PALATABILITY ENHANCERS

The present invention relates to the field of fish feed.

More precisely, the invention concerns a method for determining a coefficient of enhancement (K _e ) for standardizing palatability of fish feed pellets when coating said pellets with a palatability enhancer useful in fish feed.

Using said coefficient of enhancement K _e , the invention provides a method for standardizing palatability of fish feed pellets for use in aquaculture, a method for preparing coated fish feed pellets having standardized palatability, and a method for enhancing growth of fish in aquaculture.

BACKGROUND OF THE INVENTION

Farmers and manufacturers working in the aquaculture field are facing a major concern due to the fact that availability of marine raw materials is expected to decrease in the coming years when aquaculture is expanded more and more. In particular, availability on the world market of fish meal and fish oil - which were used to serve as the dominant protein source in dry feed for fish - is dramatically decreasing and prices rise considerably for these raw materials. As a consequence, the aquaculture industry, especially the fish feed industry, has predicted for several years that there will be a shortage of both fish meal and fish oil in the future. Alternative animal protein sources can be used for dry fish feed. For instance, it is known to use land-based raw materials (e.g., blood meal, bone meal, feather meal, and other types of meal produced from other slaughterhouse waste, for example poultry meal), as well as plant-based raw materials (e.g., soy meal, wheat meal, rapeseed meal, rice meal, and the like). These are typically cheaper and more available than fish meal and fish oil. However, in some geographic regions, including Europe, there has been a prohibition against using land-based raw materials in the production of feeds for food-producing animals and fish.

Thus, for the last 20 years, the fish feed industry has become more and more interested in finding satisfying alternatives to fish meal and fish oil to be used for preparing feed for fish farming. However, the dietary level of fish meal remains crucial for achieving feed performance, affecting both feed palatability and feed utilization. Year after year, the level of fish meal in feed designed for carnivorous fish species tends to decrease and is close to reach a critical threshold for fish growth and feed utilization. Plant- and land-based feedstuffs are now commonly proposed in fish feed formulations as an alternative to fish meal. However, balancing the dietary amino acid profile of plant-rich diets to meet the fish amino acid requirements is not efficient enough to get satisfying fish and feed performances. Feed palatability is firstly affected when substituting fish meal by plant- and land-based meals.

Unfortunately, edible ingredients dedicated to fish feed palatability are scarce. Focusing on palatability in fish and shrimp, many attractive molecules likely to be involved in olfaction and gustation could be identified. In particular, free am ino acids and nucleotides are among the most powerful palatability- enhancing compounds, and fish and squid hydrolysates or extracts appear to have a positive effect on feed attractiveness and palatability. If such special raw materials or ingredients can be used to enhance palatability of feed formulations, they also have a nutritional interest. They are thus commonly incorporated by inclusion and mixed with other raw materials for preparing palatable fish feed.

There is a need in the art for optimizing the application of a palatability- enhancing composition to fish feed in order to meet a higher palatability result at low dosage, and thus at low cost.

The feeding behaviour of fish is characterized by no feed nibbling and fast pellet ingestion. The transit time of pellets in the water is very short as they are eaten very fast by the fish when distributed. Pellets are not (or rarely) broken in the mouth of fish. A satisfying palatability enhancer needs thus to have a high contact with water to stimulate olfactory and gustatory fish receptors and to be easily sensed by the fish. The present Inventors have thus conducted a lot of trials as reported in the Examples below, to evaluate (i) the best method for applying a palatability enhancer to fish feed and (ii) the best palatability enhancer amount to be applied to the fish feed, in order to reach the highest palatability effect regardless the fish meal content of the fish feed.

As the aquaculture industry is characterized by a large quantity of fish species and feed ranges, the present invention provides a method for fitting the conditions for applying a palatability enhancer to the fish feed size.

SUMMARY OF THE INVENTION

The present invention concerns a method for determining a coefficient of enhancement (K _e ) for standardizing palatability of fish feed pellets when coating said pellets with a palatability enhancer (PE) useful in fish feed.

The present invention also relates to a method for standardizing palatability of fish feed pellets for use in aquaculture using said coefficient of enhancement K _e .

The present invention further provides a method for preparing coated fish feed pellets having standardized palatability using said coefficient of enhancement K _e .

The invention further concerns coated fish feed pellets having standardized palatability that are obtainable by the foregoing method.

Also, the present invention is related to methods for enhancing growth of fish in aquaculture.

The present invention further provides a kit useful in aquaculture comprising, in a single package, one or more PEs and one or more means for communicating information or instructions with respect to the use of said PEs for coating fish feed pellets in order to achieve standardized palatability. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Schematic representation of a typical fish trial as performed by the Inventors.

Figure 2. Influence of the method for applying the palatability enhancer (PE) on the feed intake of European seabass fed dietary treatments for 15 days.

Figure 3. Influence of the method for applying the PE on the growth of European seabass fed dietary treatments for 15 days.

Figure 4. Influence of the method for applying the PE on the feed utilization (feed conversion ratio, FCR) by European seabass fed dietary treatments for 15 days. Figure 5A. Influence of the amount of PE (Q) on the feed intake of European seabass fed dietary treatments for 15 days.

Figure 5B. Dose-response relationship between the amount of PE (Q) and the feed intake of European seabass fed dietary treatments for 15 days.

Figure 6A. Influence of the amount of PE (Q) on the growth of European seabass fed dietary treatments for 15 days.

Figure 6B. Dose-response relationship between the amount of PE (Q) and the growth rate of European seabass fed dietary treatments for 15 days.

Figure 7. Influence of the amount of PE (Q) on the feed utilization by European seabass fed dietary treatments for 15 days. Figure 8. Influence of PE coating on the feed intake of European seabass fed a fish meal free diet for 15 days - Mean, minimum and maximum responses on 27 trials. FM: fish meal.

Figure 9. Influence of PE coating on growth of European seabass fed a fish meal free diet for 15 days - Mean, minimum and maximum responses on 27 trials. FM: fish meal. Figure 10. Influence of PE coating on the feed utilization by European seabass fed a fish meal free diet for 15 days - Mean, minimum and maximum responses on 27 trials. FM: fish meal.

Figure 11. Influence of PE coating on the feed intake of European seabass fed diets containing graded amounts of fish meal for 84 days. FM: fish meal.

Figure 12. I nfluence of PE coating on growth of European seabass fed diets containing graded amounts of fish meal for 84 days. FM: fish meal.

Figure 13. Influence of PE coating on the feed utilization by European seabass fed diets containing graded amounts of fish meal for 84 days. FM: fish meal. Of note, in Figs. 2 to 13, a same letter at the top of several bars indicates a statistically non-significant difference between the results.

Figure 14. Optimal PE amount (Q _op timai) for coating feed pellets for seabass and seabream, as a function of the pellet size and based on a calculated value for K _e of 1.06 mg PE/cm ² apparent surface of the pellet. Figure 15. Optimal PE amount (Q _op timai) for coating feed pellets for salmon, as a function of the pellet size and based on a calculated value for K _e of 1 .06 mg PE/cm ² apparent surface of the pellet.

Figure 16. Optimal PE amount (Q _op timai) for coating feed pellets for turbot, as a function of the pellet size and based on a calculated value for K _e of 1.06 mg PE/cm ² apparent surface of the pellet.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

Unless specifically stated otherwise, percentages are expressed herein by weight. In the present disclosure, ranges are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.

