WO2022175557A1

WO2022175557A1 - Treatment of aquatic animals

Info

Publication number: WO2022175557A1
Application number: PCT/EP2022/054439
Authority: WO
Inventors: Inge Bruheim; Karl-Erik Slinning
Original assignee: Rimfrost Technologies As
Priority date: 2021-02-22
Filing date: 2022-02-22
Publication date: 2022-08-25
Also published as: EP4294201A1; AU2022222331A1; CA3208534A1; CL2023002425A1

Abstract

The present invention relates to a method for the treatment of aquatic animals, such as fish or prawns, in particular for the purpose of improving the bodyweight gain of the animals or for the purpose of reducing the conversion ratio of the feed used to feed the aquatic animals without reducing their bodyweight gain. The present invention further relates to feed compositions, methods for their production and uses thereof.

Description

Treatment of aquatic animals

Field of invention

Background of the invention

In aquaculture, improvements and developments have been made essentially in the breeding methods and in the rearing techniques used for increasing the body weight gain of the animals. Much emphasis is put on the body weight gain of farmed aquatic animals and the conversion ratio of the feed used to raise them. A high-calorie feed enables to achieve a lower feed conversion ratio, in particular a lower amount of feed is required to produce a certain amount of animal biomass, or other production parameters such as total roe weight for certain fish. However, it is always desirable to further reduce the feed conversion ratio in order to reduce production costs of farmed aquatic animals. When lowering the feed conversion ratio it is important that the body weight gain is not reduced by the applied treatment. In practice, it is indeed of high economic importance to be able to reduce the feed conversion ratio, i.e. the amount of feed required for 1 kg productivity, without having to use a (more expensive) feed having a higher energy or nutrient value. It is also of high economic importance to be able to increase the body weight gain so that the desired final animal weight can be achieved within a shorter period of time, i.e. so that the production cycle of the aquatic animals can be shortened. However, in recent years, farmers have been facing major concerns due to the availability of suitable raw materials for preparing appropriate aquaculture feed. In particular, the availability offish meal and fish oil, which used to be the dominant protein sources in marine feed - is dramatically decreasing. As a consequence, the aquaculture industry, and in particular the fish and prawns feed industries, has started to increase the proportion of alternative protein sources in the feed. These alternative protein sources include substitute animal-based materials (such as blood meal, bone meal, feather meal), as well as plant-based materials (such as soy meal, wheat meal, rapeseed meal, rice meal). These are typically cheaper and more available than fish meal and fish oil. However, balancing the dietary amino acid profile of plant-rich diets to meet the fish amino acid requirements is not efficient enough to obtain satisfying fish and feed performances. Indeed, the composition of marine feed remains the crucial factor for achieving feed performance, affecting both feed palatability, feed utilization, digestibility and immunity. As a result of this, there is a high need for edible ingredients which can be incorporated to feed for aquatic animals which are attractive, digestible, support gut health and digestive systems and preserve the immune system, while at the same time having nutritional interest. It should be highlighted that all of these parameters are important for assessing the performance of a feed additive, as they will all contribute to the overall performance of the feed. For instance, a highly palatable feed additive may not lead to an increase in bodyweight gain if it is not sufficiently digestible or if its protein utilization efficiency is suboptimal. The development of nutritional solutions that support overall feed performance is therefore a complex task. As krill is an abundant source of proteins of marine origin, krill meal has been used as a cheaper source of protein additives for farmed aquatic animals. The mechanisms behind the beneficial effects of krill-based protein additives on feed intake and biological performance of farmed aquatic animals are still poorly understood. Krill hydrolysates obtained by the methods described in WO 2010/030193 are one example of commercial protein additives available to feed manufacturers for the purpose of increasing feed intake and overall growth performance of farmed aquatic animals. In this case, the krill hydrolysates are obtained by contacting a disintegrated krill catch with an endoprotease (alkalase).

In view of the above, there is therefore a continuous need for improved treatment methods for aquatic animals which enable to reduce the conversion ratio of the feed used to feed these animals without reducing however the bodyweight gain, i.e. the average weight gain, or which even enable to increase the bodyweight gain. Summary of the invention

The inventors have now surprisingly found that it is possible to provide an improved method fulfilling the above mentioned needs.

Thus, there is now provided a method for the treatment of aquatic animals wherein said treatment comprises orally administering a feed to aquatic animals, wherein said feed comprises a hydrolyzed crustacean catch in an amount between 10 and 200 g/kg of dry weight of said feed, wherein said hydrolyzed crustacean catch is obtained by a method comprising providing a crustacean catch, disintegrating said crustacean catch to provide a disintegrated crustacean catch, contacting the disintegrated crustacean catch with at least one exogenous endoprotease and at least one exogenous exoprotease to provide the hydrolyzed crustacean catch, and wherein the aquatic animals are selected from anadromous fish, crustacean and mollusks.

There is also provided a method for the non-therapeutic treatment of aquatic animals wherein said treatment comprises orally administering a feed to aquatic animals, wherein said feed comprises a hydrolyzed crustacean catch in an amount between 10 and 200 g/kg of dry weight of said feed, wherein said hydrolyzed crustacean catch is obtained by a method comprising providing a crustacean catch, disintegrating said crustacean catch to provide a disintegrated crustacean catch, contacting the disintegrated crustacean catch with at least one exogenous endoprotease and at least one exogenous exoprotease to provide the hydrolyzed crustacean catch, and wherein the aquatic animals are selected from anadromous fish, crustacean and mollusks.

It is further understood that all definitions and preferences as described in detail for the method for the treatment of aquatic animals equally apply for the method for the non-therapeutic treatment of aquatic animals.

The present invention further provides the use of the hydrolyzed crustacean catch, as detailed above, for reducing the conversion ratio of feed used to feed aquatic animals, without lowering their bodyweight gain, where said hydrolyzed crustacean catch, as detailed above, is orally administered to aquatic animals in an amount between 10 and 200 g/kg of dry weight of said feed, and wherein the aquatic animals are selected from anadromous fish, crustacean and mollusks. There is further provided a method for preparing a coated pelletized feed for aquatic animals, comprising providing a hydrolyzed crustacean catch, as detailed above, and depositing the hydrolyzed crustacean catch to at least one surface of a pelletized feed to form a coated pelletized feed for aquatic animals. In another aspect the present invention further provides a feed composition comprising: a) the hydrolyzed crustacean catch, as detailed above, b) one or more plant-based ingredients in a collective amount of at least 50 dry weight percent (dry wt.%), based on the total dry weight of the feed composition, and c) one or more additional ingredients selected from the group consisting of anti-caking agents, vitamins, minerals, various amino acids, free-flowing agents, animal feed flavors or the like.

