AU2010272494B2 - Use of natural substances as feed additives for aquatic animals - Google Patents

Abstract

The present invention relates to the use of natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in the manufacture of a feed composition for aquatic animals, 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 a substance as defined above for the improvement of the feed conversion ratio and/or daily weight gain in fish, for regulating the micro flora of the gut and for protecting the animal against infections caused by pathogenic microorganisms.

Description

1 USE OF NATURAL SUBSTANCES AS FEED ADDITIVES FOR AQUATIC ANIMALS The present invention relates to the use of natural active substances selected from 5 the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, 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. 10 More particular, this invention relates to the use of at least two substances as defined above 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 microorganisms. 15 Furthermore, the present invention relates to a novel fish feed composition comprising as active ingredients at least two, preferably at least three or four active compound(s) selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene. 20 The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal. The discussion of documents, acts, materials, devices, articles and the like is included 25 in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. 30 Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the C:\poi\word\SPEC-932961.docx 2 presence of one or more other features, integers, steps or components, or group thereof. One important factor in aquaculture is the turnover rate. Turnover rate is determined 5 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. 10 It is generally described 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 15 causing wrecked fish pens. For other farm animals it is well described 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, 20 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 25 animal feed is described in the literature. For example, patent W02004/091307 describes the use of polyphenols, and other natural actives in feed to increase survival rate of Artemia after hatching. W02004/091307 is however silent with regard to the selection of compounds to be used. Moreover, the application of polyphenols to reduce mortality is in the disclosed case above only useful at time of hatching. 30 It therefore remains a need in aquaculture to prevent the development of diseases, thereby reducing mortality by any prophylactic means including antimicrobial activity at the gut level. C:\poi\word\SPEC-932961.docx 3 The inventors of the present application surprisingly found that 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 substances, which are hereinafter 5 also referred to as compounds, have also antimicrobial activity 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. 10 Therefore, in a first particular embodiment, the invention relates to methods for using at least two active compounds selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene 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 15 preventing diseases caused by pathogenic microorganisms. For example, it has been shown that selected compounds of the invention (eg.: cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent effects inhibiting the growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric Redmouth (ERM), a disease found especially in salmonids and causing high 20 mortality. In another embodiment, the present invention provides alpha-pinene, and cinnamaldehyde, in a feed composition for use in a treatment for improving feed conversion ratio and/or daily weight gain in aquatic animals. 25 In yet another embodiment, the present invention provides alpha-pinene, and cinnamaldehyde, in a feed composition for use in a treatment for regulating the micro flora of the gut in aquatic animals. 30 In a further embodiment, the present invention provides alpha-pinene, and cinnamaldehyde, in a feed composition for use in a treatment for reducing mortality in aquatic animals. C:\poi\word\SPEC-932961.docx 3a In another embodiment, the present invention provides alpha-pinene, and cinnamaldehyde, for use in the treatment and prevention of diseases caused by pathogenic microorganisms in aquatic animals. 5 In yet another embodiment, the present invention provides a feed composition or a premix composition, or a feed additive for aquatic animals thereof, comprising alpha pinene and cinnamaldehyde, as main ingredients. In yet another embodiment, the present invention provides a combination of alpha 10 pinene and cinnamaldehyde in a feed composition when used in a treatment for improving feed conversion ratio and/or daily weight gain in aquatic animals. In alternative embodiments, alpha-pinene and/or alpha-terpineol and/or cinnamaldehyde and/or dihydroeugenol and/or eugenol and/or meta-cresol and/or 15 terpinolene is/are used 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 20 terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene 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. 25 In a preferred embodiment, alpha-pinene and cinnamaldehyde are used to improve animal feed digestibility and/or maintain animal health by supporting immune system function.

