WO2012049503A1

WO2012049503A1 - Algae-derived feed additive

Info

Publication number: WO2012049503A1
Application number: PCT/GB2011/051973
Authority: WO
Inventors: Odd Helge ROMARHEIM; Margareth ØVERLAND; Liv Torunn Mydland; Anders Skrede; Thor Landsverk
Original assignee: Norwegian University Of Life Sciences; Golding, Louise
Priority date: 2010-10-12
Filing date: 2011-10-12
Publication date: 2012-04-19
Also published as: EP2627344A1; CL2013000960A1; GB201017197D0; CA2814505A1

Abstract

The invention provides a material derived from algae (e.g. of the genus Chlorella) for the prevention or amelioration of plant-induced enteritis in fish. The algae- derived material is preferably intact or ruptured cells of and/or a cell fraction of the said algae. Also provided are fish feeds comprising said algae-derived material for the prevention or amelioration of plant-induced enteritis in fish, as well as such feeds in combination with an enteritis-causing plant material, which may comprise a reduced fishmeal content and/or enhanced plant ingredient content relative to conventional fish feeds.

Description

Algae-derived feed additive

This invention relates to the use of algae or algae-derived material in the treatment and/or prevention of enteritis in fish, in particular for the prevention or amelioration of soybean-induced enteritis in carnivorous fish, e.g. salmonids such as Atlantic salmon.

Feed components account for around one third of the total cost of farming fish such as salmon and trout. Fishmeal has traditionally been the major protein source in feed for carnivorous fish. However, the price of fishmeal is steadily rising and the predicted increase in aquaculture production suggests that the world's supply of fishmeal will not be sufficient to meet future demand. Alternative sources of nutrients are therefore required to replace at least part of the fish meal fraction of aquafeeds.

The attention of the aquaculture industry has recently been drawn to plant- based ingredients, in particular plant protein sources, due to their ready availability and low price. However, such materials present a number of problems which limit their widespread use in aquaculture.

The most widely available plant protein commodity, soybean meal, is marketed as a feedstuff of high nutritional value for farmed animals, including salmon and trout, and is available as full-fat soybean meal (i.e. non-reduced oil content) and defatted soybean meal (reduced oil content) forms. In Atlantic salmon and rainbow trout, however, even at low levels in the diet soybean meal causes enteritis (a damaging inflammatory reaction of the distal intestine) which is a major ethical challenge and in turn causes impaired growth and feed utilisation

(Baeverfjord and Krogdahl, Journal of Fish Diseases (1996), 19: 375-387). This condition is often referred to as "soybean meal-induced enteritis" and is the main reason for low or no inclusion of soybean meal in diets for salmon and trout. The condition can also be induced by other plant materials, such as pea protein concentrate (Penn et ah, XIII International Symposium on Fish Nutrition and Feeding, Florianopolis, Brazil (2008), Book of Abstracts, p. 86).

Although the specific effects of alternative protein sources on the digestive physiology of fish have been most closely studied in the case of soybean products in feed for farmed salmonids, a recent report by the Norwegian Scientific Committee for Food Safety suggests there could be similar responses to other plant protein sources used by the fish feed industry. It is thus anticipated that other plant materials, such as pea protein concentrate, either alone or in combination, will show the same damaging effects as soybean meal when fed to fish as a replacement for fishmeal or other non enteritis-causing ingredients.

The severity of the morphological changes observed on feeding plant- derived products, such as soybean meal, to fish depends on the level of their inclusion in the diet. For example, enteritis of the distal intestine can be detected in salmon fed a diet containing as little as 10% by weight of soybean meal, with diets comprising 15% or more soybean meal (values based on total dietary protein) giving rise to severe morphological changes (Krogdahl et al., Aquaculture Nutrition (2003), 9: 361-371). Defatted soybean meal induces severe morphological changes in the distal intestine of Atlantic salmon and rainbow trout. Similar changes are observed on feeding soybean meal to other fish, such as gilthead bream, sea bream, common carp, Asian sea bass and channel catfish, although these are somewhat less severe.

The condition of "soybean meal induced enteritis" is not caused by the proteins in the feed, but rather by one or more alcohol-soluble components in the non-protein fraction. Protein concentrates and isolates produced from plant-based materials (e.g. soybean) following alcohol extraction can therefore be used without causing enteritis. Such products are, however, very expensive.

Thus, whilst alternative plant protein sources such as soybean meal represent promising candidates for partial replacement of fishmeal in aquaculture feeds, at present they cannot be used in any significant quantity in fish feeds due to the problem of enteritis. The present invention seeks to address this problem.