The feed composition is suitable as feed for aquatic animals, in particular for anadromous fish, crustacean and mollusks. Detailed description

As used herein and in the claims, the terms “comprising” and “including” are inclusive or open-ended and do not exclude additional unrecited elements, compositional components, or method steps. Accordingly, the terms “comprising” and “including” encompass the more restrictive terms “consisting essentially of” and “consisting of”.

In a first aspect, the present invention concerns a method for the non- therapeutic treatment of aquatic animals, which treatment comprises orally administering a feed to the aquatic animals, wherein said feed comprises a hydrolyzed crustacean catch in an amount between 10 and 200 g/kg of dry weight of said feed, wherein said hydrolyzed crustacean catch is obtained by a method comprising providing a crustacean catch, disintegrating said crustacean catch to provide a disintegrated crustacean catch, contacting the disintegrated crustacean catch with at least one exogenous endoprotease and at least one exogenous exoprotease to provide the hydrolyzed crustacean catch, wherein the aquatic animals are selected from anadromous fish, crustacean and mollusks.

As described above, such method enables to reduce the conversion ratio of the feed used to feed these animals without reducing however the bodyweight gain, i.e. the average weight gain, or even enables to increase the bodyweight gain.

Within the context of the present invention, the expression “conversion ratio of feed” refers to a measure of an animal’s efficiency in converting feed mass increased body mass (e.g. muscle or roe mass for fish).

As used herein, the expression “conversion ratio of the feed” is calculated by dividing the total weight of feed provided to the animals by the average daily bodyweight gain of the aquatic animals, all over a specified period. As discussed above, it was already described in the prior art that the addition of a hydrolyzed crustacean catch, obtained by contacting a disintegrated crustacean catch with an exogenous endoprotease, to a feed for aquatic animals, resulted in a better performance of said animals, i.e. a higher feed intake and weight gain and a lower feed conversion ratio of aquatic animals.

The inventors have now surprisingly found that when the disintegrated crustacean catch is further contacted with an exogenous exoprotease, an increase of the body weight gain or an increase of the body weight gain combined with a reduction of the feed conversion ratio can be obtained, as evidenced by the examples below. Moreover, small amounts of the hydrolyzed crustacean catch comprised between 10 and 200 g/kg of dry weight of the feed, can be used thereby resulting in a smaller cost for the supplementation of the feed with this feed additive.

Within the context of the method according to the present invention, the term “crustacean” refers to any marine organism having an exoskeleton and being classified as part of the Crustacea subphylum.

Non-limiting examples of crustaceans include krill, shrimp, lobsters, crab, water fleas, and/or barnacles.

Preferably, the crustacean used to provide the crustacean catch is krill, more preferably Antarctic krill ( Euphausia superba).

Within the context of the method according to the present invention, crustacean catches can be provided fresh, or can have been previously frozen.

Thus, in one embodiment of the method according to the present invention, the crustacean catch is a frozen crustacean catch.

In another embodiment of the method according to the present invention, the crustacean catch is a fresh crustacean catch. Preferably, the fresh crustacean catch is a crustacean catch caught within less than 24 hours, or less than 12 hours, or less than 2 hours, or less than 1 hour. Within the context of the method according to the present invention, the term “disintegrating” is intended to refer to mechanical breaking, granulation or fragmentation of the crustacean catch into smaller pieces or particles, with an average particle size of less than 25.0 millimeters (25.0 mm).

Non-limiting examples of “disintegrated crustacean catch” are crustacean catch powders, fragments, particles, flakes, granules, grains or other components obtained by grinding a crustacean catch, with an average particle size of less than 25.0 mm. Preferably, the particle sizes of the disintegrated crustacean catch can also be less than 20.0 mm, or less than 15.0 mm, or less than 10.0 mm, or less than 8.0 mm, or less than 7.0 mm, or less than 6.0 mm, or less than 5.0 mm.

In general, the average particle size can be measured by methods known in the art, for example, by particle size analyzers, methods such as measurement using light (light-scattering methods or turbidimetric methods), sedimentation methods (pipette analysis using an Andreassen pipette, sedimentation scales, photosedimentometers or sedimentation in a centrifugation force), pulse methods (Coulter counter), or sorting by means of gravitational or centrifugal force.

The disintegrated crustacean catch may be obtained by disintegrating the crustacean catch using any conventional means, such as using a knife shredder, a blender or a homogenizer.

It is further understood that the temperature at which the disintegration process takes place may be around the ambient temperature of the water in which the crustacean catch was caught when the crustacean catch is a fresh crustacean catch.

In one embodiment of the method according to the present invention, the crustacean catch is disintegrated at a temperature between -2.0 degrees Celsius (-2.0 °C) and +10.0 °C, preferably between 0.0 °C and +6.0 °C. Within the context of the method according to the present invention, the expression “hydrolyzed crustacean catch”, is intended to refer to a crustacean catch which has undergone hydrolysis. Hydrolysis is a chemical reaction which can be caused or mediated by a biological agent, such as a proteolytic enzyme (or protease), whereby the natural protein sequences become shorter (for example by breaking peptide bonds of the amino acid sequence primary structure) to form smaller peptides and/or free amino acids.