As used throughout, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references "a", "an", and "the" are generally inclusive of the plurals of the respective terms. For example, reference to "a method" o r " a feed" includes a plurality of such "methods" or "feeds". Similarly, the words "comprise", "comprises", and "comprising" are to be interpreted inclusively. Likewise the terms "include", "including" and "or" should all be construed to be inclusive. All these terms however have to be considered as encompassing exclusive embodiments that may also be referred to using words such as "consist of.

The methods and compositions and other embodiments exemplified here are not limited to the particular methodologies, protocols, and reagents that are described herein because, as the skilled artisan will appreciate, they may vary.

Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by the skilled artisan in the field(s) of the invention, or in the field(s) where the term is used. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein.

The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, more preferably ±10%, even more preferably ±5% from the specified value, as such variations are appropriate to reproduce the disclosed methods and products.

The terms "aquaculture", "fish farming", "fish breeding", and "fish rearing" are synonymous and are used herein according to their usual equivalent meaning. Fish are generally bred in "pens", "ponds", "tanks", or "cages" of different sizes, volumes and capacities, depending on the fish species to be reared, the location of the fish farm, the financial means of the fish farmer, etc.

The term "pen" will be used below to refer to anyone of a pen, a pond, a tank, and a cage. In the context of the present invention, the term "fish" refers to any fish species that can be reared for the purposes of supplying dietary fish to the population (humans and animals, in particular, companion animals).

There are 3 distinct "groups of fish" that are defined by the nutritional requirements and feeding habits of the fish: the group of carnivorous fish, the group of omnivorous fish, and the group of herbivorous fish.

As examples of carnivorous fish species, one can cite pink salmon (Oncorhynchus gorbuscha) , chum salmon (Oncorhynchus keta), coho salmon (Oncorhynchus kisutch), masu salmon (Oncorhynchus masou), rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), Atlantic salmon (Salmo salar), sea trout (Salmo trutta), brook trout (Salvelinus fontinalis), lake trout (Salvelinus namaycush), Japanese eel (Anguilla japonica), American eel (Anguilla rostrata), European eel (Anguilla Anguilla) , largemouth black bass (Micropterus salmoides), greater amberjack (Seriola dumerili), Japanese amberjack (Seriola quinqueradiata), Japanese jack mackerel (Trachurus japonicas), dark seabream (Acanthopagrus schlegeli), white seabream (Diplodus sargus), crimson seabream (Evynnis japonica) , red seabream (Pagrus major), red porgy (Pagrus pagrus), goldlined seabream (Rhabdosargus sarba), gilthead seabream (Sparus aurata), red drum (Sciaenops ocellatus), Southern bluefin tuna (Thunnus maccoyii), Northern bluefin tuna (Thunnus thynnus) , climbing perch (Anabas testudineus), turbot (Psetta maxima), bastard halibut (Paralichthys olivaceus), common sole (Solea vulgaris), Senegalese sole (Solea senegalensis), Atlantic halibut (Hippoglossus Hippoglossus), stri ped bass (Morone saxatilis), barramundi (Lates calcarifer), Nile perch (Lates niloticus), Atlantic cod (Gadus morhua), European seabass (Dicentrarchus labrax), grouper (Epinephelus sp.) spotted coralgrouper (Plectropomus maculates), European perch (Perca fluviatilis), meagre (Argyrosomus regius), cobia (Rachycentron canadum). As examples of omnivorous fish species, one can cite barb species (Puntius spp.), black carp (Mylopharyngodon piceus), Chinese mud carp (Cirrhinus molitorella), climbing perch (Anabas testudineus), common carp (Cyprinus carpio), crucian carp (Carassius carassius), pirapatinga (Piaractus brachypomus), silver or Java barb (Barbonymus gonionotus), Indian mrigal carp (Cirrhinus mrigala), Pacu (Piaractus mesopotamicus), Tilapia species [Oreochromis spp. (niloticus and mossambicus and hybrids), Sarotherodon spp., Tilapia spp.], catfish species [Clarias spp. (gariepinus, macrocephalus, hybrids), Pangasius spp. (Pangasius hypophthalmus, Pangasius pangasius), channel catfish (Ictalurus punctatus), amur catfish (Silurus asotus), Chinese longsnout catfish (Leiocassis longirostris), yellow head catfish (Pelteobagrus fulvidraco)].

As examples of herbivorous fish species, one can cite Chinese silver carp (Hypophtalmichthys molitrix), Indian catla carp (Catla catla), Indian rohu carp (Labeo rohita), milkfish (Chanos chanos), mullet (Mugil cephalus), Chinese grass carp (Ctenopharyngodon idella), Chinese "Wuchang" bream (Megalobrama amblycephala) , large gourami (Osphronemus goramy), snakeskin gourami (Trichigaster pectoralis), some Tilapia species (Tilapia rendalli and Tilapia zillii), giant gourami (Osphronemus goramy), bighead carp (Hypophthalmichthys nobilis (filter feeder)). Preferably, a fish according to the present invention is a "carnivorous" fish.

A "model fish" herein designates a fish species which is chosen for studies as being representative of other fish species in terms of physiological needs and/or physiological responses and/or behaviour. Accordingly, a "model fish" is a fish which is chosen for studies as being representative of other fish species belonging to the same group of fish as defined above. It is thus admitted that the results observed upon studying a model fish can legitimately be transposed to other fish species. In particular, a standard model for carnivorous fish is European seabass (Dicentrarchus labrax) (Altan et al., 201 1). A model for omnivorous fish is tilapia (Oreochromis niloticus). A model for herbivorous fish is Chinese grass carp (Ctenopharyngodon idella).

By the term "standardizing" or "optimizing" the palatability of a fish feed, it is meant herein that said fish feed is equally, homogeneously, uniformly and regularly palatable, and this, all along the fish rearing cycle and whatever the size of the pellets. When using a PE for coating a fish feed in the form of pellets to enhance the palatability thereof, it is important that all the pellets are equally, homogeneously, uniformly and regularly coated with said PE. Thus, the same amount (Q) of PE should be applied to each pellet by coating. By this way, the palatability-enhancing effect will be as constant and uniform as possible for all the pellets of a same batch, all along the shelf life of the pellets and all along the fish rearing cycle. If so, the pellets are thus considered as having an enhanced palatability that is "standardized" or "optimized". I n addition, industrial production costs of such "standardized" palatable pellets can advantageously be significantly reduced.

The term "feed" as used herein means a product or composition that is intended for ingestion by a fish and provides at least one nutrient to the fish. The composition of the feed depends on the group of fish that will be fed with said feed. A feed for carnivorous fish is different from a feed for omnivorous fish and from a feed for herbivorous fish, the two latter feeds being also different from each other. Typically, fish, especially carnivorous fish , need protei n, fat, mi nerals and vitamins in order to grow and to be in good health. The term "feed" according to the present invention excludes "baits" that are only used to attract fish. Originally, in the farming of carnivorous fish, there were used whole fish or ground fish to cover the nutrient requirements of the farmed fish. Ground fish mixed with dry raw materials of various kinds, such as fish meal and starch, was termed "soft feed". Gradually, the farming became industrialized and soft feed was replaced by dry feed of the pressed feed type. The pressed feed was gradually replaced by dry feed of the extruded feed type. Today, extruded feed is nearly universal in the farming of a high number of fish species such as several species of salmonids, cod, sea bass and sea bream.