The present inventors have now surprisingly found that the addition of algae to the diet of fish can prevent or ameliorate plant-induced enteritis. In particular, it has been discovered that the addition of low to moderate levels of algae, in particular from the genus Chlorella, to fish diets which include plant-based materials at levels which would otherwise cause enteritis renders such materials safe for feeding to fish. Algae are known for certain uses in aquafeeds. For example, they can be used as a feed additive in hatchery feeds. Chlorella has been suggested for use as a natural source to enhance carotenoid pigmentation in fish. However, the use of algae or algae-derived material, e.g. from the genus Chlorella, are not known for the treatment and/or prevention of enteritis in fish, e.g. soybean-induced enteritis.

Thus, in a first aspect, the present invention provides a substance derived from algae, especially from algae of the genus Chlorella, for the prevention or amelioration of plant-induced enteritis in fish. Such a substance is referred to herein as an "algae-derived material", i.e. a material containing or derived from algae, especially from algae of the genus Chlorella. In particular, the term "algae-derived material" is used to mean intact and/or ruptured algal cells and/or a cell fraction of algae, especially algae of the genus Chlorella. In particular, by the term "substance derived from algae, especially from algae of the genus Chlorella" is meant a nutrition-providing material comprising said algae, or a nutrition-providing extract or processed fraction of said algae. By "nutrition-providing" is meant that the material is suitable for use as an ingredient in fish feed, e.g. suitable for the partial replacement of conventional feed ingredients such as fishmeal. By "intact cells" is meant that the cell wall of the majority of the algal cells is largely intact; preferably the cell wall remains largely intact on at least 50%, and especially on at least 75% or at least 90%, of the algal cells in the substance. The term "intact cells" may be used to describe cells that have been treated to weaken or partially remove the cell wall, e.g. with lytic enzymes, but preferably refers to cells which have not been so treated. By "ruptured algal cells" is meant a material comprising essentially all of the constituents of the intact algal cells but wherein the cell wall of the majority of the algal cells is largely broken (e.g. the cells have been lysed); preferably the cell wall has been broken on at least 50%, and especially on at least 75% or at least 90%, of the algal cells in the substance. By "cell fraction" is meant an isolated part of the algal cell. Examples of cell fractions include cell wall material and cellular extracts, e.g. the non-cell wall fraction or the soluble cell fraction.

In one aspect the invention provides intact or ruptured cells of an algae, e.g. from the genus Chlorella, for the prevention or amelioration of plant-induced enteritis in fish. In another aspect the invention provides a cell fraction of an algae, e.g. from the genus Chlorella, for the prevention or amelioration of plant-induced enteritis in fish.

Typically, the algae-derived material will be directly incorporated into a conventional fish feed, for example a formulated fish feed, which will also comprise the plant-based material responsible for causing the enteritis. In a further aspect the invention thus provides a fish feed comprising an algae-derived material, preferably intact or ruptured cells and/or a cell fraction of algae of the genus Chlorella, in combination with an enteritis-causing plant material. As used herein, the term "fish feed" will generally be understood to be a complete food for fish, i.e. one which comprises all the necessary components of a fish diet.

In a related aspect the invention provides a fish feed component comprising (e.g. consisting essentially of) an algae-derived material, preferably intact or ruptured cells and/or a cell fraction of algae of the genus Chlorella in combination with an enteritis-causing plant material. This feed component may be provided, for example, as an admixture of said algae-derived material and said plant material for use as an ingredient (e.g. a protein-containing ingredient) in the preparation of a fish feed.

In a related aspect, the invention further provides a fish feed or fish feed component comprising intact or ruptured cells and/or a cell fraction of an algae, e.g. from the genus Chlorella, for the prevention or amelioration of plant-induced enteritis in fish.

In a further aspect the invention provides the use of an algae-derived material as herein described in the manufacture of an agent (e.g. in the manufacture of a fish feed or fish feed component) for the prevention or amelioration of plant-induced enteritis in fish.

In a yet further aspect the invention provides a method of preventing or ameliorating plant-induced enteritis in fish, said method comprising administering to said fish an algae-derived material, a fish feed or a fish feed component as herein described.

By the term "plant-induced enteritis" is meant a sub-acute inflammatory condition of the distal intestinal mucosa which arises from ingestion of a plant material. More specifically, the term is intended to refer to a condition associated with one or more of the following changes to the distal intestine: (1) widening and shortening of the intestinal folds; (2) loss of the normal supranuclear vacuolization in the absorptive cells (enterocytes) of the intestinal mucosa; (3) widening of the central lamina propria within the intestinal folds, with increased amounts of connective tissue; and (4) infiltration of a mixed leucocyte population (inflammatory cells) in the lamina propria and submucosa. These morphological changes are associated with impaired functionality of the distal intestine and are characteristic of the changes induced in fish (especially in salmon and trout) by dietary soybean meal. Such changes may readily be identified by the skilled person from a histological study of distal intestinal sections.

Plant materials which may give rise to enteritis include whole, processed or extracted plant products, in particular plant protein materials. Such materials may, for example be derived from any of the following sources: soybean, sunflower, lupin, rapeseed, canola, cottonseed, peanut, peas (e.g. field peas), beans (e.g. faba bean), grain, barley, corn and maize.