Generally speaking, the disintegrated crustacean catch needs to be hydrolyzed so that digestive enzymes, such as lipases and phospholipases, which are released from the crustacean catch upon death may be inactivated. If these digestive enzymes are not inactivated upon their release, there is a risk that they will hydrolyze the phospholipids and fatty acids within the crustacean catch. In order to inactivate the endogenous crustacean digestive enzymes, the disintegrated crustacean catch is contacted with a protease under certain conditions to form a hydrolyzed crustacean catch. The protease is generally chosen to specifically target the crustacean digestive enzymes while minimally damaging the other crustacean proteins. Within the context of the method according to the present invention, the term “protease” refers to food grade enzymes which cleaves large protein molecules into smaller molecules by hydrolyzing peptide bonds along the protein backbone. As used herein, the terms “proteolytic enzyme”, “peptidase” and “protease” can be used interchangeably. More specifically, the term “endoprotease” refers to an enzyme that catalyses the cleavage of peptide bonds within a polypeptide or protein, the internal peptide bonds of a polypeptide or protein. On the other hand, the term “exoprotease” refers to a protease that can remove the terminal amino acid(s) of a peptide or protein by cleaving peptide bonds. A terminal amino acid is an amino acid that is the N-terminal or C-terminal amino acid of a protein or peptide. Non-limiting examples of suitable exogenous endoproteases and exogenous exoproteases include serine proteases, cysteine proteases, aspartic proteases, metalloproteases, threonine proteases, glutamic proteases and asparagine proteases. Preferably, the at least one exogenous endoprotease and the at least one exogenous exoprotease are each independently selected from serine, cysteine, aspartic and metalloproteases. More preferably, the at least one exogenous endoprotease and the at least one exogenous exoprotease are independently selected form serine proteases and metalloproteases. Most preferably, the at least one exogenous endoprotease is a metalloprotease and the at least one exogenous exoprotease is a serine protease.

The at least one exogenous endoprotease and the at least one exogenous exoprotease according to the method of the present invention can also be each independently selected from acid proteases, neutral proteases and basic (or alkaline) proteases. Acid proteases are proteases that exhibit maximum activity and stability in acidic conditions (pH 2.0 - 5.0) and are typically inactive at pH values above 6.0. Acidic proteases generally have a low isoelectric point and have a low basic amino acid content. Neutral proteases are typically active in a narrow pH rage (pH 5.0 - 8.0), whereas alkaline proteases are characterized by their high activity at alkaline pH, typically above pH 9.0.

In one embodiment of the method according to the present invention, the at least one exogenous endoprotease is a neutral endoprotease or an alkaline endoprotease. Preferably, the at least one exogenous endoprotease is a neutral endoprotease.

In a further embodiment of the method according to the present invention, the at least one exogenous exoprotease is an alkaline or a neutral exoprotease. Preferably, the at least one exogenous exoprotease is a neutral exoprotease. In a preferred embodiment of the method according to the present invention, the at least one exogenous endoprotease is a neutral endoprotease and the at least one exogenous exoprotease is a neutral exoprotease.

The at least one exogenous endoprotease and the at least one exogenous exoprotease according to the present invention can further be derived from any organism, including plants, animals, bacteria, viruses or fungi.

Preferably, the at least one exogenous endoprotease and the at least one exogenous exoprotease are obtained from bacteria of the genus Bacillus. Suitable exogenous endoproteases and the exogenous exoproteases are obtained from the bacteria Bacillus Licheniformis.

Within the context of the present invention, the at least one endoprotease and the at least one exoprotease are exogenous proteases. This implies that the hydrolyzed crustacean catch results from the hydrolysis of a crustacean catch by additional proteases that are not naturally present in the crustacean catch and that are added to the catch in order to hydrolyze proteins.

In one embodiment of the method according to the present invention, the disintegrated crustacean catch will be contacted with an amount of at least 0.1 g/kg, or at least 0.2 g/kg, or at least 0.5 g/kg, or at least 1.0 g/kg, or at least 2.0 g/kg of the at least one exogenous endoprotease, relative to the total weight of said disintegrated crustacean catch.

In one embodiment of the method according to the present invention, the disintegrated crustacean catch will be contacted with an amount of at least 0.01 g/kg, or at least 0.02 g/kg, or at least 0.05 g/kg, or at least 0.1 g/kg, or at least 0.2 g/kg of the at least one exogenous exoprotease, relative to the total weight of said disintegrated crustacean catch.

Therefore, in a preferred embodiment of the method according to the present invention, the disintegrated crustacean catch will be contacted with an amount of at least 0.1 g/kg of the at least one exogenous endoprotease and an amount of at least 0.01 g/kg of the at least one exogenous exoprotease, or least 0.2 g/kg of the at least one endoprotease and an amount of at least 0.02 g/kg of the at least one exogenous exoprotease, or least 0.5 g/kg of the at least one exogenous endoprotease and an amount of at least 0.05 g/kg of the at least one exogenous exoprotease, or least 1 .0 g/kg of the at least one exogenous endoprotease and an amount of at least 0.1 g/kg of the at least one exogenous exoprotease, or least 2.0 g/kg of the at least one exogenous endoprotease and an amount of at least 0.2 g/kg of the at least one exogenous exoprotease, relative to the total weight of said disintegrated crustacean catch.

Upper limits for the amounts of exogenous endoprotease and exogenous exoprotease are not particularly critical and will be selected by the skilled in the art in view of the type of protease selected and the origin and nature of the disintegrated crustacean catch.

It is understood that, within the context of the present invention, the amounts of the at least one exogenous endoprotease and the at least one exogenous exoproteases refer either to the amount of the exogenous (endo-/exo-)protease, i.e. the exogenous (endo-/exo-)protease, when only one exogenous (endo-/exo-)protease is added to the disintegrated crustacean catch, or to the sum of the amounts of exogenous (endo-/exo- )proteases, when more than one exogenous (endo-/exo-)protease is added to the disintegrated crustacean catch.

Advantageously, the disintegrated crustacean catch can be contacted with the at least one exogenous endoprotease and the at least one exogenous exoprotease at a pH ranging from 5.0 to 9.0, preferably, ranging from 7.0 to 8.0.

Advantageously, the disintegrated crustacean catch can be contacted with the at least one exogenous endoprotease and the at least one exogenous exoprotease at a temperature ranging from 30.0 °C to 80.0 °C, or between 40.0 °C and 70.0 °C, or between 50.0 °C and 60.0 °C.

It is further understood that the disintegrated crustacean catch can be contacted either sequentially or simultaneously with the at least one exogenous endoprotease and the at least one exogenous exoprotease. Therefore, according to certain embodiments of the method according to the present invention, the disintegrated crustacean catch is contacted simultaneously with the at least one exogenous endoprotease and the at least one exogenous exoprotease. Advantageously, the disintegrated crustacean catch are contacted with the at least one exogenous endoprotease and the at least one exogenous exoprotease for a duration ranging from 1 minute to 24 hours, or from 5 minutes to 3 hours, or from 10 minutes to 2 hours.