I n its original and widest sense, "extrusion" means to create an object having a fixed cross-sectional profile. This is done by pulling or forcing a formable material through a die opening having the desired cross-section. In the food and feed industries, especial ly i n the fish feed i ndustry, the term "extrusion" is commonly used in a narrower sense. In these industries, extruders of the single screw or double screw type are used. The extruded material is a mixture of protein raw materials, starch containing raw materials, fat, and water. The water may be added to the mixture in the form of water or steam . I n addition, the mixture may comprise minerals and vitamins and possibly pigment. The mixture may be preheated in a preconditioner where the heating takes place by addition of steam to the mixture. Steam and water may also be added to the substance inside the extruder. In the extruder itself, the dough-like substance is forced by means of the screws toward a constriction in the outlet end of the extruder and on through a die plate where the substance gets a desired cross-sectional shape. On the outside of the die plate is normally a rotating knife cutting the string coming out of the die holes into desired length. Normally, the pressure on the outside of the die plate will be equal to the surrounding pressure. The extruded product is generally referred to as "extrudate". Due to the pressure created inside the extruder and the addition of steam to the substance, the temperature can be above 100°C and the pressure can be above atmospheric pressure in the substance before it is forced out through the die openings. This extrusion process is also termed "cooking extrusion". Thus, by the term "extrusion" is meant herein cooking extrusion either by means of a single screw extruder or a double screw extruder. By an "extruded feed" is meant a feed produced by cooking extrusion either by means of a single screw extruder or a double screw extruder. Extruded fish feed is typically in the form of pellets. Most of, if not all, currently-available "extruded feed" contain less than about 10% water and are oil-coated.

By a "pressed feed" is meant a feed produced by means of a feed press. This process differs from extrusion in several ways. There is used less water and steam in the process. The feed mixture is forced through a die ring from the inside out by means of rollers rotating on the inside of the die ring. Temperature and pressure are lower than in extrusion, and the product is not porous. The process entails that the starch is not as digestive as after extrusion. A "pressed feed" will normally contain less than about 10% water after pressing and any oil coating. It is not necessary to dry a pressed feed. The feed is cooled prior to optional packaging.

By a "formulated fish feed" is meant a feed composed of one or more protein sources such as, but not limited to, marine protein including, inter alia, fish meal and krill meal, vegetable protein (e.g., soy meal, rapeseed meal, wheat gluten, corn gluten, lupine meal, pea meal, sunflower seed meal, and rice meal), and slaughterhouse waste such as blood meal, bone meal, feather meal, and poultry meal. By mixing different protein sources, each having its own amino acid profile, it is possible within certain limits to achieve a desired amino acid profile in the feed adapted to the group of fish, and even to the fish species, the feed is intended for.

A "formulated feed" further contains fats such as fish oil and/or plant- based oils (e.g., rapeseed oil and soy oil), and/or land-based fats (in particular, poultry fat) as energy sources. A formulated feed also contains a binder, usually in the form of a starch-containing raw material, such as wheat or wheat flour, potato flour, rice, rice flour, pea flour, beans or tapioca flour, to give the feed the desired strength and form stability.

A typical "formulated feed" further contains minerals and vitamins for taking care of good growth and good health of the fish. The feed may also contain further additives such as pigments.

A "formulated fish feed" is thus a composite feed wherein the relative amounts between proteins, fat, carbohydrates, vitamins, minerals and any other additives is calculated to be optimally adapted to the nutritional needs of the group of fish and of the fish species based on the age of the fish, the rearing method and the environmental conditions. It is common that feeding is done with only one type of feed at once and with that every piece of feed is nutritionally adequate. Thus, a "formulated fish feed" commonly has an approximate composition of 25-60 wt% protein, 5-40 wt% lipid, and 3-15 wt% moisture. By a "dry, formulated feed" is meant a feed of the pressed or extruded type.

Both a "formulated fish feed" and a "dry, formulated fish feed" may be referred to herein as a "fish feed" or, more simply, as a "feed". Of course, exact meaning of these terms will be apparent to the reader in light of the context.

The term "pellet" used herein refers to particulate chunks or pieces formed by either a press or extrusion process. The pieces can vary in sizes and/or shapes, depending on the process or the equipment. Since the fish is farmed using a fish feed product, starting with fingerlings (having the weight of about 1 g) up to large fish having a weight of several kilograms (e.g. 4 to 5 kg), various pellet sizes are required and used in the feeding at different stages of the growth of the fish. Size and/or shape of the pellets have indeed to be adapted to the size of the fish. Thus, during one cycle of fish rearing, pellets of increasing sizes are used as the fish is growing. For example, in the farming of salmon, 6 or 7 different pellet sizes are typically needed when using the existing fish feeds (see Table 1 below). Also, in seabass farming, pellets of 5 different sizes are commonly used to comply with the fish growth (see Table 2 below). The size of the pellet to be used is determined by the size of the fish in accordance with prior practice. Examples are given in Tables 1 to 3 below.

Table 1. Fish size and corresponding illustrative feed pellet size for salmon and trout

Fish weight (g) Pellet diameter/length (mm)

<5 1.5

5-10 2

10-40 3

40-100 4

100-400 6

400-1800 8

1800-3000 10 and corresponding illustrative feed pellet siz seabream

Fish weight (g) Pellet diameter/length (mm)

<10 1.5

10-40 2.5

40-100 3.5

100-400 4.5

>400 6

Table 3. Fish size and corresponding illustrative feed pellet size for turbot

Most fish feed pellets have a cylindrical shape. Thus, the "size" of a fish feed pellet, as being herein assimilated to a cylinder, is defined by a diameter (d) and a height (h) of the cylinder. Common pellets have a diameter in the range from about 0.5 mm to about 25 mm, including particular diameter values like 1.5, 2, 3, 4, 6, 9, and 12 mm. The height of a cylindrical pellet is generally about 1 to about 1 .5 times its diameter. As illustrated in the Examples below, fish feed pellets in the form of cylinders can have a same value for both the diameter (d) and the height (h) of the cylinder. Given that a fish feed pellet is herein assimilated to a cylinder, the

"apparent area (AA)" of a pellet is defined by Equation (3):

AA = 2 x π x (d/2) x h + 2 x π x (d/2) ² wherein d and h define the size of the pellet as described above. "N" is herein defined as the number of pellets contained in a batch having a given volume or a given weight (volume or weight of the total fish feed; e.g., 1 kg). In practice, for a batch of 1 kg, N can easily be determined as follows:

N = 1000 / p wherein p is the average weight of 1 pellet (in g) as determined after weighing 100 pellets individually, and N is the number of pellets contained in said batch of 1 kg.

The term "density" means herein the weight of pellets contained in 1 unit of volume. The pellet density is expressed in g/l. The terms "total area (TA) of fish feed pellets" refer to the sum of the AA of all the pellets contained in a batch having a given volume or a given weight (volume or weight of the total fish feed). For a batch consisting of a number (N) of pellets, each pellet having an apparent area AA, the "total area (TA)" of the batch of pellets is defined by Equation (4): TA = N x AA

As used herein, the terms "feed pellets" fulfil both definitions of "feed" and "pellets" above. Thus, the terms "feed pellets" refer not only to the composition and formulation of the feed but also to the physical structure, the shape, the size, and the density of the pellets. It is meant herein by the terms "model feed pellets", fish feed pellets of reference that have a defined size that can be easily determined and that are used in a method for determining a coefficient of enhancement (K _e ) as described below. Said K _e coefficient is useful for standardizing palatability of fish feed pellets when coating said pellets with a PE. In particular, model feed pellets are used in said method together with a model fish as defined above.