Plant materials suitable for use in the invention are those which provide nutritional value in fish feeds and which are suitable for use as an ingredient in fish feed, e.g. suitable for the partial replacement of conventional feed ingredients such as fishmeal. Such products are those which give rise to enteritis in fish. Specific examples of materials which may result in enteritis or enteritis-like conditions in fish (depending on their level of inclusion in the diet) include soybean (full-fat, defatted, hulled or non-hulled), sunflower (e.g. defatted sunflower), lupin (e.g. dehulled lupin), rapeseed (e.g. defatted double-low rapeseed), whole field pea (e.g. having a crude protein content of around 24% by weight), dehulled peas (e.g. having a crude protein content of around 26% by weight), pea protein concentrate (e.g. having a crude protein content of around 35% or of around 50-60%) by weight), faba beans (e.g. whole and dehulled faba beans), co-products derived from biofuel production and the brewing industry, barley protein concentrate, wheat gluten and corn gluten. These ingredients may be provided without heat treatment, or may be pre-treated by methods such as roasting, toasting, microwaving, expansion, pelleting, extrusion or other known heat treatments. Soybean products are one example of a plant-based material known to give rise to enteritis in fish. Such products are widely available in a number of different forms and all of these are considered suitable for use in the invention. These include solvent extracted soybean meal which refers to the soybean product after the extraction of part of the oil by solvents like hexane; soybean cake derived by mechanical pressure or soybean chips; soybean meal which is ground solvent extracted soybean flake or ground soybean cake, ground soybean chips, or ground soybean flakes; soybean mill feed which is the by-product resulting from the manufacture of soybean flour or grits and is composed of soybean hulls and the offal from the tail of the mill (a typical analysis by weight is 13% crude protein, 32% crude fibre and 13% moisture); soybean mill run which is the product resulting from the manufacture of dehulled soybean meal and is composed of soybean hulls and such bean meats that adhere to the hull under normal milling operations (a typical analysis by weight is 11% crude protein, 35% crude fibre and 13% moisture);

soybean hulls which is the product consisting primarily of the outer covering of the soybean; and solvent extracted soybean flakes which is the product obtained after extracting part of the oil from soybeans by the use of hexane or homologous hydrocarbon solvents.

Soybean meal is commercially available in full-fat (i.e. non-reduced oil content) and defatted (reduced oil content) forms. Soybean meal is typically defatted by solvent extraction of oils using hexane or homologous hydrocarbon solvents (e.g. n-hexane or 2,3 dimethyl pentane) which are subsequently distilled off during the toasting process. Defatted soybean meal (also known as "extracted" soybean meal) typically has the following composition (% given by weight):

Protein (N x 6.25) min. 42 %

Fat min. 0.2 %

Crude fibre max. 8.0 %

Ash max. 7.0 %

Water max. 12 % or max. 13.5%

Total carbohydrates represent the remainder, i.e. approx. 30% (of which approximately 10% is oligosaccharides, the rest is mainly non-starch

polysaccharides (e.g. galactans, arabinan)). Analysis of soybean products may be performed according to standard testing methods as adopted by the American Oil Chemists Society (AOCS), e.g. AOCS Method Ba 2a-38 (moisture), AOCS Method Ba 4e-93 (protein), AOCS Method Ba 6-84 (crude fibre) and AOCS Method Ba 3-38 (oil). Soybean meal may be dehulled (i.e. having a lower fibre content and higher crude protein content), or not dehulled.

As used herein, the term "soybean meal" refers generally to any material derived from soybean and which is suitable for use as a fish feed ingredient (with or without further processing). Suitable soybean meal for use according to the invention may be obtained from Denofa AS, Norway. Preferably the soybean meal comprises an enteritis-causing alcohol-soluble fraction, i.e. the soybean meal is capable of causing enteritis in recipient fish.

Recent studies have suggested that plant saponins may be one of the responsible factors causing symptoms of enteritis in fish. Saponins are naturally occurring amphiphilic molecules consisting of a sugar moiety linked to a steroid or triterpenoid aglycone. They are widely distributed in wild plants and are also present in many cultivated crops including soybeans and lupin seeds. Typical saponin levels in defatted soybean meal are 5-7 g/kg.

The plant material or plant source for use in the invention is thus preferably one which naturally comprises a proportion of saponins. Preferably the plant material is a plant protein-containing material, e.g. full fat soybean meal, defatted soybean meal or pea protein concentrate (e.g. pea protein concentrate having a protein content of at least 20%, e.g. around 35% or around 50-60%) by weight). Most preferably, the material is soybean meal. In a preferred embodiment, the plant material comprises plant proteins and saponins.

In a preferred embodiment of the invention, the enteritis is induced by one of the plant materials described herein. In an especially preferred embodiment, the plant-induced enteritis is soybean-induced enteritis.