WO 2011/006993 PCT/EP2010/060325 -4 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. 5 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 %. 10 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. 15 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 20 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. 25 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 30 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 WO 2011/006993 PCT/EP2010/060325 -5 untreated group by at least 5 %, preferably at least, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, or at least 50 %. In particular, the inventors of the present application surprisingly found that the 5 compounds according to the invention and mixtures thereof are effective against a number pathogenic microorganisms of cold and warm water fish. Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, meta-cresol and terpinolene were shown to exhibit inhibitory effect against Vibrio anguillarum, a shrimp patho gen causing vibriosis. 10 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. 15 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 20 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 0C in summer months. 25 Cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent in hibitory 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: 30 (i) Vibrio salmonicida which is a psychrophilic bacterium that is the causative agent of cold-water vibriosis in Atlantic salmon.

WO 2011/006993 PCT/EP2010/060325 -6 (ii) Aeromonas hydrophila causing ulcers and hemorrhagic septicae mia. This pathogen is very resistant to conventional simple antim icrobials like chlorine. (iii) Photobacterium damselae formerly Pasteurella piscicida: a pathogen 5 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. (iv) Streptococcus iniae which is highly pathogenic in marine fish and is highly lethal: outbreaks may be associated with 30-50 % mortality. 10 Other aquatic pathogens such as (i) Piscirickettsia salmonis the causative agent of piscirickettsiosis or salmonid rickettsial septicaemia (SRS), (ii) Vibrio viscosus recently renamed Moritella viscosa etiologically re 15 sponsible for the disease referred to as "winter ulcer", (iii) Ich (parasite) one of the most prevalent protozoan parasites of fish, (iv) 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. 20 In a second aspect, the present invention provides a fish feed composition com prising at least two active compound 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 25 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 30 mixtures thereof, in the form of natural available extracts or extract-mixtures or in the form of a natural substance.

WO 2011/006993 PCT/EP2010/060325 -7 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. 5 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. 10 To the active 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 compounds as specified above is preferred, cinnamal 15 dehyde and/or meta-cresol are used as a major component of the mixture. Suita bly the mixture contains 10-90 % by weight of meta-cresol and/or cinnamalde hyde, 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 components. The total amount of these active ingredients may vary within wide limits but is fi 20 nally 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 compounds as specified above com prises, cinnamaldehyde and alpha-pinene as a major component of the mixture. 25 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 di hydro-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 between 30 100 and 1000 ppm, calculated on the dry weight of the fish feed. All compounds defined herein above (active compounds and additional ingredi ents) may be used in combination with an emulsifying surfactant. The emulsifying WO 2011/006993 PCT/EP2010/060325 -8 agent can be selected advantageously from those of a rather hydrophilic nature, for example among polyglycerol esters of fatty acids such as esterified ricinoleic acid or propylene glycol esters of fatty acids, saccharo-esters or saccharo glycerides, polyethylene glycol, lecithins etc. 5 In a preferred embodiment of the invention, a mixture of active compounds 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. 10 In another preferred embodiment of the invention, a mixture of 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.-% fur furyl alcohol. 15 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 ac tive 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. 20 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 ac tive 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 25 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, 30 such as gum Arabic, 5-50 % by weight of sugar and/or starch and 5-15 % by weight of water.

WO 2011/006993 PCT/EP2010/060325 -9 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 nu cleotides, 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 5 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 to gether with yeast extract containing nucleotides, and glucan. Further, optional, feed-additive ingredients are coloring agents, e.g. carotenoids 10 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 Al (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); 15 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 20 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. 25 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), 30 - 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), WO 2011/006993 PCT/EP2010/060325 - 10 - 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 5 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 10 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 spe cies 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. 15 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 20 the invention in addition to those shown and described herein will become appar ent 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 25 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 30 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.

WO 2011/006993 PCT/EP2010/060325 - 11 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 5 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 10 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 15 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 20 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 25 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) 30 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 WO 2011/006993 PCT/EP2010/060325 - 12 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 5 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 10 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. 15 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 20 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) 25 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 Yersinia ruckeri, Vibrio anguillarum, Vibrio sal 30 monicida, Aeromonas salmonicida, Aeromonas hydrophila, Edwardsellia tarda, Photobacterium damselae, Lactococcus garvieae, Streptococcus iniae.