The term "palatability" means a preference of a fish for one feed to another. Palatability refers to the overall willingness of a fish to eat a certain feed. Advantageously but not necessarily, palatability further refers to the capacity of the eaten feed to satisfy the fish. Whenever a fish shows a preference, for example, for one of two or more feeds, the preferred feed is more "palatable" and has "enhanced palatability". The palatability of one feed compared to one or more other feeds can be determined, for example, by testing consumption of the feeds by fish. Such preference can arise from any of the fish senses, but typically is related to, inter alia, taste, smell, aroma, flavour, texture, and/or mouth feel. A fish feed stated herein to have "enhanced palatability" is one for which a fish exhibits preference compared to a control feed composition.

The terms "palatability enhancers" (PEs), "palatants", "flavours", "palatability agents", "appetizing factors", "flavour compositions", "palatability- enhancing compositions", "flavour enhancers", and any other similar terms mean any material that enhances the palatability of a feed composition to a fish. A PE may be a single material or a blend of materials, and it may be natural , processed or unprocessed, synthetic, or part of natural and part of synthetic materials. Typically, a PE useful in fish feed is an edible composition that provides a taste, smell, aroma, flavour, texture, mouth feel, and/or organoleptic sensation that is appealing or pleasing to the fish.

Examples of commercially-available PEs useful in fish feed belong to the ACTIPAL™ product line (Aquativ, SPF, France).

As used herein, a "palatability-enhancing composition ingredient" is any compound, composition or material that is suitable for fish consumption. Non- limiting examples of palatability-enhancing composition ingredients are animal meals and/or hydrolysates, fish meals and/or hydrolysates, krill meals and/or hydrolysates, crustacean meals and/or hydrolysates, mollusk meals and/or hydrolysates (including worms), feather meals and/or hydrolysates, yeasts and/or hydrolysates and/or extracts , plant hydrolysates, algae meals and/or hydrolysates and/or extracts, nitrogen compounds [e.g. , proteins, peptides, and amino acids (in particular, free amino acids), amino acid derivatives (betaine, taurine)], nucleosides, nucleotides, carbohydrates, fats, and the like. Also is encompassed by the terms "palatability-enhancing composition ingredient", any functional, active or bioactive edible ingredient that cannot undergo extrusion and thus has to be added by coating (e.g., vitamins, enzymes, pigments, etc.) The term "amino acid" means a molecule containing both an amino group and a carboxyl group. In some embodiments, the amino acids are α-, β-, γ- or δ- amino acids, including their stereoisomers and racemates.

By the term "peptide", it is meant herein a short chain of amino acids. By "short chain of amino acids", it is typically referred to a chain having from 2 to about 6 amino acids. I n particular, a "peptide" in the present context has a molecular weight (MW) equal to or less than about 1000 Da.

Since it is difficult, or even impossible, to determine the exact composition of protein hydrolysates, manufacturers rather commonly use undirect information such as the MW profile (using methods that are typically based on size-exclusion chromatography) and/or the degree of hydrolysis (DH) of peptide bonds of proteins. DH is represented by the: (i) ratio of amino nitrogen over total nitrogen (AN/AT Ratio) in the resulting hydrolysate; (ii) presence of amines in the hydrolysate; and (iii) osmolarity of the hydrolysate. Many conventional methods can be used to determine the DH. Methods used in the food and feed industries, and more specifically in the fish feed industry, for quantifying the DH are typically based on one of the following principles: (1 ) determination of soluble nitrogen in the presence of a precipitating agent such as trichloroacetic acid (TCA); or (2) determination of free alpha amino groups by colorometric methods (e.g., titration with trinitrobenzenesulfonic acid, TNBS), or pH titration of the released protons. DH may advantageously be determined by an OPA (o-phthaldialdehyde)-based method as described in Nielsen (2001). Alternatively, TCA-soluble nitrogen may be determined by the Kjeldhal assay (A.O.A.C. 1995) or the Biuret reaction (Hung et al. 1984). Such methods are described in detail in Silvestre (1997). A "nucleoside" is a compound containing a purine or pyrimidine base linked to a sugar (usually, ribose or deoxyribose), such as adenosine, inosine, uridine, guanosine, cytidine, ribothymidine, deoxyadenosine, deoxyinosine, deoxythymidine, deoxyuridine, deoxyguanosine, deoxycytidine, and the like.

A "nucleotide" is any compound consisting of a nucleoside combined with a phosphate group such as adenosine monophosphate, inosine monophosphate, thymidine monophosphate, uridine monophosphate, guanosine monophosphate, cytidine monophosphate, adenosine diphosphate, inosine diphosphate, thymidine diphosphate, uridine diphosphate, guanosine diphosphate, cytidine diphosphate, adenosine triphosphate, inosine triphosphate, thymidine triphosphate, uridine triphosphate, guanosine triphosphate, cytidine triphosphate, deoxyadenosine monophosphate, deoxyinosine monophosphate, deoxythymidine monophosphate, deoxyuridine monophosphate, deoxyguanosine monophosphate, deoxycytidine monophosphate, deoxyadenosine diphosphate, deoxyinosine diphosphate, deoxythymidine diphosphate, deoxyuridine diphosphate, deoxyguanosine diphosphate, deoxycytidine diphosphate, deoxyadenosine triphosphate, deoxyinosine triphosphate, deoxythymidine triphosphate, deoxyuridine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and the like.

Examples of lipids include tallow, oils, fats from any origin such as animal, plant (including vegetable), dairy or marine oils, all of these being indifferently herein referred to as "lipids", "fats" or "oils". Plant oils which are available in large quantities are typically canola oil, soybean oil, corn oil, olive oil, sunflower oil, rapeseed oil, linseed oil, palm oil, safflower oil, and the like, as well as byproducts thereof. Typical animal fats are tallow, lard, poultry fat, beef fat, and the like, as well as by-products thereof. Marine oils are typically tuna oil, sardine oil, salmon oil, anchovy oil, other pelagic fish oil, and the like, as well as by-products thereof. Also are encompassed herein the fats that are derived from animal, plant, marine sources, or that are produced by fish, animals and plants. Preferably, fish feed contains marine oils.

As reflected by the definitions provided above, palatability-enhancing composition ingredients include more particularly "nutrients" or "macronutrients", and "micronutrients".

Examples of "nutrients" or "macronutrients" are, without limitation, fish-, plant- and land-based raw materials, nitrogen compounds (e.g. , proteins, peptides, amino acids, especially free amino acids, amino acid derivatives), carbohydrates, fats, and the like. Examples of "micronutrients" include, without limitation, vitamins, minerals and oligoelements, such as vitam ins A, C, E, B12 , D3, folic acid , D-biotin, cyanocobalamin, niacinamide, thiamine, riboflavin, pyridoxine, menadione, beta- carotene, calcium pantothenate, choline, inositol, calcium, phosphorus, potassium, sodium, zinc, iron, manganese, copper, iodine, and the like.

In the present text, the term "nutrients" may be used to refer to nutrients or to micronutrients or to both. Of course, the exact meaning of this term will be made clear by the context to the skilled artisan.

"Preservatives" are in particular used for ensuring a long shelf life of feed, in particular of fish feed. Preservatives comprise natural or synthetic anti-oxidants (such as etoxyquine, BHA, BHT, propyl gallate, octyl gallate, tocopherols, rosemary extracts, and the like); chelatants (e.g., citric acid); as well as sorbic acid or sorbic salts (e.g., potassium sorbate), and other acids like phosphoric acid, and the like.

