifting nets, or due to bad weather causing wrecked fish pens.

For other farm animals it is well known to use antibiotics and vaccines to prevent the development of diseases. In aquaculture, antibiotics are not so much used - at least in cold water aquaculture - due to the fact that disease spread very quickly, diseased fish do not eat much and also due to the negative impact on the environment of the wasted medicated feed. Vaccines are widely used when available but they are not developed for all diseases.

As an alternative to synthetic drugs, the use of plant extracts and essential oils in animal feed is described in the literature. For example, patent WO201 1/006993 describes the use of essential oils in fish feed. WO201 1/006993 is however silent with regard to the protection against infections caused by gram+ microorganisms. Moreover, the application of essential oils has in the disclosed case above only limited efficiency. It therefore remains a need in aquaculture to prevent the development of diseases, thereby reducing mortality by any prophylactic means including antimicrobial activity against gram+ microorganisms.

The inventors of the present application surprisingly found that the combination of substances as defined above have a great potential for use in fish feed, e.g. for improving the feed conversion ratio (FCR) and/or weight gain and/or for the modulation of the gut flora. Further, the inventors surprisingly found that the novel fish feed compositions have also antimicrobial activity against gram+ microorganisms resulting in a reduced mortality. The unique selection of active compounds of the present invention allows for the first time controlling a number of fish diseases caused by a number of different pathogens.

Therefore, in a first particular embodiment, the invention relates to methods for using at least one, preferably at least two active compounds selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeuge- nol, eugenol, meta-cresol and terpinolene in combination with a microbial lyso- zyme in fish feed for improving the Feed Conversion Ratio (FCR) and/or weight gain and/or for reducing mortality by modulation of the gut microflora and/or by preventing diseases caused by pathogenic microorganisms, preferably caused by gram+ microorgansims. For example, it has been shown that selected compounds of the invention (eg.: cinnamaldehyde and alpha-pinene in combination with a bacterial lysozyme) exhibit excellent effects in inhibiting the growth of Lactococcus garvieae a disease found especially in cold water fish. The combination according to the present invention also exhibits excellent effects against Streptococcus iniae, a disease found especially in warm water fish.

In alternative embodiments, alpha-pinene and/or alpha-terpineol and/or cinnamaldehyde and/or dihydroeugenol and/or eugenol and/or meta-cresol and/or terpinolene is/are used in combination with at least one microbial polypeptide having lysozyme activity to improve animal feed digestibility and/or maintain animal health by supporting immune system function.

In another embodiment, the invention relates to methods for using at least three active compounds selected from the group consisting of alpha-pinene, alpha- terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in combination with at least one microbial polypeptide having lysozyme activity in fish feed for improving the Feed Conversion Ratio (FCR) and/or weight gain and/or for reducing mortality by modulation of the gut microflora and/or by preventing diseases caused by pathogenic microorganisms. In a preferred embodiment, alpha-pinene and cinnamaldehyde are used in combination with a bacterial lysozyme to improve animal feed digestibility and/or maintain animal health by supporting immune system function.

DEFINITIONS The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal.

The FCR may be determined on the basis of a fish growth trial comprising a first treatment in which a mixture of at least two compounds according to the invention is added to the animal feed in a suitable concentration per kg feed, and a second treatment (control) with no addition of the compound(s) to the animal feed.

As it is generally known, an improved FCR is lower than the control FCR. In particular embodiments, the FCR is improved (i.e., reduced) as compared to the control by at least 1 .0 %, preferably at least 1 .5 %, 1 .6 %, 1 .7 %, 1 .8 %, 1 .9 %, 2.0 %, 2.1 %, 2.2 %, 2.3 %, 2.4 %, or at least 2.5 %. The term "gut" as used herein designates the gastrointestinal or digestive tract (also referred to as the alimentary canal) and it refers to the system of organs within multi-cellular animals which takes in food, digests it to extract energy and nutrients, and expels the remaining waste.

The term gut "microflora" as used herein refers to the natural microbial cultures residing in the gut and maintaining health by aiding in proper digestion.

The term "modulate" as used herein in connection with the gut microflora generally means to change, manipulate, alter, or adjust the function or status thereof in a healthy and normally functioning animal, i.e. a non-therapeutic use.