WO 2011/006993 PCT/EP2010/060325 - 13 The antimicrobial activity of the composition of the invention towards Yersinia ruckeri, Vibrio anguillarum, Vibrio salmonicida, Aeromonas salmonicida, Aeromo nas hydrophila, Edwardsellia tarda, Photobacterium damselae, Lactococcus gar vieae, and Streptococcus iniae were determined in vitro. 5 In the tests the following organisms, growth media, culture conditions and evalua tion method were used: Bacteria: All tested pathogenic strains belong to the strain collection of the Cen 10 tre 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 15 ferred to sterile NaCl until a McFarland value of 0.5 was obtained. From this solu tion 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 pl. After 24 hours at 22 0C 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. 20 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 25 growth. Concentration of test substances: The in vitro dose range was estimated con sidering 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 30 0.1 pl/100 pl. A serial 2 dilution was then tested leading to final concentrations of the substance of 0.85 pg/ml; 0.42 pg/ml and 0.21 pg/ml when adjusted to average essential oil density.

WO 2011/006993 PCT/EP2010/060325 - 14 From each substance a stock solution consisting of 2 pl substance, 18 pl ETOH and 180 pl PBS was prepared. Preparation of Plates: Triplicates of three concentrations (0.21, 0.42 and 0.85 5 pg/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 0C the plates were read 10 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 Table 1: Suitable concentration of bacteria for test: A concentration of 1.5 % of 0.5 15 McFarland value resulted in a clearly visible spot covering 2/3 of a round bottom well. This concentration was therefore considered as suitable for the tests. Effect of ETOH on bacterial growth: No effect of any of the applied alcohol con centrations (1 %, 0.5 % and 0.05 %) could be found. 20 Effect of tested substances: The results are summarized in Table 1. No major differences within triplicates nor between triplicates on different plates could be seen. All positive controls showed clearly visible growth of bacteria within 48 hours. As expected from the preliminary trials no negative effect of alcohol could 25 be determined. Broadest spectrum effects on the bacteria were found with cinna maldehyde, and alpha-pinene followed by meta-cresol and dihydro-eugenol. Table 1: Effect of different substances on growth of Yersinia ruckeri, Vibrio anguil larum, Vibrio salmonicida, Aeromonas salmonicida, Aeromonas hydrophila, Ed 30 wardsellia tarda, Photobacterium damselae, Lactococcus garvieae, and Strepto coccus iniae after 48 hours incubation at 22 C. Values represent means of 5 indi vidual triplicates. nd: not determined, 3: no effect, 0: maximum growth inhibition.