A "pigment" means herein any substance of natural origin or any synthetic colour that is suitable (preferably, approved or certified) for use in fish feed. Pigments are useful to, inter alia, give an appetizing colour to a fish feed and/or give an appetizing colour to the fish intended for consumption by animals and/or humans. Examples of commonly used pigments in aquaculture are carotenoids (such as canthaxanthin, astaxanthin, etc.), and the like.

A "variable" as used herein is one of the following: initial biomass, final biomass, initial fish number, final fish number, feed consumption, and dead fish weight. Those variables are illustrated in Fig. 1 .

A cycle of fish rearing includes a period of time D of feeding the fish (Fig. 1).

The "initial fish number" variable (n, in Fig. 1 ) is defined as the total number of fish that are initially contained in the pen, i.e., at the beginning of said period of time D.

The "initial biomass" variable (B, in Fig. 1 ) is defined as the total weight of the n, fish initially contained in the pen.

The "final fish number" variable (n _f in Fig. 1) is defined as the total number of living fish that are finally contained in the pen, i.e., at the end of said period of time D. The "final biomass" variable (B _f in Fig. 1) is defined as the total weight of the n _f fish finally contained in the pen.

During said period of time D, a number of fish n _d is dead. Thus, the "dead fish weight" variable (B _d in Fig. 1 ) is defined as the total weight of the n _d fish. The variable "feed consumption" or "feed intake" (Fc) is defined as the weight of the feed really consumed by the fish in the pen during said period of time D. As shown in Fig. 1 , Fc can be calculated using Equation (5):

Fc = Fd - Fu, wherein Fd is the dry weight of the total feed that is distributed in excess in the pen during said period of time D, and Fu is the dry weight of the total uneaten feed that is recovered daily from the pen during said period of time D.

A "parameter" as used herein is one of the following: initial mean fish weight, final mean fish weight, biomass gain, feed consumption, specific growth rate, and feed conversion ratio. In particular, reference is made in the Examples below to "zootechnical parameters" which include feed intake, specific growth rate, and feed conversion ratio. An "optimal value" of a parameter is the best value of said parameter that is achieved during said period of time D. Depending on the parameter under consideration, said "best" value can be the highest or the lowest value that is observed during said period D. The "initial mean fish weight" parameter (W,) is defined by Equation (6):

The "final mean fish weight" parameter (W _f ) is defined by Equation (7):

The "biomass gain" parameter (B+) is defined by Equation (8):

The "specific growth rate" parameter (SGR) is expressed as percentage increase in fish biomass from day to day. It can be calculated using Equation (9): SGR = [ In(W _f ) - In(Wi) ] / D x 100

SGR (expressed, e.g., in %/day) does not take into account the amount of feed fed to obtain growth. It is a measure of growth rate only. SGR depends on the digestibility of the fish feed and its profile in terms of protein and fat ratio, as well as of amino acid and fatty acid compositions.

The "feed intake" parameter (Fi) is defined by Equation (10):

Fi = Fc / [(Bi + B _f + B _d )/2] / D

Fi can be expressed in g feed / kg of average biomass of fish / day.

The "feed conversion ratio" parameter (FCR) is an economic parameter indicating how efficiently the fish grows on the feed. Fish growth actually corresponds to protein, fat and water deposition in the muscle. FCR thus reflects the "feed utilization" by the fish, or the "feed efficiency". FCR is defined by Equation (1 1 ):

FCR = Fc / B+ FCR varies between fish species and also with the size of the fish. As an example, in Atlantic salmon, FCR may typically be from about 0.7 to about 2. Industrial fish feed in the form of pressed feed and extruded feed contains low amounts of water, typically from about 5 to about 10%. The fish body has a higher water content. This is the reason why the FCR of different feeds should theoretically take into account the water content of the feeds as water does not contribute to growth. More precisely, the FCR should theoretically be calculated on a dry matter basis. However, as the water content is within a narrow range and as it is cumbersome for the fish farmer to calculate dry matter FCR, FCR is usually calculated on the feed including water content. Unless otherwise specified, weights are herein expressed in grams.

"Coating" or "top-coating", as used herein, refers to topical deposition of a palatability-enhancing composition onto the surface of a fish feed, such as by spraying, dusting, and the like. "Inclusion" as used herein, refers to addition of a palatability-enhancing composition internally to a fish feed preparation, by mixing it with the fish feed preparation, before further processing steps for obtaining the final fish feed.

The term "long shelf life" as used herein means a shelf life from about 6 to about 12 months, provided appropriate storage conditions are fulfilled. "Appropriate conditions" for storage and long shelf life of fish feed pellets are well known in the art. For instance, as most foodstuffs and feedstuffs, fish feed pellets should be protected from moisture and heat in order to preserve organoleptic and/or physical properties thereof. Fish feed pellets can be stored either under a packaged form in appropriate packaging units (e.g., micropunched plastic bags) or loose, in silos for example.

When referri ng to a kit, the term "single package" means that the components of said kit are physically associated in or with one or more containers and considered as a unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, pouches, packages of any type or design or material, over-wrap, shrink-wrap, stapled or otherwise affixed components, or combinations thereof. A single package may be containers of individual components physically associated such that they are considered as forming a unit for manufacture, distribution, sale, or use. As used herein, a "means for communicating information or instructions" is a kit component under any form suitable for providing information, instructions, recommendations, and/or warranties, etc. Such a means can comprise a document, digital storage media, optical storage media, audio presentation, visual display containing information. The means of communication can be a displayed web site, brochure, product label, package insert, advertisement, visual display, etc.

The term "database" means an organized collection of information that may be available under any appropriate form and on any appropriate supporting material (paper, electronic, and the like). In the context of the present invention, a "database" more specifically relates to a collection of one or more values of a coefficient of enhancement K _e as a function of PEs: a value K _e 1 specific to one PE (PE1 ), a value K _e 2 specific to another PE (PE2), a value K _e 3 specific to yet another PE (PE3), etc.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

Aiming at providing means and methods for improving the effect of PEs in fish feeds, the Inventors performed a lot of experiments that are reported in the Examples below.

As a first lever of improvement, the Inventors aimed at identifying the most efficient method to be used for applying a PE in fish feeds.

To apply PEs in fish feeds, two methods can be contemplated: either a conventional incorporation by inclusion or a more original application by top- coating.

As shown in paragraph 2.1 of the Examples below, the Inventors could demonstrate that top-coating represents the best solution to apply PEs in fish feeds. Indeed, as illustrated in Fig. 3 and 4, it is necessary to increase the level of inclusion of the PE to 2% to reach the same effect as that of a PE applied by top- coating at 1 %. It comes that top-coating is the most efficient and the least costly solution to apply PEs in fish feeds. Noticeably, this conclusion goes against the conventional practice of manufacturers of fish feeds who usually incorporate PEs by inclusion. Thus, in all aspects of the present invention described below, PEs are applied onto fish feeds by top-coating.