The term "supporting immune system function" as used herein refers to the immune stimulation effect obtained by the compounds.

The term "mortality" as used herein refers to the ratio of life animals at the end of the growth phase versus the number of animals originally included into the pond. It may be determined on the basis of a fish challenge trial comprising two groups of fish challenged by a particular fish pathogen with the aim to provoke a mortality of 40 to 80 % of the animals in the untreated group. However, in the challenge group fed with a suitable concentration per Kg of feed of a mixture of at least two compounds according to the invention, the mortality is reduced compared to the untreated group by at least 5 %, preferably at least, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, or at least 50 %.

Animal feed: The term "animal feed" refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a monogas- trie animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and/or other feed ingre- dients (such as in a premix).

Antimicrobial activity: The term "antimicrobial activity" is defined herein as an activity that kills or inhibits the growth of microorganisms, such as, algae, archea, bacteria, fungi and/or protozoans. The antimicrobial activity can for example be bactericidal meaning the killing of bacteria or bacteriostatic meaning the preven- tion of bacterial growth. The antimicrobial activity can include catalyzing the hydrolysis of 1 ,4-beta-linkages between /V-acetylmuramic acid and /V-acetyl-D- glucosamine residues in a peptidoglycan and between /V-acetyl-D-glucosamine residues in chitodextrins. Antimicrobial activity can also include the lysozyme binding to the surface of the microorganism and inhibiting its growth. The antimi- crobial effect can also include the use of the lysozymes of the present invention for activation of bacterial autolysins, as an immunostimulator, by inhibiting or reducing bacterial toxins and by an opsonin effect.

For the purpose of the present invention, antimicrobial activity is determined according to the antimicrobial assay described in Example 6 ("Determination of an- timicrobial activity"). Antimicrobial activity is determined by the diameter of the clearing zone and is evaluated as significant if the clearing zone is more than 2 mm using 50% Mueller-Hinton broth, pH 6.

Concentrates: The term "concentrates" means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)). European Production Efficacy Factor (EPEF): The European Production Efficacy Factor is a way of comparing the performance of animals. This single-figure facilitates comparison of performance within and among farms and can be used to assess environmental, climatic and managemental variables. The EPEF is calcu- lated as [(liveability (%) x Liveweight (kg)) / (Age at depletion (days) x FCR)] x 100, wherein livability is the percentage of animals alive at slaughter, Liveweight is the average weight of the animals at slaughter, age of depletion is the age of the animals at slaughter and FCR is the feed conversion ratio at slaughter.

Feed Conversion Ratio (FCR): FCR is a measure of an animal's efficiency in converting feed mass into increases of the desired output. Animals raised for meat - such as swine, poultry and fish - the output is the mass gained by the animal. Specifically FCR is calculated as feed intake divided by weight gain, all over a specified period. Improvement in FCR means reduction of the FCR value. A FCR improvement of 2% means that the FCR was reduced by 2%. Fragment: The term "fragment" means a polypeptide or a catalytic domain having one or more {e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has lysozyme or phytase activity. In one aspect, a fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino ac- ids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids or at least 200 amino acids of SEQ ID NO: 1 and has lysozyme activity.

In another aspect, a fragment comprises at least 210 amino acids, such as at least 215 amino acids, at least 220 amino acids, at least 225 amino acids, at least 230 amino acids, at least 235 amino acids or at least 240 amino acids of SEQ ID NO: 4 and has lysozyme activity.

Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated {e.g., multiple copies of a gene encoding the sub- stance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

Lysozyme activity: The term "lysozyme activity" means the hydrolysis of the 1 ,4- beta-linkages between /V-acetylmuramic acid and /V-acetyl-D-glucosamine resi- dues in a peptidoglycan or between /V-acetyl-D-glucosamine residues in chitodex- trins, resulting in bacteriolysis. Lysozyme belongs to the enzyme class EC

3.2.1 .17. Lysozyme activity is typically measured by turbidimetric determination. The method is based on the changes in turbidity of a suspension of Micrococcus luteus ATCC 4698 induced by the lytic action of lysozyme. In appropriate experi- mental conditions these changes are proportional to the amount of lysozyme in the medium (c.f. INS 1 105 of the Combined Compendium of Food Additive Specifications of the Food and Agriculture Organisation of the UN (www.fao.org)). For the purpose of the present invention, lysozyme activity is determined according to the turbidity assay described in example 5 ("Determination of Lysozyme Activity"). In one aspect, the polypeptides of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the lysozyme activity of SEQ ID NO: 1 . In another aspect, the polypeptides of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the lysozyme activity of SEQ ID NO: 4.

Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

Microbial lysozyme: The term "microbial lysozyme" means a polypeptide having lysozyme activity which is obtained or obtainable from a microbial source. Examples of microbial sources are fungi. In particular, the the microbial lysozyme is obtained or obtainable from the phylum Ascomycota, such as the sub-phylum

Pezizomycotina.

Sequence identity: The relatedness between two amino acid sequences or be- tween two nucleotide sequences is described by the parameter "sequence identity".

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needle- man and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et ai, 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Substantially pure polypeptide: The term "substantially pure polypeptide" means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1 %, and at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. Preferably, the polypeptide is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well known recombinant methods or by classical purification methods. Variant: The term "variant" means a polypeptide having lysozyme or phytase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, of one or more (several) amino acid residues at one or more {e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a posi- tion; and an insertion means adding 1 , 2, or 3 amino acids adjacent to and immediately following the amino acid occupying the position.

In one aspect, a lysozyme variant according to the invention may comprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1 to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1 -50, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 alterations and have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the phytase activity of the parent phytase, such as SEQ ID NO: 1 . In another aspect, a lysozyme variant according to the invention may comprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1 to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1 -50, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 alterations and have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the lysozyme activity of the parent lysozyme, such as SEQ ID NO: 4.

DETAILED DESCRIPTION OF THE INVENTION

In particular, the inventors of the present application surprisingly found that the compound combinations according to the invention are effective against a number pathogenic gram+ microorganisms of cold and warm water fish. Compositions according to the invention were shown to exhibit inhibitory effect against Lactococ- cus garvieae a disease found especially in cold water fish and against Streptococcus iniae, a disease found especially in warm water fish.

Furthermore, the use of at least one microbial polypeptide having lysozyme activi- ty in combination as defined above further improves existing inhibitory effects of natural essential oil actives.

Known inhibitory effects of essential oils are:

Alpha-pinene, cinnamaldehyde, dihydroeugenol, eugenol, limonene, and meta- cresol were shown to exhibit inhibitory effect against Aeromonas salmonicida which is the pathogen causing a disease known as furunculosis.

Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta- cresol and terpinolene were shown to exhibit inhibitory effect against Edwardsellia tarda causing systemic infection in fish.

Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, and terpinolene were shown to exhibit inhibitory effect against Lactococcus garvieae which is the etiological agent of Latococcosis, an emergent disease which affects many fish species and causes important economic losses both in marine and freshwater aquaculture when water temperature increases over 16 °C in summer months. Cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent inhibitory effects on the growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric Redmouth (ERM), a disease found especially in salmonids. Cinnamaldehyde and meta-cresol were shown to exhibit inhibitory effect against:

- Vibrio salmonicida which is a psychrophilic bacterium that is the causative

agent of cold-water vibriosis in Atlantic salmon.

- Aeromonas hydrophila causing ulcers and hemorrhagic septicaemia. This

pathogen is very resistant to conventional simple antimicrobials like chlorine.

- Photobacterium damselae formerly Pasteurella piscicida: a pathogen causing high losses in the culture industry of economically important marine fishes such as seriola and red grouper in Japan and striped bass and white perch in the United States. - Streptococcus iniae which is highly pathogenic in marine fish and is highly lethal: outbreaks may be associated with 30-50 % mortality.

Other aquatic pathogens such as

- Piscirickettsia salmonis the causative agent of piscirickettsiosis or salmonid rickettsial septicaemia (SRS), - Vibrio viscosus recently renamed Moritella viscosa etiologically responsible for the disease referred to as "winter ulcer",

- Ich (parasite) one of the most prevalent protozoan parasites of fish,

- Vibrio harveyi, responsible for luminous vibriosis, a disease that affects commercially-farmed prawns will also be inhibited by the compound mixture described in the present invention.