The algae-derived material for use according to the invention is a substance derived from or comprising algae, especially algae of the genus Chlorella. A number of strains of Chlorella are known and have previously been investigated for use in nutrition. Examples of Chlorella strains which may be used according to the invention include Chlorella minutissima, Chlorella pyrenoidosa, Chlorella variabilis and Chlorella vulgaris. Chlorella vulgaris is preferred for use in the present invention.

When the algae-derived material comprises intact cells, these may be active (capable of reproduction) or inactivated (incapable of reproduction). In a preferred embodiment, especially where the cells are active, the algae-derived material is derived from a non-pathogenic algae, especially from an algae which is not a pathogen of an animal to which the material is to be fed (e.g. fish such as salmon) and/or which is not a pathogen of an animal which will ultimately eat the animal grown on the algae material-containing diet (e.g. humans or livestock such as cows, sheep, pigs etc.). One especially preferred algae-derived material comprises ruptured algal cells.

The algae-derived material for use in the methods herein described may be formed by growth of the algae on or in a suitable medium or substrate. The exact nature of the growth medium used to produce the algae-derived material is not critical and a variety of suitable substrates may be used. The algae for use according to the invention may be obtained from commercial sources, such as Synergy Natural Products Pty Ltd (Sydney, Australia) and may be used directly or further processed as described herein. Alternatively, algae for use according to the invention may be obtained from an organism depository, e.g. the American Type Culture Collection (ATCC), and can be cultured under standard conditions using known media (e.g. Bold's Basal Medium), for example as recommended by the organism depository.

Typically, the algae produced after initial culture may comprise (dry weight, i.e. at around 4-7% moisture content): about 50-60%, e.g. 58%, by weight protein; around 5-8% by weight ash; around 3-12% by weight lipids; around 8-12 g/kg phosphorus; around 3-4 g/kg magnesium; and around 2-3 g/kg calcium. The amino acid profile of the protein content can be expected to be nutritionally favourable with a high proportion of the more important amino acids. Typically these amino acids may be present in amounts of 45-55 g/kg of leucine, 35-40 g/kg of lysine, 25- 35 g/kg of threonine, 25-35 g/kg of valine, around 30 g/kg of isoleucine, around 30 g/kg of phenylalanine, 8-30 g/kg of tryptophan, 8-10 g/kg of methionine and about 4 g/kg of cysteine. Typically, the resulting algae culture will be produced in the form of a flowable aqueous paste or slurry. Generally this will consist essentially of whole cell material, although a proportion of ruptured cell material may also be present. This culture may be used directly (i.e. without further processing) or subjected to further processing steps before use as the algae-derived material component of the invention. Further processing steps include, in addition to centrifugation and/or filtration (e.g. ultrafiltration) processes whereby to reduce the water content prior to use. Suitable processing methods are known in the art.

Following production of the algal culture, cells may be concentrated from the growth medium, for example by conventional centrifugation and/or filtration methods, e.g. microfiltration or ultrafiltration. The size exclusion used during ultrafiltration will generally be in the range of about 100 kD. However, filters having a molecular weight cut-off in the range of from 10 to 100 kD, e.g. about 20 kD, may also be used. Microfiltration will generally be carried out using filters in the range of 0.2 μιη to 0.4 μιη.

Concentration of the algae material may be effected by centrifugation alone. If necessary, or indeed desirable, filtration (e.g. microfiltration and/or ultrafiltration) methods may be used to further increase the solids content of the algae material.

Following centrifugation and/or ultrafiltration the algae-derived material will be a relatively viscous protein slurry or paste. Although this may be used directly in the products and methods herein described, this will usually be further processed whereby to remove excess water from the product. The choice of any additional drying step or steps will depend on the water content of the material and the desired moisture content of the final product and could be determined by the skilled person. Typically, the product will be further processed in accordance with spray drying techniques well known in the art.

Cell fractions of the algae of the present invention may be produced by known means. Generally these will involve enzymatic and/or physical disruption of the algae cells, optionally followed by one or more steps to isolate the desired cell fraction. Algal cell fractions may be obtained by disruption of the algal cell (e.g. using pressure drop, enzyme treatment and/or homogenisation) followed by isolation of the desired fraction, such as by centrifugal separation of a solid (e.g. cell wall) fraction from the algae extract. In the pressure drop process, the Chlorella is stored in a pressurised holding vessel. Sudden release of the pressure in the holding vessel results in the breaking open of the cell wall of the algae. This process can break open the majority of cells in the culture, e.g. more than 95% of the cells. US patent No. 6,000,551 describes several alternative methods for rupturing microalgae. Cell fractions, e.g. the cell wall fraction, may be solvent extracted, washed, dried and/or pasteurised, e.g. on a steam drum drier, according to known methods. Preferably the algal cells are disrupted using pressure treatment and the ruptured cells are optionally spray dried to provide an algae-derived material for use according to the invention. A preferred processing technique involves drying of the cells (e.g. by spray drying) followed by cell lysis (e.g. using a pressure drop process).