As a second lever of improvement, the Inventors aimed at determining the optimum level of PE to be applied on fish feeds by top-coating so as to observe the highest effect of said PE. The Inventors thus propose to rely on a coefficient of enhancement (K _e ) to manufacture in a simple, easy, and costless way, standardized coated fish feed pellets (e.g., a same amount of PE coats each pellet of a batch). Thanks to this K _e coefficient, the Inventors make it now possible to expedite determination of the optimal amount of a given PE to apply onto pellets by top-coating. Actually, since the K _e coefficient depends on the PE that is used for coating fish feed pellets, the Inventors provide herein a method to obtain a database of coefficients of enhancement K _e as a function of PEs.

Thus, the present invention addresses for the first time an important issue due to the risk of a lack of efficiency of palatability enhancement upon coating fish feed with PEs . In particular, when the size and/or density of the pellets is(are) changed, then the apparent area of the pellets is changed, resulting in a risk of an heterogeneous top-coating of the pellets. Indeed, the pellets cannot be coated efficiently with the same amount of PE when the apparent area of the pellets changes. Clearly, if the apparent area of the pellets changes, then the amount of PE used for coating the pellets has to be adapted. Thus, the present invention makes it possible to have a same amount of PE coating fish feed pellets in a batch, whatever the size and/or density of the pellets. This means that, with the present invention, all pellets of a batch are coated with the same and optimal amount of PE per unit of apparent area.

A first aspect of the present invention concerns a method for determining a coefficient of enhancement (K _e ) for standardizing palatability of fish feed pellets when coating said pellets with a PE useful in fish feed, comprising: a) providing a model fish and model feed pellets having a defined size; b) coating said model feed pellets with increasing amounts (Q) of said PE; c) feeding said coated model feed pellets to said model fish during a period of time D; d) determining, for each amount Q, the following variables: B,, n,, B _f , n _f , Fc, and B _d over said period D; e) calculating at least one parameter selected from Fi, SGR and FCR, as a function of the Q amount; f) selecting the Q _op timai-mo _d ei amount achieving an optimal value of said calculated parameter; g) determining apparent area (AA) of one model feed pellet and number (N) of model feed pellets per weight of the total fish feed; and h) determining said coefficient K _e as the ratio of Q _op timai-modei to total area of model feed pellets, wherein said coefficient K _e is defined by Equation (1 ): K _e = Q _op timai- _mode i / (N x AA).

As the K _e coefficient depends on the PE that is used for coating the fish feed pellets, the method above wherein a given PE (e.g., PE1 ) is used will in fact enable to determine a value K _e 1 which is the value of the K _e coefficient specific to PEL Preferably, K _e is from about 0.4 to about 3. In particular, the K _e coefficient varies depending on the physical form of the PE (dry or liquid). As an example, for dry PEs, K _e is preferably from about 0.4 to about 2, with preferred values from about 1.0 to about 1 .2. Alternatively, for liquid PEs, K _e is preferably from about 1.6 to about 3, with preferred values from about 2.2 to about 2.4. It is of note that variable and parameter determination, as set forth above, is important in aquaculture, at least for the following reasons. There are typically numerous individual fish in a pen or a pond (e.g., 10000 to 30000 individuals), so that feeding cannot be observed on the individual level. In addition, some feed is commonly lost because feed pellets break apart in the feeding system and the broken pieces are too small to be eaten or are so small that they are recognized as dust. Also, some feed pellets are not eaten by the fish but just sink through the water column (Fu as defined above). Moreover, some feed is lost because the fish are fed to satiation, while feeding continues (known as "overfeeding" or feeding "in excess"). Alternatively, underfeeding may occur, in which case the FCR increases because a higher proportion of the feed nutrients (fish-, plant- or land-based raw materials, nitrogen compounds, fat, carbohydrates, and the like) will be used for metabolic purposes instead of being used for muscle deposition.

Importantly, a desirable fish feed will lead to a high SGR and a low FCR. In this prospect, fish feed typically comprises one or more of: - sources of protein, carbohydrate and lipid [for example, fish meal, fish oil, animal meal (for example blood meal, feather meal, poultry meal and/or other types of meal produced from other slaughterhouse waste), animal fat (for example poultry oil), vegetable meal (e.g., soya meal, lupine meal, pea meal, bean meal, rape meal, sunflower meal), vegetable oil (e.g., rapeseed oil, soya oil), gluten (e.g., wheat gluten, corn gluten) and added amino acids (e.g., lysine, methionine)]; and/or

- vitamin premix; and/or

mineral premix; and/or

phosphorus sou rces (e. g . , m onocalci um phosphate, d ical ci u m phosphate); and/or

- pigments (e.g., canthaxanthin, astaxanthin).

Preferably, said parameter calculated in step e) is SGR.

K _e further depends on the group of fish under consideration (carnivorous, omnivorous, or herbivorous) since the model fish is representative of any other fish belonging to the same group. Said model fish is selected from a model carnivorous fish, a model omnivorous fish, and a model herbivorous fish. In particular, said model fish is a model carnivorous fish, preferably European sea bass Dicentrarchus labrax. Alternatively, said model fish is a model omnivorous fish, preferably Tilapia Oreochromis niloticus. Yet alternatively, said model fish is a model herbivorous fish, preferably Chinese grass carp Ctenopharyngodon idella.

According to step a) of the method above, one K _e value (K _e 1 ) is determined for (1 ) a model fish representative of a group of fish (carnivorous, omnivorous, or herbivorous), (2) model feed pellets, and (3) one given PE (PE1 ).

By reproducing the method above using one or more other PEs (PE2, PE3, PE4, etc.), one or more corresponding K _e values (K _e 2, K _e 3, K _e 4, etc.) can be determined so that a database can be obtained. Such a database will thus contain K _e values as a function of PEs (K _e 2 specific to PE2, K _e 3 specific to PE3, K _e 4 specific to PE4, etc.), for a given group of fish (carnivorous, omnivorous, or herbivorous). So, advantageously, the method above further comprises one or more steps i) of re-performing steps a) to h) using another PE (if more than one step i) is performed, then a different PE is used for each step i) so that a different K _e value is determined). Yet advantageously, the method above further comprises a step j) of obtaining a database including K _e values specific to PEs for a given group of fish (carnivorous, omnivorous, or herbivorous).

Practically, in order to ensure reliability of the method above, it is advantageous to be able to observe a dose-response effect as the amount Q of PE increases according to step b) . Th is can be done for i nstance by experimentally testing a sufficient number of amounts Q (at least 5, preferably at least 6, yet preferably at least 7, yet more preferably at least 8 different values for Q) so that one can observe an increasing slope followed by a plateau in the values of the zootechnical parameters (as illustrated by, e.g., Figs. 5B and 6B).

As i ndicated , the database is obtained usi ng a model model fish representative of a group of fish (carnivorous, omnivorous, or herbivorous) and model feed pellets. The K _e value specific to a given PE can then be used to standardize palatability of any other feed pellets (different from model feed pellets) intended to be consumed by any other fish (of the same group of fish but different from the model fish).