In alternative embodiments of the method according to the present invention, the disintegrated crustacean catch is contacted sequentially with the at least one exogenous endoprotease and the at least one exogenous exoprotease. Advantageously, the disintegrated crustacean catch is first contacted with the at least one exogenous endoprotease, and subsequently with the at least one exogenous exoprotease. In such cases, the inventors have found that the disintegrated crustacean catch could be advantageously contacted with the at least one exogenous exoprotease for a longer time period than with the at least one exogenous endoprotease. Preferably, the duration of the contacting step with the at least one exoprotease can range from 30 minutes to 24 hours, or from 1 hour to 3 hours, while the duration of the contacting step with the at least one endoprotease can be reduced to less than 2 hours, or less than 1 hour, or less than 30 minutes, or less than 15 minutes, or less than 10 minutes or even less than 5 minutes.

It is further understood that, within the context of the method according to the present invention, the term “sequentially” is intended to refer to two separate steps occurring either directly after one another, or to a second step occurring substantially later than a first step. In particular, within the context of the method according to the present invention, the contacting of the disintegrated crustacean catch with the at least one exogenous exoprotease can take place at any point in time after the contacting with the at least one exogenous endoprotease. According to certain embodiments of the method according to the present invention, the hydrolysis may be stopped by inactivating the proteases. This may advantageously be achieved by raising the pH and/or the temperature. As discussed above, it was already described in the prior art that the addition of a crustacean hydrolysate obtained by contacting a disintegrated crustacean catch with one or several exogenous endoprotease(s), to a control diet, resulted in a better performance of aquatic animals, such as fish (i.e. a higher feed intake and weight gain and/ora lower feed conversion ratio).

The inventors have surprisingly found that by performing the hydrolysis in the presence of at least one exogenous endoprotease and at least one exogenous exoprotease, a hydrolyzed crustacean catch having improved properties could be obtained. It was observed that the hydrolyzed crustacean catch according to the method of the present invention, as detailed above, resulted in increased bodyweight gain as well as specific growth rate (SGR) as compared to conventional hydrolysates when orally administered to aquatic animals, wherein the aquatic animals are selected from anadromous fish, crustacean and mollusks. Although not wishing to be bound by theory, it appears that the hydrolyzed crustacean catch, as detailed above, comprises a modified distribution of total amino acids, free amino acids, as well as a different distribution of molecular weight peptide size, as compared to conventional hydrolyzed crustacean catches. In particular, the hydrolyzed crustacean catch, as detailed above, comprises a higher proportion of small molecular weight peptides (< 200 Da). In addition, distribution of many amino acids (total and free amino acids) is altered in the hydrolyzed crustacean catch, as detailed above, as compared with conventional hydrolyzed crustacean catches. The combination of these factors appears to have a beneficial effect on the overall performance of aquatic animals to which a feed comprising the hydrolyzed crustacean catch according to the method of the present invention, as detailed above, is orally administered.

According to certain embodiments, the method according to the present invention comprises administration of a feed which is in the form of a pelletized feed.

Non-limiting examples of suitable pelletized feeds include feeds in the form of particulate chunks or pieces formed by either a press or extrusion process. The pellets can vary in size and/or shape, depending on the process or the equipment. Typically, fish pelleted feeds have a cylindrical shape. The size of the pelleted feed to be used can be determined by the skilled person based on the size of the aquatic animal.

Preferably, the pelletized feed is a coated pelletized feed comprising a core, and at least one coating layer. More preferably, at least part of the hydrolyzed crustacean catch according to the present invention is comprised in said coating layer. More preferably, the hydrolyzed crustacean catch is substantially only present in the coating layer, and this in an amount between 10 and 200 g/kg of dry weight of the feed.

The inventors have indeed surprisingly found that when the hydrolyzed crustacean catch is present in the coating layer, improved effects in terms of bodyweight gain and feed conversion ratio could be overserved. Without being bound by theory, it seems that when the hydrolyzed crustacean catch is present in the coating layer, it will dissolve more efficiently in water, thereby increasing the overall attractiveness of the feed. As mentioned above, the inventors have found that the upper limit for the amount of hydrolyzed crustacean catch comprised in the feed should be at most 200 g/kg of dry weight of said feed.

In one embodiment of the method according to the present invention, the feed comprises the hydrolyzed crustacean catch in an amount of less than 180, preferably less than 150, more preferably less than 120, even more preferably less than 100, most preferably less than 80 g/kg dry weight of said feed.

It is further understood that the amount of hydrolyzed crustacean catch, as detailed above, in the feed is at least 12, preferably at least 15, more preferably at least 20 g/kg dry weight of said feed.

Therefore, in most preferred embodiments of the method according to the present invention, the feed comprises the hydrolyzed crustacean catch in an amount between 12 and 180, preferably between 15 and 150, more preferably between 20 and 100 g/kg dry weight of said feed.

Within the context of the method according to the present invention, the expression “aquatic animal” refers to any animal, either vertebrate or invertebrate, which lives in water for the most or all of its lifetime.

The method according to the present invention is applicable for the treatment of aquatic animals selected from anadromous fish, crustacean and mollusks.

In a preferred embodiment of the method according to the present invention, the aquatic animal is a crustacean.

Non-limiting examples of crustaceans include prawns, shrimp, lobsters, crabs, water fleas, and/or barnacles.

Preferably, the aquatic animal is a prawn or a shrimp. Non-limiting examples of farmed species suitable for the method according to the present invention include whiteleg shrimp (also known as Pacific white shrimp or king prawn, Litopenaeus vannamei, formerly known as Penaeus vannamei ), giant tiger prawn (also known as Asian tiger shrimp or black tiger shrimp, Penaeus monodon), Indian white prawn (also known as Indian prawn, Fenneropenaeus indicus, formerly known as Penaeus indicus) and giant river prawn (also known as giant freshwater prawn, Macrobrachium rosenbergii).