Further processing steps which may be performed include enzymatic hydrolysis of the partially-purified algae fraction. The partially or fully purified fraction may also be processed by centrifugation and/or ultrafiltration as described above. The choice of process conditions (e.g. centrifugation speed and/or filter molecular weight cut-off) will depend on the fraction to be isolated and may be readily determined by the skilled person using the knowledge in the art and routine optimisation. For example, a hydrolysate may be produced by the action of one or more enzymes capable of hydro lysing (e.g. hydrolytically degrading) the cell structure and/or intracellular components of the algal cells, preferably an enzyme or enzyme system capable of hydrolysing the nucleic acid content of the cells.

Autolysates may similarly be prepared by incubation of the algal cells under carefully controlled conditions to allow the endogenous enzymes contained within the cells, such as nucleases and proteases, to digest the components of the cell. This "self-digestion" process results in the production of various degradation products of the cell which may include peptides, amino acids, nucleotides, phospholipids, fatty acids, etc. Suitable reaction conditions for hydrolysis and autolysis of the algal cells may be determined by one skilled in the art.

The algae-derived material for use in the invention may also be a permeate, i.e. the soluble fraction obtained following filtration, e.g. microfiltration or ultrafiltration. One preferred permeate is obtained by homogenisation and then filtration of the algal cells. To improve the yield of product, the homogenisate (which may be hydrolysed or autolysed) may be washed repeatedly (e. g. up to 5 times, e. g. up to 3 times) with water followed by ultrafiltration steps. Following separation of the permeate from the solid fraction retained by the filter (herein called the retentate) the solids content of the permeate may be expected to be about 1 to 10% by weight, e.g. in the range of from 2 to 5% by weight.

The algae-derived material herein described may be treated to reduce the nucleic acid content of the material. General methods for the reduction in nucleic acid content of a cellular material are known in the art and include heat shock treatments. An algae-derived material, treated to reduce the nucleic acid content is referred to herein as a "nucleic acid-reduced" material. A nucleic acid-reduced material preferably comprises nucleic acids (measured as the content of DNA and RNA in the material relative to the other, non-solvent components) at a level of less than 40%, preferably less than 30% and especially preferably less than 20%, e.g. at a level of from 5 to 25%, of the content of nucleic acids in the algal cells before nucleic acid reduction treatment.

In a preferred embodiment, the invention therefore provides a algae-derived material or fish feed as herein defined, wherein said material is derived from a culture comprising an algae, e.g. from the genus Chlorella, by one or more of the following processes: centrifugation, filtration (e.g. ultrafiltration), pressure drop, homogenisation, hydrolysis and/or autolysis. Other suitable fractions include concentrates (e.g. a material enriched fraction following centrifugation and/or ultrafiltration), permeates (i.e. the soluble fraction obtained after filtration of the algae-derived material or fraction thereof) and nucleic acid-reduced fractions. The algae-derived material or fish feed of the invention is suitable for the prevention or amelioration of plant-induced enteritis in fish that suffer from said condition.

Typically, the algae-derived material herein described will be fed to fish in combination with the enteritis-inducing plant material, e.g. in a formulated fish feed, and is thus preferably incorporated into a conventional feed containing a plant protein source, e.g. soybean meal. Methods for mixing feed components and providing fish feeds (e.g. in extruded or pellet form) are well known in the art.

Preferred forms for fish feeds of the invention include dry pelleted, expanded and extruded forms and also include moist and semi-moist forms. Alternatively, the algae-derived material may be fed to fish separately from the enteritis-inducing plant material as a supplement to the diet.

A process for preparing a fish feed or feed component according to the invention, e.g. as described above, will generally include the admixture of the algae- derived material with an enteritis-causing plant material (e.g. soybean meal) and, optionally, with one or more conventional fish feed ingredients. This mixture may then be further processed. Suitable conventional ingredients and methods for preparing fish feeds and feed components are well known in the art.

Thus, viewed from a further aspect, the invention provides a process for preparing a fish feed or feed component according to the invention, which process comprises admixing an algae-derived material as defined herein and an enteritis- causing plant material (e.g. soybean meal). In a preferred embodiment, the process comprises admixing said algae-derived material with one or more conventional feed ingredients.

Fish which may suffer from plant-induced enteritis include both carnivorous and omnivorous fish (although the extent to which they suffer from the condition will depend on several factors, including the fish species, inclusion level and duration of feeding the plant material, etc.). The condition is typically observed within 1 day to 4 weeks of feeding the fish on plant-derived feedstuff^'s. Typical levels of dietary inclusion of plant-based materials which give rise to enteritis in fish are at least 5% by weight, especially at least 8% by weight and particularly at least 10% by weight (based on the total weight of the diet). Such values may also be given based on the total weight of dietary protein and may be at least 5% by weight, preferably at least 8% by weight, e.g. in the range of 8 to 40%, preferably 10 to 25%. Plant-derived feedstuff^'s which can induce enteritis are as herein defined, but include soybean meal (full-fat and defatted) as well as pea protein concentrates and other enteritis-causing materials.