WO 2011/006993 PCT/EP2010/060325 - 15 Table 1: alpha-pinene 1 nd 0 nd 0 nd 0 nd 0.22 nd alpha-terpineol 3 nd 0 nd 1.44 nd 0 nd 0.33 nd cinnamaldehyde0 0.02 0 0.02 0 0 0 0 0 0 dihydro-eugenol0 nd 0 nd 0 nd 0 nd 0 nd LO eugenol 0 nd .0.56 nd 10 nd .0 nd 10.56 Ind 6 limonene 3 nd 1 nd 0 nd 0 nd 1 nd meta-cresol 0 0.02 0 0.02 0 0 0 0 1 0 terpinolene -3 nd 0 nd 1.44 nd 0 nd 0.33 nd alpha-pinene -3 nd .0 nd 0 nd .0 nd .2.67 nd alpha-terpineol 3 nd 0 nd 3 nd 0 nd 2.67 nd cinnamaldehyde0.27 2.33 0 0.02 0 0 0 0 2.93 0.27 0 dihydro-eugenol3 nd 1 nd 1.33 nd 2.6 nd 1 nd CN eugenol 2.89 nd 1.67 nd 0.78 nd 1.9 nd 1.67 nd limonene 3 nd 2.67 nd 0.67 nd 0.1 nd 2.67 nd meta-cresol 2.13 0.73 0.6 0.02 0 0 0 0 3 1.13 terpinolene 3 nd 0 nd 3 nd 0 nd 2.67 nd alpha-pinene 3 Ind 0 nd 10 nd .0 nd .3 nd alpha-terpineol 3 nd 3 nd 3 nd 0.11 nd 3 nd cinnamaldehyde2.8 3 0.2 0.02 0.73 0 0.47 0 3 3 0 dihydro-eugenol3 nd 2 nd 2.56 nd 3 nd 2 nd eugenol 3 nd 3 nd 2 nd 3 nd 3 nd 6 limonene 3 Ind 13 Ind 12 Ind 11.44 Ind 13 Ind meta-cresol 3 1 1.53 0.2 10.13 0O 11.93 1.4 13 13 terpinolene 3 nd .3 Ind 13 Ind 10.11 Ind 13 Ind Example 4: Evaluation of the effect of the active compounds according to the invention on survival of Vibrio anguillarum, and Aeromonas salmonicida. 5 The effect of combinations of different substances was tested for growth inhibition of Aeromonas salmonicida and Vibrio anguillarum after 48 hours of incubation at 22 OC. The conditions of the tests were as described in example 3 and concentra tions tested were (0.21 pg/ml, 0.42pg/ml and 0.85pg/ml).Values of Table 2 and 3 WO 2011/006993 PCT/EP2010/060325 - 16 represent the mean of 3 individual triplicates on three different plates for Aeromo nas salmonicida and Vibrio anguillarum, respectively. 3: means no effect, 0: means maximum growth inhibition. 5 The substances tested either alone or in combinations are meta-cresol, cinnamal dehyde, alpha-pinene, dihydroeugenol. Mixtures of the substances were prepared in proportions 1:1 for two substances, 1:1:1 for three substances and 1:1:1:1 for the four substances. io Results: All compounds and combinations of compounds have shown some in hibitory effect against the two pathogens tested at the highest concentration, and a clear dose dependence of the effects against the two bacterium species was observed and is shown in Table 2 and Table 3. 15 WO 2011/006993 PCT/EP2010/060325 - 17 Table 2: Results of inhibition assay of Aeromonas salmonicida Product Concentrations 0.85 pg/ml 0.42 pg/ml 0.21 pg/ml meta-cresol 0.02 0.02 0.33 cinnamaldehyde 0.02 0.02 0.33 alpha-pinene 0.02 0.22 2 dihydroeugenol 0.02 1.33 3 meta-cresol / cinnamaldehyde 1:1 0.02 0.22 2 meta-cresol / alpha-pinene 1:1 0.02 0.22 1.67 meta-cresol / dihydroeugenol 1:1 0.02 1.11 2.33 cinnamaldehyde / alpha-pinene 1:1 0.02 0.11 1.78 cinnamaldehyde / dihydroeugenol 1:1 0.02 0.89 3 alpha-pinene / dihydroeugenol 1:1 0.02 1.78 3 meta-cresol / cinnamaldehyde / alpha-pinene 0.02 0.33 2 1:1:1 meta-cresol / cinnamaldehyde / dihydroeugenol 0.02 0.67 2 1:1:1 meta-cresol / alpha-pinene / dihydroeugenol 0.02 0.89 2.33 1:1:1 cinnamaldehyde / alpha-pinene / dihydroeugenol 0.02 1.22 2.44 1:1:1 meta-cresol / cinnamaldehyde / alpha-pinene / 0.02 1.33 2.33 dihydroeugenol 1:1:1:1 ETOH 3 3 3 Positive control 3 Negative control 0.02 WO 2011/006993 PCT/EP2010/060325 - 18 Table 3: Inhibition of Vibrio anguillarum Product Concentrations 0.85 pg/ml 0.42 pg/ml 0.21 pg/ml meta-cresol 0.02 2.67 3 cinnamaldehyde 0.02 0.02 1 alpha-pinene 0.02 0.02 0.02 dihydroeugenol 0.02 1.56 2.67 meta-cresol / cinnamaldehyde 1:1 0.02 0.89 2.67 meta-cresol / alpha-pinene 1:1 0.02 0.02 2.67 meta-cresol / dihydroeugenol 1:1 0.02 2.22 3 cinnamaldehyde / alpha-pinene 1:1 0.02 0.02 0.02 cinnamaldehyde / dihydroeugenol 1:1 0.02 0.11 2.89 alpha-pinene / dihydroeugenol 1:1 0.02 0.02 1.78 meta-cresol / cinnamaldehyde / alpha-pinene 0.02 0.02 2.78 1:1:1 meta-cresol / cinnamaldehyde / dihydroeugenol 0.02 2.33 3 1:1:1 meta-cresol / alpha-pinene / dihydroeugenol 0.02 2.33 2.89 1:1:1 cinnamaldehyde / alpha-pinene / dihydroeugenol 0.02 0.67 3 1:1:1 meta-cresol / cinnamaldehyde / alpha-pinene / 0.02 2.22 3 dihydroeugenol 1:1:1:1 ETOH 3 3 3 Positive control 3 Negative control 0.02 Example 5: Effect of a combination of essential oils comprising alpha 5 pinene, cinnamaldehyde, dihydroeugenol and meta-cresol on the mortality of rainbow trout challenged with Aeromonas salmonicida. A challenge experiment with Aeromonas salmonicida has been set up with juve nile rainbow trout in order to test the effect of three doses of a combination of four 10 essential oils supplemented to the feed. Fish: 600 young of the year rainbow trout were obtained from a commercial fish farm in Switzerland.