In a second aspect, the present invention provides a fish feed composition comprising at least one microbial peptide having lysozyme activity in combination with at least two active compounds selected from the group consisting of alpha-pinene (CAS 99-86-5), alpha-terpineol (CAS 98-55-5), cinnamaldehyde (CAS 14371 -10-9 / 104-55-2), dihydroeugenol (CAS 2785-87-7), eugenol (CAS 97-53-0), meta- cresol (CAS 108-39-4) and terpinolene (CAS 554-61 -0). The compounds according to the invention are commercially available or can easily be prepared by a skilled person using processes and methods well-known in the prior art. As fish feed composition, the compounds of the invention can be used alone or in mixtures thereof, in the form of natural available extracts or extract-mixtures or in the form of a natural substance.

The term "extract" as used herein includes compositions obtained by solvent ex- traction (which are also known as "extracted oils"), steam distillation (which are also known as "essential oils") or other methods known to the skilled person. Suitable extraction solvents include alcohols such as ethanol.

The term "natural" is in this context understood a substance which consists of compounds occurring in nature and obtained from natural products or through synthesis.

To the active essential oil compound(s) or natural substance or extract further ingredients may be added in minor amounts. Examples of such ingredients are: capsaicin, tannin, piperin, trimethylamine, 3,4,xylenol, furfuryl alcohol and mixtures thereof. If a mixture of at least two essential oil compounds as specified above is preferred, cinnamaldehyde and/or meta-cresol are used as a major component of the mixture. Suitably the mixture contains 10-90 % by weight of meta-cresol and/or cinnamaldehyde, 1 -50 % by weight of alpha-pinene, 1 -50 % by weight of dihydro- eugenol, wherein the amounts being calculated on the total amount of said com- ponents. The total amount of these active ingredients may vary within wide limits but is finally used in the fish feed from 10 to 5000 ppm, preferably between 100 and 1000 ppm, calculated on the dry weight of the fish feed.

The most preferred mixture of at least two essential oil compounds as specified above comprises, cinnamaldehyde and alpha-pinene as a major component of the mixture. Suitably the mixture contains 30-70 % by weight of alpha-pinene, 30-70 % by weight cinnamaldehyde, 1 -20 % by weight of metacresol, 1 -20 % by weight of dihydro-eugenol, wherein the amounts being calculated on the total amount of said components. The total amount of these active ingredients may vary within wide limits but is finally used in the fish feed from 10 to 5000 ppm, preferably be- tween 100 and 1000 ppm, calculated on the dry weight of the fish feed.

All essential oil compounds defined herein above (active compounds and additional ingredients) may be used in combination with an emulsifying surfactant. The emulsifying agent can be selected advantageously from those of a rather hydro- philic nature, for example among polyglycerol esters of fatty acids such as esteri- fied ricinoleic acid or propylene glycol esters of fatty acids, saccharo-esters or saccharo-glycerides, polyethylene glycol, lecithins etc.

In a preferred embodiment of the invention, a mixture of essential oils may contain 20 % by weight of cinnamaldehyde, 20 % by weight of meta-cresol, 20 % by weight of dihydro-eugenol, 20 % by weight of alpha-pinene, 3 % by weight of trimethylamine, 1 .8 % by weight of piperin and 4 % by weight of furfuryl alcohol.

In another preferred embodiment of the invention, a mixture of essential oil active compounds contains 40 to 60 wt.-% alpha-pinene, 40 to 60 wt.-% cinnamaldehyde, and may further contain 1 to 5 wt.-% trimethylamine, 1 to 5 % piperin, and 3 to 8 wt.-% furfuryl alcohol.

As mentioned above, it has been surprisingly found that supplementing an animal feed with a microbial lysozyme results in a significant performance benefit in aquatic animals including fish and shrimp. This is surprising since improved animal performance using a microbial lysozyme has never previously been demon- strated.