The maximum tolerable level of plant protein in the feed according to the invention will be dependent on the species of fish and on the level of other components in the feed, especially on the level of algae-derived material in the feed, but may readily be determined by those skilled in the art. A conventional feed (e.g. for salmonids) may, for example, comprise (by weight): 5-50%, e.g. about 25%, fishmeal; 5-35%, e.g. about 20%, fish oil; 0-40%, e.g. about 25%, non enteritis-causing plant protein ingredients; 5-20%, e.g. about 10%), plant oils; 5-15%, e.g. about 10%, starch ingredients (e.g. wheat); and about 1% of other components (e.g. minerals, vitamins, colouring agents etc.). A preferred source of fishmeal is Norse LT-94^®, low-temperature dried fishmeal from

Norsildmel (Bergen, Norway). A preferred source of soybean meal is extracted and toasted soybean meal from Denofa AS (Norway). A preferred source of fish oil is Silfas AS (Karmsund, Norway). Other components may be obtained from Rousselot SAS (Courbevoie, France), e.g. Rousselot^® 250 PS gelatin, and from Lyckeby Culinar (Fjalkinge, Sweden), e.g. Lygel F 60 potato starch.

In one embodiment, the fish feed according to the invention may comprise (by total weight of feed): 0-25%, e.g. 5-15%, preferably about 12% fishmeal; 1- 50%), preferably 5-40%, especially 10-25%), e.g. about 15%, algae-derived material as herein defined; and 5-50%, e.g. 10-40% or 15-30%, preferably about 20%, enteritis-causing plant material (e.g. soybean meal). In a further embodiment, the fish feed according to the invention may comprise (by total weight of feed): 0-25%, e.g. 5-15%), preferably about 10% fishmeal; 2.5-30%), preferably 5-20%, especially 10-15%), algae-derived material as herein defined; and 5-20%, preferably 10-15%), enteritis-causing plant material (e.g. soybean meal). The fish feed of the invention will typically comprise other components for the health and nutrition of fish, such as are listed above. The quantities of these additional components may be determined by the skilled person.

Where the fish feed is intended for use in feeding salmon this may contain at least 5% by weight soybean meal, e.g. at least 10%, at least 15%, at least 20% or at least 25% by weight (based on the total weight of the diet). Where the fish feed is intended for use in feeding trout, the feed may contain at least 10% by weight soybean meal, e.g. at least 20%, at least 30% or at least 35% by weight (based on the total weight of the diet).

As will be appreciated, where other protein ingredients are present in the final feed and the algae-derived material is not intended to provide any significant contribution to the protein content of the fish diet, it is preferable that this should be used in relatively low amounts provided that this is capable of achieving the necessary enteritis-reducing effect. On the other hand, where the algae-derived material is intended to contribute to the protein content of the diet, this may be used in higher amounts. The actual amount of algae-derived material which may be used will naturally be dependent on several factors, including the nature of the enteritis- causing plant material (i.e. the extent to which this is responsible for enteritis) and the amount in which this is present in the feed, the nature of the fish species, etc. Taking into account these factors, suitable amounts of the algae-derived material may readily be determined by those skilled in the art.

In one embodiment, a fish feed of the present invention may comprise at least 0.5% by weight of algae-derived material (as herein defined), preferably at least 0.75%) by weight, e.g. at least 1 , 1.2, 1.5, 2, 2.5, 3, 4 or 5% by weight. A fish feed comprising between 1.5 and 2.5% by weight of algae-derived material is particularly preferred. Alternatively, a fish feed comprising between 1 and 50% by weight, especially between 2 and 40%> by weight, e.g. between 2.5 and 30%>, between 5 and 20%>, e.g. about 10%> or about 20%>, or between 10 and 15%> by weight, is also preferred. A fish feed comprising between 20 and 50%> by weight, especially between 30 and 40%> by weight, algae-derived material is also preferred. In a further embodiment, the fish feed of the present invention may comprise up to 40% by weight of enteritis-causing plant material as herein defined (e.g. soybean meal), preferably up to 30%> by weight. A fish feed comprising between 5 and 40%> by weight plant material, especially between 10 and 35%>, e.g. from 10 to 15%>, by weight or 20 and 30% by weight plant material is preferred. A fish feed comprising between 2.5 and 15% by weight of algae-derived material and between 10 and 20% by weight of enteritis-causing plant material as herein defined (e.g. soybean meal) is especially preferred.