WO 2011/006993 PCT/EP2010/060325 - 19 Fish rearing conditions: For the acclimatization period and the experimental feeding period before infection, fish from the same dietary treatment are kept in 130 I glass aquaria equipped with a tap water flow through system and constant 5 aeration. Water temperature is maintained at 17 ± 1.0 *C. At the time of infection, fish from each treatment are transferred into four 38 1 glass aquaria equipped with a flow through system with tap water and constant aeration. Water temperature is maintained at 17.5 ± 1.0 *C. 10 Diets and feeding: One basal diet for juvenile rainbow trout was produced in 4 mm diameter. From these pellets, crumbles adapted to the size of the fish were produced and ca 5-kg batch of feed were produced by coating the essential oil combination at the different doses mixed with the oil onto the crumbles. Coating 15 was performed using a peristaltic pump and a concrete mixer to insure homoge nous distribution of the products. Fish are fed daily at a rate of 3.3 % of the body weight. The diet rate was regularly adjusted to the weight gain of fish. 20 The four experimental diets were produced: - Diet A: a control diet not supplemented with essential oil combination - Diet B: basal diet supplemented with 500 ppm of the essential oil combination - Diet C basal diet supplemented with 2000 ppm of the essential oil combination 25 - Diet D: basal diet supplemented with 4000 ppm of the essential oil combination The essential oil combination was the following one: alpha-pinene, cinnamalde hyde, dihydroeugenol and meta-cresol at equivalent doses. The experimental di ets were fed for four weeks prior to challenge and after challenge until the end of 30 the experiment. Fish weight was recorded at the start of experimental feeding, be fore challenge and at the end of the experiment on the remaining fish.