Thus the invention also relates to a method of improving the European Production Efficiency Factor (EPEF) comprising administering an animal feed or animal feed additive comprising at least two essential oil compounds and one or more microbial lysozymes to the animal. In a preferred embodiment, the improvement is compared to an animal feed or animal feed additive wherein the microbial lysozyme is not present (herein referred to as the control).

In one embodiment, the EPEF is improved by at least 1 %, such as by at least 1 .5%, at least 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or at least 5% compared to the control. In another embodiment, the EPEF is improved by between 1 % and 15%, such as between 1 % and 12%, between 1 % and 10%, 1 .5% and 8%, 2.0% and 7% compared to the control, or any combination of these intervals.

In one embodiment, the microbial lysozyme is dosed at a level of 8 to 250 ppm enzyme protein per kg animal feed, such as 9 to 200 ppm, 10 to 150 ppm, 1 1 to 125 ppm, 12 to 100 ppm, 13 to 75 ppm, 15 to 50 ppm, 17.5 to 40 ppm, 25 to 75 ppm or 30 to 60 ppm enzyme protein per kg animal feed, or any combination of these intervals. In one embodiment, the microbial lysozyme is of fungal origin. In an embodiment, the microbial lysozyme is obtained or obtainable from the phylum Ascomycota, such as the sub-phylum Pezizomycotina.

In one embodiment, the microbial lysozyme comprises one or more domains se- lected from the list consisting of GH24 and GH25.

In a preferred embodiment, the invention relates to a method of improving the European Production Efficiency Factor (EPEF) and/or feed conversion ratio (FCR) comprising administering an animal feed or animal feed additive comprising one or more microbial lysozymes to the fish, wherein: (a) the microbial lysozyme is a microbial lysozyme comprising one or more domains selected from the list consisting of GH24 and GH25, is dosed at a level of 10 to 150 ppm enzyme protein per kg animal feed;

(b) the aquatic animal is a selected from shrimp, for cold water fish as for example salmon, bream, bass and for warm water fish as for example carp, tilapia, catfish and;

(c) European Production Efficiency Factor (EPEF) and/or feed conversion ratio (FCR) is improved by at least 1 % compared to control.

In one embodiment, the microbial lysozyme is of fungal origin. In an embodiment, the microbial lysozyme is obtained or obtainable from the phylum Ascomycota, such as the sub-phylum Pezizomycotina.

In one embodiment, the microbial lysozyme has at least 50%, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 .

In one embodiment, the microbial lysozyme comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an allelic variant thereof; or is a fragment thereof having lysozyme activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids or at least 200 amino acids. In another embodiment, the microbial lysozyme comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an allelic variant thereof and a N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 208 of SEQ ID NO: 1 .

In another embodiment, the microbial lysozyme is a variant of SEQ ID NO: 1 wherein the variant has lysozyme activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions or any combination thereof in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions. In another embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 1 is between 1 and 45, such as 1 -40, 1 -35, 1 -30, 1 -25, 1 -20, 1 -15, 1 -10 or 1 -5 positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 1 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions, and/or insertions in SEQ ID NO: 1 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 1 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the number of conservative substitutions in SEQ ID NO: 1 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activ- ity of the protein; small deletions, typically of 1 -30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methio- nine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA/al, Ala/Glu, and Asp/Gly.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for lysozyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et ai, 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et ai, 1992, Science 255: 306-312; Smith et ai, 1992, J. Moi Biol. 224: 899-904;

Wlodaver et ai, 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

The crystal structure of the Acremonium alcalophilum CBS1 14.92 lysozyme was solved at a resolution of 1 .3 A as disclosed in WO 2013/076253. These atomic coordinates can be used to generate a three dimensional model depicting the structure of the Acremonium alcalophilum CBS1 14.92 lysozyme or homologous structures (such as the variants of the present invention). Using the x/ray structure, amino acid residues D95 and E97 (using SEQ ID NO: 1 for numbering) were identified as catalytic residues. In one embodiment, the microbial lysozyme has at least 50%, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In one embodiment, the microbial lysozyme comprises or consists of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or is a fragment thereof having lysozyme activity, wherein the fragment comprises at least 210 amino acids, such as at least 215 amino acids, at least 220 amino acids, at least 225 amino acids, at least 230 amino acids, at least 235 amino acids or at least 240 amino acids. In another embodiment, the microbial lysozyme comprises or consists of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof and a N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 245 of SEQ ID NO: 4.