The ratio of plant material (e.g. soybean meal) to algae-derived material in the feeds herein described is preferably between 1 : 10 and 10: 1 , preferably between 1 :8 and 8: 1 , between 1 :5 and 5: 1 , between 1 :3 to 3 : 1 or 1 :2 to 2: 1 , especially preferably about 1 : 1. In an alternative embodiment, the ratio of plant material to algae-derived material in the feeds herein described may be less than 80: 1 , preferably less than 40: 1 , e.g. less than 16: 1. For example, suitable ratios may be 8: 1 to 2:3, preferably 4: 1 to 2:3, especially 3: 1 to 1 : 1. In an alternative embodiment, the ratio of plant material to algae-derived material in the feeds herein described may be 25 : 1 to 2: 1 , especially around 4: 1. A preferred ratio of plant material (e.g. soybean meal) to algae-derived material in the feeds herein described is less than 8: 1, especially less than 4: 1 , e.g. between 4: 1 and 1 : 1, especially about 2: 1.

In the embodiment of the invention where the algae-derived material is provided as part of a feed component, the relative amounts of algae-derived material and enteritis-causing plant material may be the same as those set out above in respect of the above feeds, i.e. where no further algae-derived material or plant material are provided in the final feed (and thus the ratio of said components in the final feed will be the same as that in the feed component). Alternatively, the relative amounts of algae-derived material and plant material in the feed component may be such that incorporation of said component into the final feed results in the ratios described above. In a particularly preferred embodiment, the ratio of plant material (e.g. soybean meal) to algae-derived material in the feed component is 20: 1 to 1 :4, preferably 8: 1 to 1 :3. Preferred feed component may comprise a ratio of enteritis- causing plant material to algae-derived material of around 10: 1 or of around 4: 1, a ratio of 10: 1 is particularly preferred. In an alternative embodiment, a preferred feed component comprises a ratio of enteritis-causing plant material to algae-derived material of between 4: 1 and 1 : 1, e.g. around 4: 1, around 2: 1 or around 3:2.

Particularly preferred in accordance with the invention is a feed component which comprises about 10% by weight algae-derived material and about 90% by weight enteritis-causing plant material. Also preferred in accordance with the invention is a feed component which comprises about 20% by weight algae-derived material and about 80% by weight enteritis-causing plant (e.g. soybean meal), or which comprises about 35 > by algae-derived material and about 65 % by weight enteritis-causing plant material.

It is envisaged that the plant-based material herein described will be used as a replacement for fishmeal or other non enteritis-causing protein sources in fish feeds. Accordingly, the invention further provides a fish feed having a reduced content of non enteritis-causing protein sources, e.g. having a reduced fishmeal content, characterised in that some or all of the non enteritis-causing protein source (e.g. fishmeal) is replaced by an enteritis-causing plant material and an algae- derived material as herein defined. Preferably the plant material is as hereinbefore defined and is especially preferably soybean meal. Preferably at least 50% of the non enteritis-causing protein source (e.g. fishmeal) may be replaced, e.g. at least 75%, at least 85% or at least 95%.

Whilst the methods herein described are applicable to any fish susceptible to plant-induced enteritis, the present invention is directed particularly at carnivorous fish, especially salmonids (of the family Salmonidae) and particularly salmon, e.g. Atlantic salmon (Salmo salar), chinook salmon (Oncorhynchus tshawytscha) or Coho salmon (Oncorhynchus kisutch); trout, e.g. rainbow trout (Oncorhynchus mykiss); char, e.g. Arctic char (Salvelinus alpinus); whitefish, e.g. common whitefish (Coregonus lavaretus); grayling, e.g. Thymallus thymallus; and amberjack, e.g. Seriola dumerili. The invention is also applicable to bream, e.g. gilthead bream (Sparus aurata Linn.) and sea bream; carp, e.g. common carp (Cyprinus carpio); cod, e.g. Atlantic cod (Gadus morhua); halibut, e.g. Atlantic halibut (Hippoglossus hippoglossus); turbot, e.g. European turbot (Psetta maxima); sea bass (e.g. Asian sea bass); tilapia, e.g. of the genus Oreochromis; and catfish, e.g. channel catfish (Ictalurus punctatus) and members of the family Pangasiidae (e.g. Pangasius bocourti).