The method according to the present invention is particularly suitable for the treatment of farmed shrimp or prawn as these animals are typically grown under high density conditions which induce high stress and reduced appetite. It is therefore essential to use feed ingredients that can trigger feed intake and initiate body weight gain in order to obtain efficient growth.

In another preferred embodiment of the method according to the present invention, the aquatic animal is an anadromous fish. Anadromous fish are those that spawn in freshwater, migrate to the ocean to forage and mature, and return to freshwater to spawn and begin the cycle again. More preferably, the aquatic animal is an anadromous carnivorous fish or an anadromous omnivorous fish.

Non-limiting examples of anadromous carnivorous fish or anadromous omnivorous fish include 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 morpha trutta), striped bass (Morone saxatilis), and sturgeon (Acipenseridae spp.).

The method according to the present invention is particularly suitable for the treatment of juvenile anadromous fish which have been transferred from a freshwater environment to a marine environment for farming purposes. This is indeed a critical stage in the farming process, where most of the body weight gain of the animals will occur. It is also a period where the fish will typically be under substantial stress. As a result, juvenile fish (such as salmon smolt) often have poor appetite after transfer and it can take weeks before the fish start eating properly. Moreover, as is well known, normal aquaculture growing conditions include substantial density in the enclosure. It is therefore essential to use feed ingredients that can trigger feed intake and initiate body weight gain to obtain efficient growth.

In a preferred embodiment of the method according to the present invention, the aquatic animal is an anadromous carnivorous fish from the Salmonidae family (salmonids), most preferably an anadromous salmonid selected from 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) and sea trout (Salmo trutta morpha trutta).

In a more preferred embodiment of the method according to the present invention, the aquatic animal is an anadromous salmonid from the genus Salmo, even more preferably a salmonid from the species Salmo salar (Atlantic salmon).

Salmonids are among the most important fish species used in aquaculture worldwide, particularly in Europe. Globally, Salmonid aquaculture production grew over ten-fold between 1982 and 2007, reaching a 10.7 billion USD globally. Anadromous salmonids are usually farmed in two stages. First the salmonids are hatched from eggs and raised on land in freshwater tanks. After 12 to 18 months, the smolts are then transferred to floating sea cages or net pens, typically anchored in sheltered bays or fjords along a coast. After transfer, the smolt are fed (pelleted) feed for another 12 to 24 months, before they develop into adult stage and are considered ready to be harvested.

Such farming practices reflect the natural life cycle of anadromous salmonids, involving long migrations to novel environments and challenging physiological transformations when moving between salt-free and salt-rich waters. In the wild, the juvenile freshwater stage of Atlantic salmon ( Samos salar) takes 24-36 months (as compared to 12-18 months in farms), developing from egg through alevin, fry and parr stages, up to the smolt stage which is then ready to move to seawater (Klemetsen et al. (2003). Atlantic salmon Salmo salar L, brown trout Salmo trutta L. and Arctic charr Salvelinus alpinus (L.): a review of aspects of their life histories. Ecology of Freshwater Fish 12, 1-59).

Smoltification is a complex developmental transformation involving morphological, biochemical, physiological and behavioral changes that preadapt young parr for life in high salinity water (Hogasen, HR (1998). Physiological changes associated with the diadromous migration of salmonids. Canadian Special Publication of Fisheries and Aquatic Sciences 127, 128 p.). Light and temperature regimes can be manipulated artificially to induce early smoltification. The morphological changes include a slimmer body form and alterations in body coloration (darkened fins, dark back, white belly and silver sides) that help to conceal with the pelagic environment. Atlantic salmon smolts originally weigh 50-80 grams and can be grown up to 1 kg before being transferred to seawater.

After transfer from freshwater to seawater, smolts will develop into adult stage. On farms it takes 12-24 months for smolts to reach adult stage. Thus, as used herein, the term “smolt” refers to anadromous salmonids which have undergone smoltification but have not yet reached full adult size (adult stage). The term “smolt” thus encompasses fish which a grown in seawater up to the moment they reach their full adult size, typically 12-24 months on farms, sometimes referred to as “post-smolts”. In a preferred embodiment of the method according to the present invention, the aquatic animal is a smolt, most preferably an Atlantic salmon ( Salmo salar) smolt.

Another aspect of the present invention is a feed composition for aquatic animals, in particular the aquatic animals, as detailed above, comprising the hydrolyzed crustacean catch as detailed above.

The choice of the other ingredients of the feed according to the method of the present invention, which comprises the hydrolyzed crustacean catch as detailed above, is not particularly limited and any suitable ingredient that is compatible for use in a feed for aquatic animals may be used.

It is understood that all definitions and preferences, as described above for the method, equally apply to all further embodiments and aspects of the feed composition, as described below.

The feed composition, which is suitable as a feed for aquatic animals, in particular the aquatic animals, as detailed above, comprises: a) the hydrolyzed crustacean catch, as detailed above, in an amount of between 10 and 200 g/kg of dry weight of said feed composition; b) one or more plant-based food ingredients in a collective amount of at least 50 dry weight percent (dry wt.%), based on the dry weight of the feed composition; and c) one or more additional ingredients comprising anti-caking agents, vitamins, minerals, various amino acids, free-flowing agents, animal feed flavors or the like.

The plant-based food ingredients may be derived from any plant source. Non-limiting examples of suitable plant sources include soy, rapeseed, wheat, corn, lupine, pea, sunflower, rice and the like.

It is further understood that the feed composition does not contain any added ingredients or food contaminant that are poisons or toxins, e.g. substances that have an inherent property and in amounts to induce death or illness in animals, including the aquatic animals.

In one embodiment of the feed composition according to the present invention, the feed composition is in a solid form.

Preferably, the feed composition is in the form of a pellet.

In some embodiments of the feed composition according to the present invention, the hydrolyzed crustacean catch can be distributed within and throughout the pellets.

Yet, in other embodiments of the feed composition according to the present invention, the pellet is a coated pellet, comprising a core and at least one coating layer.

Preferably, at least part of the hydrolyzed crustacean catch is comprised in the coating layer. More preferably, the core of the coated pellet comprises said one or more plant-based ingredients, whereas the at least one coating layer comprises said hydrolyzed crustacean catch.