In another embodiment, the microbial lysozyme is a variant of SEQ ID NO: 4 wherein the variant has lysozyme activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions or any combination thereof in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions. In another embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 4 is between 1 and 45, such as 1 -40, 1 -35, 1 -30, 1 -25, 1 -20, 1 -15, 1 -10 or 1 -5 positions. In an embodiment, the number of positions comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 4 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions, and/or insertions in SEQ ID NO: 4 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 4 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In a further embodiment, the number of conservative substitutions in SEQ ID NO: 4 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Examples of amino acid changes, conservative substitutions and N- and/or C- terminal linkers are described above. The enzyme of the invention may be formulated as a liquid or a solid. For a liquid formulation, the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol). Thus in one embodiment, the composition is a liquid composition comprising the enzyme(s) of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1 ,2-propylene glycol, 1 ,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol. The liquid formulation may be sprayed onto the feed after it has been pelleted or may be added to drink- ing water given to the animals. For a solid formulation, the formulation may be for example as a granule, spray dried powder or agglomerate. The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).

In an embodiment, the solid composition is in granulated form. The granule may have a matrix structure where the components are mixed homogeneously. However, the granule typically comprises a core particle and one or more coatings, which typically are salt and/or wax coatings. The core particle can either be a homogeneous blend of the enzyme(s) of the invention optionally combined with one or more additional enzymes and optionally together with one or more salts or an inert particle with the enzyme(s) of the invention optionally combined with one or more additional enzymes applied onto it.

In an embodiment, the material of the core particles are selected from the group consisting of inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals. The salt coating is typically at least 1 μιτι thick and can either be one particular salt or a mixture of salts, such as Na2SO ₄ , K ₂ SO ₄ , MgSO ₄ and/or sodium citrate. Other examples are those described in e.g. WO 2008/017659, WO 2006/034710, WO 1997/05245, WO 1998/54980, WO 1998/55599, WO 2000/70034 or polymer coating such as described in WO 2001/00042. In another embodiment, the composition is a solid composition comprising the en- zyme(s) of the invention and one or more formulating agents selected from the list consisting of sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium car- bonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate. In a preferred embodiment, the solid composition is in granulated form. In an embodiment, the solid composition is in granulated form and comprises a core particle, an enzyme layer comprising the enzyme(s) of the invention and a salt coating.

In a further embodiment, the formulating agent is selected from one or more of the following compounds: glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3- propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate. The incorporation of the fish feed composition containing the active compound(s) into the fish feed may be performed as described in example 1 . The mixture of active compounds is then prepared directly as an oil which is then mixed with the oil sprayed onto the feed pellets as described in example 1 .

The incorporation of the fish feed composition containing the active compound(s) into the fish feed may alternatively be carried out by preparing a premix of the active ingredients and other suitable additives. Such a premix may comprise 2-10 % by weight of the active mixture or natural substance or extract, 0-40 % by weight of other conventional additives, such as flavorings, and 50-98 % by weight of any conventional absorbing support. The support may contain, for example, 40-50 % by weight of wood fibers, 8-10 % by weight of stearin, 4-5 % by weight of curcuma powder, 4-5 % by weight of rosemary powder, 22-28 % by weight of limestone, 1 -3 % by weight of a gum, such as gum Arabic, 5-50 % by weight of sugar and/or starch and 5-15 % by weight of water. This premix is then mixed with vitamins, as for example vitamin C, mineral salts and other feed additive ingredients, as for example yeast extracts containing nucleotides, glucan and other gut microflora modulators and then finally added to the feed in such quantities that the feed will contain 10-5000 ppm, preferably 100- 1000 ppm or 100-500 ppm of the active ingredients according to the invention.

Moreover, the composition of the present invention will be preferably used together with yeast extract containing nucleotides, and glucan.