The thus obtained database makes it possible to optimize top-coating of any feed pellets to be consumed by any fish of a given group of fish (carnivorous, omnivorous, or herbivorous), using a particular PE. Indeed, the K _e value specific to said particular PE that is known as a result of the method above (and that is advantageously included in a database) can be used to optimally coat any feed pellets intended to be consumed by any fish of a given group of fish (carnivorous, omnivorous, or herbivorous), so that palatability of the thus obtained coated feed pellets is standardized. Another aspect of the present invention is related to a method for standardizing palatability of fish feed pellets (candidate feed pellets, different from model feed pellets) for use in aquaculture, comprising: a) coating fish feed pellets with a PE useful in fish feed using a coefficient of enhancement (K _e ) determined by the aforementioned method (using model feed pellets and a model fish); and b) obtaining said fish feed pellets having standardized palatability. For clarity purposes, fish feed pellets that are different from the model feed pellets and that will be coated with an optimal amount of PE for achieving a standardized palatability of the coated feed pellets, can be referred to as "candidate feed pellets" in the present description. Yet another aspect of the present invention is directed to a method for preparing coated fish feed pellets (candidate feed pellets, different from model feed pellets) having standardized palatability, comprising at least: a) providing a PE useful in fish feed; b) determining apparent area (AA) of one fish feed pellet and number (N) of feed pellets per weight of the total fish feed; c) determining an amount (Q _op timai) of said PE as defined by Equation (2): Q _op timai = K _e x N x AA, using a coefficient K _e determined by the method described above (using model feed pellets and a model fish); d) coating the feed pellets with said Q _op timai amount of said PE; and e) obtaining said coated fish feed pellets having standardized palatability.

With respect of the two aspects above, the candidate feed pellets and/or the fish that is expected to consume these pellets is(are) different from the model feed pellets and/or the model fish used in the method for determining K _e according to the first aspect of the present invention, respectively. However, the fish that is expected to consume the candidate feed pellets belongs to the same group (carnivorous, omnivorous, or herbivorous) as the model fish.

In addition, the Q _op timai amount determined in step c) of the method above is different from the Q _op timai-modei amount selected in step f) of the method for determining K _e according to the first aspect of the present invention. Advantageously, this method for preparing coated candidate fish feed pellets further comprises a step f) after said step e), of packaging said coated fish feed pellets having standardized palatability under appropriate conditions. Additionally or alternatively, the method for preparing coated candidate fish feed pellets further comprises a step g) after said step e) or f), of storing said coated fish feed pellets having standardized palatability under appropriate conditions.

In all methods disclosed herein, said PE useful in fish feed preferably comprises a protein hydrolysate. Appropriate PEs preferably have a high content of free amino acids and/or of peptides and/or of nucleotides.

Advantageously, in all the methods described herein, fat can be sprayed on the feed pellets before coating with the PE. Alternatively, fat can be mixed with the PE before coating the feed pellets.

As indicated in the definitions above, fish feed pellets preferably have a size defined by a diameter (d) and a height (h) of a cylinder. Fish feed pellets are commonly obtained by a cooking extrusion process. Cooking extrusion of material containing starch causes the starch granules to swell such that the crystalline starch in the granules is released and may unfold. This is referred to as gelatinization of the starch. The starch molecules will form a network contributing to bind the extrudate together. Particularly in the feed for carnivorous fish, starch-containing raw materials are added due to their binding ability in the final fish feed. The natural prey for carnivorous fish does not contain starch. Carnivorous fish thus have small amounts of enzymes able to change starch to digestible sugar. Cooking of the starch makes it more digestible. This is partly due to the starch no longer being in a raw, crystalline form, and partly that the cooking process starts a decomposition of starch into smaller sugar units, which are easier to digest. Another effect of cooking extrusion on the mixture of protein, carbohydrates and fat, is that these will form complexes and bindings that may have both positive effects on the digestibility of the mixture. A further effect of cooking extrusion is that the extrudate become porous. This is due to the pressure drop and the temperature drop over the die opening. The water in the extrudate will immediately expand and be liberated as steam leaving a porous structure in the extrudate. This porous structure may be filled with oil in a later process step. An extruded feed will typically contain between 18 and 30% water after extrusion. After extrusion, this feed usually goes through a drying step and, advantageously, a following step of oil coating. The end product generally contains about 10% of water or less and will thus have a long shelf life as the water activity is so low i n such feeds , that g rowth of fungus and mould is prevented and also that bacterial decay is avoided. In particular, before or after coating with oil, the feed is cooled and can be packaged.

A further aspect of the present invention concerns coated fish feed pellets having standardized palatability that can be obtained by a method of preparation as described above.

Yet another aspect of the present invention relates to a method for enhancing growth of fish in aquaculture.

In one embodiment, said method comprises at least: a) preparing coated fish feed pellets having standardized palatability according to the method of preparation described above; and b) feeding said coated feed pellets to fish so as to enhance growth thereof.

In another embodiment, said method comprises at least: a) feeding coated feed pellets as herein described to fish so as to enhance growth thereof.

I n particular, in these embodiments, the feed pellets (candidate feed pellets) and/or the fish that is expected to consume these pellets is(are) different from the model feed pel lets and/or the model fish used in the method for determining K _e according to the first aspect of the present invention. However, the fish that is expected to consume the candidate feed pellets belongs to the same group (carnivorous, omnivorous, or herbivorous) as the model fish.

Yet a further aspect of the present invention provides a kit useful in aquaculture comprising, in a single package, one or more PEs and, optionally, one or more means for communicating information or instructions with respect to the use of said PEs for coating fish feed pellets (candidate feed pellets) in order to achieve standardized palatability. In all aspects of the present invention, a preferred fish is a carnivorous fish, e.g., sea bream, sea bass, salmon, amberjack, trout, turbot, plaice, and the like.

The following Examples illustrate some embodiments and advantages of the present invention.

EXAMPLES

I- Materials and methods

1.1. Fish species

For the purposes of illustrating the present invention, European seabass (Dicentrarchus labrax) was selected as being considered as an appropriate model carnivorous fish (Altan et al., 201 1 ).

1.2. Feed composition and feed preparation

For determining the effect of the method for applying the PE and of the dosage thereof, an experimental fish meal (FM) free diet was formulated. This type of formula appears to be unpalatable for carnivorous fish and is thus considered as a good negative control for screening PEs because of its neutrality regarding olfaction and taste.

Four other diets were formulated to contain graded level of FM (10%, 20%, 30%, and 40%). The diets containing 40% FM was considered as a positive control. The dietary FM was substituted by a mixture of plant-based meal, free amino acids and a phosphorous source.

All the formulated diets met the theoretical nutritional requirements of European seabass excepted for palatability. The nutritional composition of the diets was as follows: crude protei n : 45%, crude fat: 16%, ash: 7-9% , crude energy: 4950Kcal/kg. Table 4. Feed composition

1.3. Fish trials

Two types of trials were performed: - Short term trial: 15 day duration, to study the fish feeding behavior in response to a method for applying a PE and to its dosage

Long-term study: 84 day duration, to confirm the reliability of the effect on feeds having different levels of FM, more representative of the fish feed market than the FM free diet used during the screening step. All the trials were conducted in the experimental flow-through facilities of IFREMER (Centre de Brest, France). Seawater (salinity: 35 g/l) was filtered (high pressure sand filter) and thermoregulated (water temperature: 20 ± 1 °C) . Triplicate groups of 40 juvenile European seabass (D. labrax) each initial mean body weight: 5.0-20.0 g, (depending on the trial) were reared in 24 tanks of 80 I capacity (flow rate: 3 l/min; photoperiod: 12 h light / 12 h dark). Three tanks were allotted at random to each diet.

The diets were distributed in excess to the fish, by automatic feeder (Arvotech, Finland), 10 feed distributions per day. Uneaten feed was collected every day by home-made feed waste collectors, pooled and kept frozen till the end of the trial. At the end, the frozen uneaten feed was dried by evaporation and weighed. Depending on the fish sizes, pellet sizes were ranging from 1.5mm to 3mm (value of both d and h). Fish were counted and weighted at the beginning of the trial (15 day duration) and every 4 weeks (84-day duration). Before every weighing, fish were fasted for 24 h.