As mentioned above, the inventors have found that the upper limit for the amount of hydrolyzed crustacean catch comprised in the feed composition should be at most 200 g/kg of dry weight of said feed. ln one embodiment of the feed composition according to the present invention, the feed composition comprises the hydrolyzed crustacean catch in an amount of less than 180, preferably less than 150, more preferably less than 120, even more preferably less than 100, most preferably less than 80 g/kg dry weight of said feed.

It is further understood that the amount of hydrolyzed crustacean catch, as detailed above, in the feed composition is at least 12, preferably at least 15, more preferably at least 20 g/kg dry weight of said feed.

Therefore, in most preferred embodiments of the feed composition according to the present invention, the feed composition comprises the hydrolyzed crustacean catch in an amount between 12 and 180, preferably between 15 and 150, more preferably between 20 and 100 g/kg dry weight of said feed.

The methods to manufacture a coated pelletized feed is also an aspect of the present invention.

It is understood that all definitions and preferences, as described above, equally apply to all further embodiments, as described below.

The coated pelletized feed, suitable for aquatic animals, in particular the aquatic animals, as detailed above, can be prepared by a variety of methods known in the art.

In one embodiment of the present invention, the method for preparing the coated pelletized feed, as detailed above, comprises: a) providing a hydrolyzed crustacean catch, as detailed above; and b) depositing said hydrolyzed crustacean catch to at least one surface of a pelletized feed to form a coated pelletized feed for aquatic animals.

It is understood that the skilled person in the art will carry out said depositing according to general practice such as notably using optimal times, speeds, weights, volumes and batch quantities. The skilled person will also select the pelletized feed to which the coating is to be deposited according to the target aquatic animal. Examples

The invention will be now described in more details with reference to the following examples, whose purpose is merely illustrative and not intended to limit the scope of the invention. Anadromous fish

Example 1. Materials and methods.

A total of 1440 fertilized Atlantic salmon ( Salmo salar) eggs were hatched and raised in freshwater aquaria for 12 months. All fish were fed a commercial freshwater diet prior to the start of the trial. The fish were then starved for one day and randomly distributed over 18 fiberglass tanks (1 x 1 m²) comprising each 80 animals and assigned to one out of the six test diets in triplicate. The fish were fed in excess appetite by automatic feeders at intervals of 38 seconds duration, every 32 minutes, which amounts to 48 meals per day on average, for a feeding period of 42 days. Unconsumed feed was collected daily, dried (40 °C, 24 h) and weighed to record the daily feed consumption. Recovery tests were performed for each test diet in order to correct for variable loss of dry matter. The fish were exposed to continuous light (12:12h) throughout the trial and kept at ambient water temperature of average 12 °C for the duration of the trial. All tanks were supplied with running seawater at a flow of 24 L/min. Oxygen content of the outlet water from each tank ranged from 83% to 95% saturation throughout the trail. The total fish biomass was recorded at the start and end of the trail. The total biomass and number of fish per tank were used to calculate the fish body weight.

The composition of the diets was designed according to meet energy and nutrient requirements of salmon feed. The composition of the basal formulation of these diets is shown in Table 1. Table 1. Ingredient composition of the basal formulation.

*Microingredients include vitamins, minerals, amino acids, an ingestible marker and astaxanthin. The basal formulation was used to produce the seven experimental diets. The diets, as described in Table 1 , were supplemented with fish meal, wheat, fish oil and rapeseed oil on top of the basal formulation. In addition, the krill hydrolysate diets contained 25 g/kg or 50 g/kg of different types of krill hydrolysates. For each diet, the amount of fish meal, wheat, fish oil, rapeseed oil and water was adjuster to maintain a constant overall nutritional composition (iso-caloric, constant protein and lipid levels) throughout each of the diets, as described in Table 3. The weight percentage of each of the components is relative to the total weight of the test diets. All diets were produced as pelleted feed in a commercial feed mill.

The characteristics of the krill hydrolysates 1 to 3 (KH1 to KH3) are summarized in Table 2. The krill hydrolysates were deposited as coating layer to the feed pellets. Table 2. Overview of the characteristics of the krill hydrolysates 1 to 3.

The krill hydrolysates KH1, KH2 and KH3 were obtained as follows. 500 kg of frozen krill was shredded using a knife cutter to a particle size comprised between 3 and 6 mm. The temperature of the krill was comprises between 1 and 2 °C. A mixture of 500 kg of fresh water and protease was added and incubated at 55 °C. In krill hydrolysate 1 (KH1), 0.15 wt.% of a neutral endoprotease (metalloprotease) was added. In krill hydrolysate 2 (KH2), 0.45 wt.% of a neutral endoprotease (metalloprotease) was added. Finally, in krill hydrolysate 3 (KH3), 0.45 wt.% of a neutral endoprotease (metalloprotease) was added in combination with 0.04 wt.% of a neutral exoprotease (serine protease). The hydrolysis reaction was run for one hour for KH1 and for two hours for KH2 and KH3. After hydrolysis, the shells were separated from the main process line using a decanter. The hydrolyzed krill in the main process line was then pasteurized to ensure hygienic quality and to inactivate the proteases. Pasteurization was performed at 90 °C for 10 minutes. The krill hydrolysates were subsequently dried using a sedicanter to mechanically separate the excess water, followed by low temperature vacuum drying.

Each of the three krill hydrolysates were added to the basal formulation in combination with other additives, so as to maintain a stable nutritional balance throughout the test diet compositions, as detailed above.

Table 3. Overview of the test diets with the amount of additives added on top of the basal formulation.

Table 4. Nutritional balance of the test diets

From day 1 until day 42, the fish body weight was monitored, together with the feed intake per smolt salmon. Example 2. Effect of krill hydrolysate supplementation on daily feed intake.

Table 5 demonstrates that supplementation with krill hydrolysates increases feed intake relative to the control diet. Most importantly, it was observed that supplementation with the either 2.5 wt.% or 5.0 wt.% of the krill hydrolysates according to the invention (Examples 4 - 5, Ex 4 - 5) results in an increase in feed intake as compared to conventional krill hydrolysates (Comparative Examples 1 - 3, CEx 1 - 3) after both 15 and 42 days in smolts.

Table 5. Effect of krill hydrolysate supplementation on daily feed intake in smolt salmon after 15 and 42 days.