WO 2011/006993 PCT/EP2010/060325 - 20 Challenge experiment: The challenge dose was determined in a preliminary ex periment, on the same fish population and under the same conditions. After a pe riod of 4 weeks of experimental feeding, 25 fish were randomly distributed into four 38 1 glass aquaria. Fish from each experimental tank were then anaesthetized 5 and challenged by i.p. injection of Aeromonas salmonicida at a dose of 1.2*102 cfu/fish determined by spectrophotometry. The daily mortality was then recorded over a period of three weeks. First dead fish from each glass aquarium were sub jected to a bacteriological investigation to confirm the bacteriological aetiology causing death. At the end of the experiment, all surviving fish were assessed for 10 external signs of infection and in addition from 6 fish per diet group a necropsy and bacteriology were performed. Results of cumulative mortality are presented in Table 4: 15 Table 4: Mean + SD of cumulative mortality in relation to essential oil combi nation dose challe Control 500 ppm 2000 ppm 4000 ppm Mean Stdev Mean Stdev Mean Stdev Mean Stdev 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 4 7.0 2.0 8.0 7.3 2.0 2.3 0.0 0.0 5 14.0 7.7 15.1 6.8 10.0 9.5 6.0 6.9 6 16.0 8.6 22.2 11.5 11.0 10.5 7.0 6.8 7 16.0 8.6 23.3 11.5 11.0 10.5 8.0 6.5 8 17.0 8.9 23.3 11.5 12.0 11.8 9.0 8.2 9 20.0 11.3 23.3 11.5 12.0 11.8 10.0 10.1 10 20.0 11.3 23.3 11.5 12.0 11.8 10.0 10.1 11 20.0 11.3 23.3 11.5 12.0 11.8 12.0 13.9 12 20.0 11.3 23.3 11.5 12.0 11.8 12.0 13.9 13 21.0 12.4 23.3 11.5 12.0 11.8 14.0 17.7 14 21.0 12.4 23.3 11.5 12.0 11.8 14.0 17.7 WO 2011/006993 PCT/EP2010/060325 -21 15 21.0 12.4 23.3 11.5 12.0 11.8 15.0 19.7 16 21.0 12.4 23.3 11.5 12.0 11.8 15.0 19.7 17 21.0 12.4 23.3 11.5 12.0 11.8 15.0 19.7 18 21.0 12.4 23.3 11.5 12.0 11.8 15.0 19.7 19 21.0 12.4 23.3 11.5 12.0 11.8 15.0 19.7 Results: Feed intake was normal and fish grew from 4 to ca 14 g during the ex periment. 5 The results reveal a dose dependent effect of essential oils in rainbow trout chal lenged with A. salmonicida. Lowest mean values were found in the two groups re ceiving the higher doses, while in the group receiving the lowest dose a slightly higher cumulative mortality than in the control was found. These results suggest that the mixture of 4 essential oils in the diet have a beneficial effect on rainbow 10 trout infected with A. salmonicida. Differences were found in cumulative mortality and in the presence of bacteria in surviving fish at the end of the experiment. Example 6: Effect of an essential oil combination comprising alpha-pinene and cinnamaldehyde on the mortality of rainbow trout challenged with 15 Aeromonas salmonicida. Aim: A challenge experiment with Aeromonas salmonicida has been set up with juvenile rainbow trout in order to test the effect of alpha-pinene and cinnamalde hyde supplemented to the feed. 20 Fish: 600 young of the year rainbow trout were obtained from DSM Nutritional Products Fish rearing conditions: For the acclimatization period and the experimental 25 feeding period before infection, fish from the same dietary treatment are kept in 130 1 glass aquaria equipped with a tap water flow through system and constant aeration. Water temperature is maintained at 14.5 ± 1.0 *C.