Further, optional, feed-additive ingredients are coloring agents, e.g. carotenoids such as beta-carotene, astaxanthin, and lutein; aroma compounds; stabilisers; antimicrobial peptides; polyunsaturated fatty acids; and/or at least one enzyme selected from amongst phytase (EC 3.1 .3.8 or 3.1 .3.26); xylanase (EC 3.2.1 .8); galactanase (EC 3.2.1 .89); alpha-galactosidase (EC 3.2.1 .22); protease (EC 3.4.), phospholipase A1 (EC 3.1 .1 .32); phospholipase A2 (EC 3.1 .1 .4);

lysophospholipase (EC 3.1 .1 .5); phospholipase C (EC 3.1 .4.3); phospholipase D (EC 3.1 .4.4); amylase such as, for example, alpha-amylase (EC 3.2.1 .1 ); and/or beta-glucanase (EC 3.2.1 .4 or EC 3.2.1 .6).

Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. The fish feed as described herein has a proximate composition of 20-60 wt.-% protein, and 1 -45 wt.-% moisture and lipid.

In some specific examples, the fish feed comprises one or more of sources of:

- protein, carbohydrate and lipid (for example, fish meal, fish oil, blood meal, feather meal, poultry meal, chicken meal and/or other types of meal produced from other slaughterhouse waste),

- animal fat (for example poultry oil),

- vegetable meal (e.g. soya meal, lupin meal, pea meal, bean meal, rape meal and/or sunflower meal),

- vegetable oil (e.g. rapeseed oil, soya oil),

- gluten (e.g. wheat gluten or corn gluten) and

- added amino acids (e.g. lysine)

The term "fish feed" as used herein includes a fish feed composition according to the invention and components as described above. Typically, fish feed includes fish meal as a component. Suitably, fish feed is in the form of flakes or pellets, for example extruded pellets.

In a third aspect, the invention relates to a feed composition for aquatic animals and to the use of this composition for feeding fish. The feed is particularly suitable for feeding salmonids, including Atlantic salmon (Salmo salar), other salmon species and trout, and non-salmonids such as cod, sea bass, sea bream and eel. However, it can be fed to all types of fish, for example turbot, halibut, yellow tail and tuna.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1 : Preparation of Pressed Fish Feed

The main raw materials are ground and mixed. Microingredients are then added to the mixer and the homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. This starts a cooking process in the starch fraction (the binding component). The mass is fed into a pellet mill. The mass is forced through the mill's die and the strings are broken into pellets on the outside of the die. The moisture content is low and drying of the feed is not necessary.

Additional oil including a fish feed composition according to the present invention is then sprayed onto the surface of pellets, but as the pellets are rather compact, the total lipid content rarely exceeds 24 %. The added oil may be fish oil or vegetable oils, for example rape seed oil or soy oil, or a mixture of vegetable oils or a mixture of fish oil and vegetable oils. After oil coating, the pellets are cooled in a cooler and bagged. The final pressed fish feed contains 10 to 5000 ppm of the composition as described in the invention. Example 2: Method for Preparation of Extruded Fish Feed

The main raw materials are ground and mixed. Micro ingredients incl. a fish feed composition according to the invention are added to the mixer. The homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. Additional oil may also be added to the mass at this stage. This starts a cooking process in the starch fraction (the binding component). The mass is fed into an extruder. The extruder may be of the single screw or the twin-screw type. Due to the rotational movement of the mass in the extruder, the mass is further mixed. Additional oil, water and steam may be added to the mass in the extruder. At the end of the extruder, the mass has a temperature above 100 °C and a pressure above ambient pressure. The mass is forced through the openings in the extruder's die plate. Due to the relief in temperature and pressure, some of the moisture will evaporate immediately (flash off) and the extruded mass becomes porous. The strings are cut into pellets by a rotating knife. The water content is rather high (18-28 %) and the pellets are therefore immediately dried to

approximately 10 % water content in a dryer.

After the dryer, more oil may be added to the feed by spraying oil onto the surface of the feed, or by dipping the feed in oil. It is advantageous to add the oil to the feed in a closed vessel where the air pressure is below ambient (vacuum coating) so that the porous feed pellets absorb more oil. Feed containing more than 40 % lipid may be produced this way. After the coater, the feed is cooled and bagged. Oil may be added at several places in the process as explained above, and may be fish oil or vegetable oils, by example rape seed oil or soy oil, or a mixture of vegetable oils or a mixture of fish oil and vegetable oils.