Example 3. Effect of krill hydrolysate supplementation on body weight and daily specific growth rate

Table 6 demonstrates that supplementation with krill hydrolysates increases final body weight and daily specific growth rate (SGR) relative to the control diet. Most importantly, it was observed that supplementation with the either 2.5 wt.% or 5.0 wt.% of the krill hydrolysates according to the invention

(Examples 4 - 5) results in an increase in final body weight as well as in daily specific growth rate (SGR) as compared to conventional krill hydrolysates (Comparative Examples 1 - 3) after both 15 and 42 days in smolts.

Table 6. Effect of krill hydrolysate supplementation on smolt salmon performance during a 42 days experiment.

Based on the results of Table 5 and Table 6, it can be concluded that the dietary treatments prepared according to the invention (Examples 4 and 5) perform best. When the dietary preparations comprise the krill hydrolysate KH4 at concentrations of 2.5 wt.% (Example 4) or 5.0 wt.% (Example 5), smolt salmons perform better in terms of final growth and growth rate. Also, the feed intake response was found to be increased.

Crustacean

Example 4. Materials and methods.

Penaeus vannamei are imported as postlarvae and reared into a water recirculation system containing artificial seawater at a salinity of 20 g L ¹. They are raised on an artificial diet and after weaned onto a pelleted feed. Both feed are distributed 12 times a day. Water temperature is kept constant at 27°C by means of an automatic heating system. A complete biological/mechanical filter and regular water changes keep the total ammonia at <0.05 mg L·¹ and nitrites at <0.8 mg L·¹.

For the growth performance trial, duplicate/triplicate groups of shrimp are fed with the experimental diets during 28 days. This procedure assesses the effects for the experimental diets on the growth performance of the shrimp. During the feeding trial, faeces are collected for the analysis of protein and fat digestibility. After this period, the feed palatability is assessed. The daily feeding rate for each group is calculated based on shrimp’s weight and adjusted daily according to the expected shrimp growth and mortality. In addition, shrimp are fed at the maximum feeding ratio (per treatment), to ensure differences in palatability and therefore feed intake are taken into account.

Specifically, a total of 980 shrimp weighing 0.5-2 grams are used to randomly compose 14 groups of 70 individuals. Each group is housed in a feeding unit. Each experimental diet is randomly assigned to 3 tanks. During the feeding trial period, the groups of shrimp receive the respective diets at the predetermined percentages of their initial mean body weight (MBW). This is adjusted daily according to the expected growth, and observed mortality and feed consumption per group. The total weight of the groups is measured at the start and at the end of the trials. A subsampling is performed every two weeks and growth curves are adjusted accordingly.

After completion of the growth performance trial, the number of shrimp is reduced to 30 shrimp per tank for performing the palatability trial. Feeding tanks are kept in full recirculation except during the periods of when palatability feed samples will be collected. At this time, tanks are placed in individual circulation to avoid exchange of dietary attractants in between treatments. Once a day for 7 days, feed portions are added to the respective tanks. Thirty minutes after feeding, the leftover portions are recovered and dried. Weights of each feed leftover portions are measured and percentage of feed intake per group is calculated. During the completion of the trials, shrimp faeces are collected suing a faeces precipitation system coupled to the shrimp feeding tank. The daily samples are stored at -20°C until at least the minimal wet weight of the faeces has been collected from each replicate tank. The frozen feaces are free-dried before analysis. The analysis is performed for protein (Kjeldahl) and T1O2 based on UV-spectroscopy. The outcome in each experimental group is compared with the control group and with every other experimental group using a two-way ANOVA statistical analysis followed by a Turkey’s multiple comparisons test.

The composition of the diets are designed to meet energy and nutrient requirements of shrimp feed. A basal formulation are used to produce five experimental diets. Four of the diets are supplemented with 40 or 80g/kg of different types of krill hydrolysates. For each diet, the composition of the basal formulation is adjusted to maintain a constant overall nutrition composition (iso-caloric, constant protein and lipid levels) throughout each of the diets. The weight percentage of each of the components is relative to the total weight of the test diets. All diets were produced as pelleted feed in a commercial feed mill. As indicated in Table 7, 4.00 wt.% of krill hydrolysate is added through topcoating in Examples 6 and 7 (Ex 6 and 7). In Examples 7 and 8 and Comparative Example 9 (Ex 7, 8 and CEx 9), 4.00 wt.% of krill hydrolysate is added by inclusion. Titanium oxide is added as a tracer for the digestibility analysis.

The characteristics of the krill hydrolysates 2 and 3 (KFI2 and KFI3) are summarized in Table 2. Table 7. Overview of the test diets.

^* 4.00 wt.% of KH3 is added as a top-coating (not inclusion) Table 8. Nutritional balance of the test diets

Example 5. Effect of krill hydrolysate supplementation on growth performance Supplementation with krill hydrolysates increases feed intake, relative to the control diet. Most importantly, supplementation the krill hydrolysates according to the invention (Examples 6 - 8) results in an increase in feed intake as compared to conventional krill hydrolysates (Comparative Example 9). Applying the krill hydrolysate as a top-coating (Example 6) also increases feed intake as compared to the same amount of krill hydrolysate added by inclusion into the diet (Example 8).

Example 6. Effect of krill hydrolysate supplementation on body weight and daily specific growth rate Supplementation with krill hydrolysates increases final body weight and daily specific growth rate (SGR) relative to the control diet. Most importantly, supplementation with krill hydrolysates according to the invention (Examples 6 - 8) results in an increase in final body weight as well as in daily specific growth rate (SGR) as compared to conventional krill hydrolysates (Comparative Example 9) in shrimp. Applying the krill hydrolysate as a top-coating (Example 6) also increases final body weight as well as in daily specific growth rate (SGR) as compared to the same amount of krill hydrolysate added by inclusion into the diet (Example 8). Example 7. Effect of krill hydrolysate supplementation on feed palatability

Supplementation with krill hydrolysates increases feed palatability relative to the control diet. Most importantly, supplementation with krill hydrolysates according to the invention (Examples 6 - 8) results in an increase in feed palatability as compared to conventional krill hydrolysates (Comparative Example 9) in shrimp. Applying the krill hydrolysate as a top-coating (Example 6) also increases feed palatability as compared to the same amount of krill hydrolysate added by inclusion into the diet (Example 8).