Survival was daily checked. Dead fish were counted and weighed. Over the trial period, the following zootechnical parameters were calculated: specific growth rate (SGR), feed intake (Fi) and feed efficiency (FCR).

All data were analyzed by one-way analysis of variance (ANOVA) followed by Fisher test. Differences were considered significant at P<0.05.

1.4. Application of PEs

1.4.1. Palatability enhancers

A dry PE named AP31 , belonging to the ACTIPAL™ product line developed and commercialized by SPF (on behalf of its activity AQUATIV), was used for the studies. This PE is characterized by a high level of protein hydrolysis, free amino acid and nucleotides. 1.4.2. Method for applying PEs

1.4.2.1. Inclusion

The PE was mixed with the other raw materials before grinding and then extruded.

1.4.2.2. Top-coating Top-coating (or coating) was performed in a 2-kg Forberg mixer (Forberg International, Larvik, Norway) with a speed of about 50 rpm. Fish oil was first applied at a level of 1 % (duration of the application: about 30 s) then the dry PE was dusted for about 60 s while mixing, and then the mixer was kept switch on for about 60s for retention. II- Results

2.1. Demonstration of the impact of the method for applying a PE - Comparison coating versus inclusion

The objective of the trial was to compare the effect of a PE when applied either by inclusion in or by top-coating of a FM free diet. A dosage of 1 % was arbitrarily selected for the application by coating and two dosages were tested for the application by inclusion (1 and 2%). The duration of the trial was 15 days. The size of pellets was 2mm (value of both d and h) and the feed ration was the same for all the treatments excepted for the negative control which was reduced after the first day of trial, due to a high level of uneaten feed.

At the end of the trial, a significant increase of feed intake for experimental diets was recorded , compared to the negative control (fig 2) , but no statistical difference was observed between coating application and inclusion application.

A significant increase of fish growth was recorded when adding the PE by inclusion and coating compared to the negative control (fig. 3). The application method influenced the effect of the PE, the coating method enabling to observed a better effect than inclusion (P<0.05). To reach the same result, it appears that the amount of a PE applied by inclusion would have to be two-fold higher than that of the same PE applied by coating. The feed utilization data gave evidence for the better results of a coating application compared to inclusion (fig. 4). At the same PE amount, the coating method allowed to significantly improve feed utilization compared to inclusion. The high FCR calculated for the fish fed the negative control and the feed containing 1 % PE by inclusion could be explained by a high level of uneaten feed having a low palatability.

2.2. Determination of the optimal PE amount (Q _o timai-modei) and of the coefficient of enhancement (K _e )

The objective of the trial was to study the dose response of a PE applied by coating and to define the optimal PE amount (Q _op timai-modei) allowing to reach the optimal values for the zootechnical parameters Fi, SGR, and FCR. Here, model feed pellets and a model fish were used.

Seven dosages were evaluated: 0.5%, 1 %, 2%, 3%, 4%, 5%, and 6% on a FM free diet. Feed were overfed for 15 days with daily collection of uneaten feed. Feed intake, growth rate and feed utilization were calculated. The size of the feed pellets was 2mm (value of both d and h).

From 1 % of application, the coating of a PE on the surface of the pellet allowed to increase significantly the feed intake of seabass (figs. 5A and 5B) but the growth rate was not significantly improved until 2% of PE was used for coating (figs. 6A and 6B). The application of PE did not strongly influence the feed utilization (fig. 7).

Based on these results, the K _e calculations for a PE dosage of 2% (Q _op timai-mo _d ei) and a model feed pellet of 2mm length and 2mm diameter, with N=100022 (given an average weight of 1 pellet p=0.0099978g), gave a value of 1 .06 mg PE/cm ² apparent surface of the pellet. This value was then considered in the next trials. 2.3. Reliability of the effect of a PE applied by coating

The data presented herein are a compilation of results from 27 trials conducted with seabass fed a FM free diet made of 2mm pellets coated or not with 2% PE (corresponding to 1.06 mg PE/cm ² ). The duration of each trial was 15 days and the fish were overfed with daily collection of uneaten feed. The data thus collected during these 27 trials showed a significant positive effect of the PE coating on feed intake and growth of fish fed the FM free diet (figs. 8 and 9). The application of the PE by coating also allowed to improve significantly the feed utilization (fig. 10). The analysis of the minimum and maximum values collected during the 27 trials shows that coating fish feed with a PE is an appropriate solution to reduce any deviation of the zootechnical parameters Fi, SGR, and FCR, that could be observed trial by trial.

In conclusion, application of a PE by coating at a selected amount enables to increase significantly the palatability of FM free feed for fish, especially feed designed for carnivorous fish species. 2.4. Effect of coating fish feed diets containing graded amounts of FM with PEs

The objective of the trial was to evaluate the effect of a PE applied by coating on diets containing different amounts of FM. The fish were overfed for 84 days with collection of uneaten feeds. The PE was applied at 2% on a 2mm pellet during the first 56 days of trial then at 1 .6% on a 2.5mm pellet (value of both d and h) during the last 28 days of trials, each equivalent to a K _e of 1 .06 mg PE/cm ² .

At the end of the trial, a significant reduction of feed intake was recorded while the dietary FM decreased (fig. 1 1 ). Applying a PE by coating allowed to significantly increase the feed i ntake of fish fed 1 0% FM + PE treatment compared to 10% FM treatment. At the other amounts of dietary FM, no significant positive effect of the PE was recorded on the feed intake.

The final growth rate was significantly affected by the dietary amount of FM (fig. 12). At 10%, 20% and 30% FM, applying a PE by coating allowed to improve the fish growth rate, but this result was not significant (P>0.05). At 10% FM, applying a PE by coating allowed to reach the result observed with 20% FM and was thus sufficient to compensate a lack of 10% FM. At 20% FM, applying a PE by coating al lowed to reach the same result as that obtained with 40% FM d iet, compensating a lack of 20% FM. No increase was recorded in the effect when the PE was coated on a 40% FM diet.

No strong impact of the PE was observed on feed utilization (fig. 13).

In conclusion, coating fish feed with an appropriately selected amount of PE is shown to be a very advantageous solution to enhance palatability of feed designed for fish, in particular for carnivorous fish, containing low amounts of FM (<40%).

2.5. Illustrative determination of the optimal PE amount (Q _o timai) for coating feed pellets for different carnivorous fish species, based on the coefficient of enhancement (K _e ) calculated in Section 2.2. above

Figures 14 to 16 illustrate the variation of Q _op timai as a function of the pellet size for seabass and seabream (Fig. 14), for salmon (Fig. 15), and for turbot (Fig. 16), based on a calculated value for K _e of 1.06 mg PE/cm ² apparent surface of the pellet.

Here, the fish is different from the model fish but belongs to the same group (carnivorous). The feed pellets are candidate feed pellets, different from model feed pellets.

REFERENCES

Altan et al., 201 1. Turkish Journal of Fisheries and Aquatic Sciences 1 1 :87-92.

A.O.A.C. 1995. Official methods of analysis. 16 ^th ed. , Ch . 12 H orowitz, Washington, DC, pp 7-9 Hung et al. 1984. J. Food Sci. 49: 1535-1542

Silvestre. 1997. Food Chem. 60:263-273

Nielsen et al. 2001. J. Food Sci. 66:642-646