Fish need protein, fat, minerals and vitamins in order to grow and to be in good health. The diet of carnivorous fish is particularly important. Originally in the farming of carnivorous fish, whole fish or ground fish were used to meet the nutritional requirements of the farmed fish. Ground fish mixed with dry raw materials of various kinds, such as fish meal and starch, was termed soft or semi- moist feed. As farming became industrialized, soft or semi-moist feed was replaced by pressed dry feed. This was itself gradually replaced by extruded dry feed.

Today, extruded feed is nearly universal in the farming of a number of fish species such as various types of salmonid, cod, sea bass and sea bream.

The dominant protein source in dry feed for fish has been fish meal of different qualities. Other animal protein sources are also used for dry fish feed. Thus, it is known to use blood meal, bone meal, feather meal and other types of meal produced from other slaughterhouse waste, for example chicken meal. These are typically cheaper than fish meal and fish oil. However, in some geographic regions, there has been a prohibition against using such raw materials in the production of feeds for food-producing animals and fish.

It is also known to use vegetable protein such as wheat gluten, maize (corn) gluten, soya protein, lupin meal, pea meal, bean meal, rape meal, sunflower meal and rice flour.

Example 3: Evaluation of the effect of the active compounds according to the invention on survival of Lactococcus garvieae and Streptococcus iniae.

The antimicrobial activity of the composition of the invention towards of Lactococcus garvieae and Streptococcus iniae were determined in vitro. Feeding regime:

1 . lysozyme alone for 48h

2. lysozyme for 24 hours and essential oils added on top for the next 24h

3. Lysozyme and essential oils mixed together and added for 48h

- Lysozyme: LBF 007 batch 9 PPL 38353 T.reesei at 52 g EP/kg

- Essential oil combination at 1/2:

• 50% alpha pinene

• 50% cinnamaldehyde

In the tests the following organisms, growth media, culture conditions and evaluation method were used: Bacteria: All tested pathogenic strains belong to the strain collection of the Centre for fish and wildlife health, Institute of Animal Pathology, University of Bern (Switzerland).

Determination of suitable bacteria dilution: From a 24 hour old subculture of bacteria on blood sheep agar (Biomerieux, Geneva) a small amount was trans- ferred to sterile NaCI until a McFarland value of 0.5 was obtained. From this solution 3, 1 .5 and 0.75 % dilutions in TSB were made on 96 well plates with round bottoms. Each well received a total volume of 100 μΙ. After 24 hours at 22 °C the growth of bacteria was assessed. The dilution resulting in a well demarcated spot covering half of the round bottom well was selected for the experiments. Determination of solvent effect: To solve test substances in TSB agar, alcohol (ETOH) was used. To determine a possible effect of alcohol, the calculated final concentrations of alcohol in the test wells (0.1 , 0.05 and 0.025 %) were tested with different concentrations of bacteria, and did not show any effect on bacterial growth.

Concentration of test substances: The in vitro dose range was estimated considering a probable dietary concentration of at least 1000 ppm and a daily feeding rate of 2 %. The potential concentration in the gut was established at maximum 0.1 μΙ/100 μΙ. A serial 2 dilution was then tested leading to final concentrations of the substance of 0.85 μg ml; 0.42 μg ml and 0.21 μg ml when adjusted to average essential oil density.

From each substance a stock solution consisting of 2 μΙ substance, 18 μΙ ETOH and 180 μΙ PBS was prepared.

Preparation of Plates: Triplicates of three concentrations (0.21 , 0.42 and 0.85 μg ml) of each substance were tested. On each plate a positive control consisting of bacteria in TSB and a blank control (PBS) was included. Further Triplicates of three dilutions of ETOH were also included on each plate.

Reading of plates: After an incubation of 24 hours at 22 °C the plates were read using a score of 0 (no bacterial growth = no dot on the bottom of the well) to 3 (normal growth = size of dot comparable to dot of positive control).

The following results were obtained and are summarized in Figures 1 and 2.