WO 2011/006993 PCT/EP2010/060325 - 22 At the time of infection, fish from each treatment are transferred into four 38 1 glass aquaria equipped with a flow through system with tap water and constant aeration. Water temperature is maintained at 13.5 ± 1.0 *C. 5 Diets and feeding: One basal diet for juvenile rainbow trout was produced in 4 mm diameter. From these pellets, crumbles adapted to the size of the fish were produced and ca 5-kg batch of feed were produced by coating the essential oil combination at the different doses mixed with the oil onto the crumbles. Coating was performed using a peristaltic pump and a concrete mixer to insure homoge 10 nous distribution of the products. Fish are fed daily at a rate of 3.3 % of the body weight. The diet rate was regularly adjusted to the weight gain of fish. 15 Three experimental diets were produced: - Diet A: a control diet not supplemented with essential oil combination - Diet B: basal diet supplemented with 4000 ppm of alpha-pinene - Diet C: basal diet supplemented with 4000 ppm of alpha-pinene + cinnamalde hyde at equal doses 20 The experimental diets were fed for four weeks prior to challenge and after chal lenge until the end of the experiment. Fish weight was recorded at the start of experimental feeding, before challenge 25 and at the end of the experiment on the remaining fish. Challenge experiment: The challenge dose was determined in a preliminary ex periment, on the same fish population and under the same conditions. After a pe riod of 4 weeks of experimental feeding, 25 fish were randomly distributed into 30 four 38 1 glass aquaria. Fish from each experimental tank were then anaesthetized and challenged by i.p. injection of Aeromonas salmonicida at a dose of 5*103 cfu/fish determined by spectrophotometry. The daily mortality was then recorded over a period of three weeks. First dead fish from each glass aquarium were sub- WO 2011/006993 PCT/EP2010/060325 - 23 jected to a bacteriological investigation to confirm the bacteriological aetiology causing death. At the end of the experiment, all surviving fish were assessed for external signs of infection and in addition from 6 fish per diet group a necropsy and bacteriology were performed. 5 Results: Feed intake was normal and fish grew from 1 to ca 8 g during the ex periment. The results reveal no effect of alpha-pinene alone but there is a positive effect of 10 the combination of alpha-pinene + cinnamaldehyde in reducing mortality by 15 % against the control treatment. Results of cumulative mortality are presented in Table 5: Table 5: Mean + SD of cumulative mortality in relation to essential oil combi 15 nation Day after Alpha-pinene Alpha-pinene + cin challenge Control (0 p namaldehyde (2000 ppm each) Mean Stdev Mean Stdev Mean Stdev 0 0 0 0 0 0 0 1 0 0 0 0 0 0 2 0 0 0 0 0 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 5 0 0 0 0 1 2 6 4.1 5.9 5.0 3.8 2.0 2.3 7 16.2 9.9 21.1 14.3 11.0 3.8 8 25.3 8.6 37.3 11.2 22.0 5.2 9 28.3 10.0 40.3 9.5 24.0 5.7 10 30.4 12.3 41.4 8.6 25.0 6.8 11 33.4 13.5 43.4 9.1 27.0 3.8 12 33.4 13.5 43.4 9.1 27.0 3.8 13 34.4 12.7 43.4 9.1 28.0 3.3 14 34.4 12.7 43.4 9.1 29.0 3.8

Claims (10)

1. A combination of alpha-pinene and cinnamaldehyde in a feed composition when used in a treatment for improving feed conversion ratio and/or daily weight gain 5 in aquatic animals.

2. Combination according to claim 1, wherein the aquatic animal is salmon, trout, bream or bass. 10

3. Combination according to claim 1, wherein the aquatic animal is carp, tilapia or catfish.

4. Combination according to any one of claims 1 to 3, wherein the feed composition further contains dihydro-eugenol and meta-cresol. 15

5. Combination according to claim 4, wherein the feed composition contains 30 70% by weight of alpha-pinene, 30-70% by weight of cinnamaldehyde, 1-20% by weight of dihydro-eugenol and 1-20% by weight of meta-cresol, wherein the amounts being calculated on the total amount of said components. 20

6. Combination according to claim 4, wherein the feed composition contains alpha pinene, cinnamaldehyde, dihydro-eugenol and meta-cresol each in a concentration between 0.1 g and 1 g per Kg of feed. 25

7. A feed composition or a premix composition, or a feed additive for aquatic animals thereof, comprising alpha-pinene and cinnamaldehyde, as main ingredients.

8. A method of treating and preventing diseases caused by pathogenic 30 microorganisms in aquatic animals comprising administering a therapeutically effective amount of the feed composition according to claim 7.

9. The combination according to claim 1, substantially as hereinbefore described with reference to the Examples. 25

10. The feed composition according to claim 7, substantially as hereinbefore described, with reference to the Examples.