Example 8. Effect of krill hydrolysate supplementation on feed digestibility

Supplementation with krill hydrolysates increases feed digestibility relative to the control diet. Most importantly, supplementation with krill hydrolysates according to the invention (Examples 6 - 8) results in an increase in feed digestibility as compared to conventional krill hydrolysates (Comparative Example 9) in shrimp. Applying the krill hydrolysate as a top-coating (Example 6) also increases feed digestibility as compared to the same amount of krill hydrolysate added by inclusion into the diet (Example 8).

It can be concluded that the dietary treatments prepared according to the invention (Examples 6 - 8) perform best. When the dietary preparations comprise the krill hydrolysate KH3 as a topcoating (Examples 6 and 7), shrimp perform better.

Claims

1. A method for the non-therapeutic treatment of aquatic animals, wherein said treatment comprises orally administering a feed to aquatic animals, wherein said feed comprises a hydrolyzed crustacean catch in an amount between 10 and 200 g/kg of dry weight of said feed, wherein said hydrolyzed crustacean catch is obtained by a method comprising providing a crustacean catch, disintegrating said crustacean catch to provide a disintegrated crustacean catch, contacting the disintegrated crustacean catch with at least one exogenous endoprotease and at least one exogenous exoprotease to provide the hydrolyzed crustacean catch, and wherein the aquatic animals are selected from anadromous fish, crustacean and mollusks.

2. The method according to claim 1, wherein the anadromous fish are salmonids, preferably smolt.

3. The method according to claim 1 or claim 2, wherein the crustacean are prawn or shrimp.

4. The method according to any one of the preceding claims, wherein the disintegrated crustacean catch is a disintegrated krill catch.

5. The method according to any one of the preceding claims, wherein the disintegrated crustacean catch is contacted simultaneously with the at least one exogenous endoprotease and at least one exogenous exoprotease.

6. The method according to any one of the preceding claims, wherein the disintegrated crustacean catch is contacted with an amount of at least 0.1 g/kg, or at least 0.2 g/kg, or at least 0.5 g/kg, or at least 1.0 g/kg, or at least 2.0 g/kg of the at least one exogenous endoprotease, relative to the total weight of said disintegrated crustacean catch.

7. The method according to any one of the preceding claims, wherein the disintegrated crustacean catch is contacted with an amount of at least 0.01 g/kg, or at least 0.02 g/kg, or at least 0.05 g/kg, or at least 0.1 g/kg, or at least 0.2 g/kg of the at least one exogenous exoprotease, relative to the total weight of said disintegrated crustacean catch.

8. The method according to any one of the preceding claims, wherein the feed is a pelletized feed.

9. The method according to claim 8, wherein the pelletized feed is a coated pelletized feed comprising a core and at least one coating layer, and wherein at least part of the hydrolyzed crustacean catch is comprised in said coating layer.

10. The method according to any one of the preceding claims, wherein the feed comprises the hydrolyzed crustacean catch in an amount of less than 180, preferably less than 150, more preferably less than 120, even more preferably less than 100, most preferably less than 80 g/kg dry weight of said feed.

11. The method according to any one of the preceding claims, wherein the feed comprises the hydrolyzed crustacean catch in an amount of at least 12, preferably at least 15, more preferably at least 20 g/kg dry weight of said feed.

12. The method according to any one of the preceding claims, wherein the hydrolyzed crustacean catch is orally administered to aquatic animals for the purpose of reducing the conversion ratio of the feed used to feed aquatic animals without lowering their bodyweight gain.

13. The method according to any one of the preceding claims, wherein the hydrolyzed crustacean catch is orally administered to aquatic animals for the purpose of increasing their bodyweight gain.

14. Use of a hydrolyzed crustacean catch for reducing the conversion ratio of feed used to feed anadromous fish, crustacean or mollusks without lowering their bodyweight gain, or for increasing the bodyweight gain of the fish, which hydrolyzed crustacean catch is orally administered to said fish in an amount between 10 and 200 g/kg of dry weight of said feed, wherein said hydrolyzed crustacean catch is obtained by a method comprising providing a crustacean catch, disintegrating said crustacean catch to provide a disintegrated crustacean catch, contacting the disintegrated crustacean catch with at least one exogenous endoprotease and at least one exogenous exoprotease to provide the hydrolyzed crustacean catch.

15. A method for preparing a coated pelletized feed for aquatic animals, comprising a) providing a hydrolyzed crustacean, wherein said hydrolyzed crustacean catch is obtained by a method comprising providing a crustacean catch, disintegrating said crustacean catch to provide a disintegrated crustacean catch, contacting the disintegrated crustacean catch with at least one endoprotease and at least one exoprotease to provide the hydrolyzed crustacean catch; b) depositing the hydrolyzed crustacean catch to at least one surface of a pelletized feed to form a coated pelletized feed for aquatic animals.

16. A feed composition for aquatic animals comprising a) a hydrolyzed crustacean catch in an amount of between 10 and 200 g/kg of dry weight of said feed composition, said hydrolyzed crustacean catch being obtained by a method comprising comprising providing a crustacean catch, disintegrating said crustacean catch to provide a disintegrated crustacean catch, contacting the disintegrated crustacean catch with at least one endoprotease and at least one exoprotease to provide the hydrolyzed crustacean catch; b) one or more plant-based food ingredients in a collective amount of at least 50 dry weight percent based on the dry weight of the feed composition; and c) one or more additional ingredients selected from the group consisting of anti-caking agents, vitamins, minerals, various amino acids, free- flowing agents, animal feed flavors or the like.

17. The feed composition according to claim 16, wherein said feed comprises the hydrolyzed crustacean catch in an amount of between 20 and 80 g/kg of dry weight of said feed.

18. The feed composition according to claim 16 or claim 17, wherein the disintegrated crustacean catch is a disintegrated krill catch.

19. The feed composition according to claim 16, 17 or 18, wherein said feed composition is a coated pelletized feed composition comprising a core and at least one coating layer, wherein said core comprises said one or more plant-based food ingredients and the at least one coating layer comprises said hydrolyzed crustacean catch.