The invention will now be described in more detail by reference to the following non-limiting Examples and Figures, in which:

Figure 1 shows the effect of three different diets on the degree of enteritis in Atlantic Salmon. Example 1 - Fish feed components

Table 1 shows typical compositions of various ingredients which may be formulated to produce fish feeds: Table 1

Fishmeal Wheat Soybean

meal

Ingredients, g kg^"1

Crude protein 677.50 134.63 454.56

Dry matter 913.24 861.95 881.36

Ash 127.48 13.18 58.02

Crude lipids 80.48 13.43 9.30

Starch 5.80 598.00 4.00

Example 2 - Complete fish feed Table 2 shows the formulation of a fish diet. The diet contains soybean meal

(SBM) at a level of 200 g/kg and Chlorella vulgaris (Synergy Natural Products Pty Ltd - Sydney, Australia) at a level of 100 g/kg. The diet is in accordance with the present invention. Table 2

Main ingredients (g/kg) Fish diet

Fishmeal 355

Soybean meal 200

Chlorella vulgaris 100

Fish oil 140

Gelatine 100

Potato starch 100

Vitamin, mineral, marker

and astaxanthin mixture¹ 5

¹ Per kg diet: vitamin A: 2500 IU; vitamin D₃: 1500 IU; vitamin E: 200 mg; vitamin K₃: 10 mg; vitamin 15 mg; vitamin B₂: 25 mg; vitamin B₃: 75 mg;

vitamin B5 : 30 mg; vitamin B₆: 15 mg; vitamin B9: 5 mg; vitamin Bi₂: 0.02 mg; vitamin C: 125 mg; biotin: 0.25 mg; Ca: l . lg; Zn: 120 mg; Mn: 15 mg; Cu: 5 mg; Co: 1 mg; I: 3 mg; astaxanthin: 175 mg (F. Hoffmann-La Roche, Basel,

Switzerland); Yttrium oxide 99,9 % purity: 100 mg (Metall Rare Earth Limited, Shenzhen, Guangdong, China). Example 3 - Feeding study Diets

A diet with 200 g/kg soybean meal in combination with 100 g/kg of algae was prepared as defined in Table 2. The ingredients were mixed and then cold pelleted through a pasta machine (P35 SP, Italgi S.r.L, Carasco, Italy) with a 3 mm dye and rotating cutting knives at the end of the dye. The diets were kept frozen until feeding. Fish and rearing conditions

Atlantic salmon (Salmo salar) with 26g mean initial weight were randomly distributed into a tank with 50 fish in the tank, and the diet was fed to the fish. The tanks were supplied with fresh water, and the fish were fed continuously by automatic feeders for 31 days. The mean fish weight was 33 g per fish at termination of the experiment. Oxygen levels and temperatures were recorded daily according to standard routines.

Weighing and sampling for analysis

At termination of the experiment, five fish from the tank were picked at random for sampling of organs for histochemical evaluation. The fish were anaesthetised with MS222 and killed by a sharp blow to the head. Organs were sampled for morphological evaluation.

The intestinal tracts of the five fish were dissected for histological examination of wall tissue from distal intestine. Samples of about 5 x 5 mm were obtained from the mid part of the intestinal sections and fixed in 4% phosphate buffered formaldehyde (10% formalin). Formalin fixed tissue was routinely dehydrated in ethanol 48 hours pre-sampling, equilibrated in xylene and embedded in paraffin according to standard histological techniques. Sections of approximately

5 μιη were cut and stained with haematoxylin and eosin before examination under a light microscope. Intestinal morphology was evaluated according to the following criteria described for soybean meal-induced enteritis in Atlantic salmon

(Baeverfjord and Krogdahl, supra): 1) Accumulation of leukocytes (lymphocytes, granulocytes and granular cells) in the lamina propria;

2) Changes in epithelium including a) Reduced vacuolization; b)

Cytoplasmic basophilia (RNA staining); and c) Reduced cellular height

3) Atrophy, i.e. reduced height of the intestinal folds; and

4) Oedema, i.e. accumulation of protein-rich fluid in the lamina propria.

Individual histological sections were evaluated and graded as follows, according to criteria (1) to (4) above:

0 = Normal intestine

1 = Slight changes associated with enteritis

2 = Moderate changes associated with enteritis

3 = Severe changes associated with enteritis.

The average score was calculated as the mean of individual scores for each fish. A score of 1 or more in any of the four criteria examined is indicative of soybean-induced enteritis in the fish from which the sample was taken.

Results

Table 3 shows the results of a visual classification from histology of distal intestinal sections. Scores are given as average values across the five fish.

Table 3

Average classification Accumulation Changes in Atrophy Oedema of distal intestine of leukocytes epithelium

Fish diet 0.6 0.8 0.6 0.2 The fish fed the diet containing the algae generally show healthy intestines with histological scores in the range expected of normal fish. Conclusions

Morphological studies of the distal intestine of fish fed a diet with sufficient soybean meal to cause significant levels of enteritis show that inclusion in the diet of 10% by weight of Chlorella can prevent the soybean meal induced enteritis.

Example 4 - Complete fish feeds

Table 4 shows the formulation of three fish diets. Diets 1 and 2 are comparative diets containing no algal material and either soybean meal (SBM) at a level of 200 g/kg, or no SBM, respectively. Diet 3 contains SBM and Chlorella at a level of 200 g/kg.

Table 4

Main ingredients (g/kg) Diet 1 Diet 2 Diet 3

Fishmeal 510 710 295

Soybean meal 200 - 200

Chlorella vulgaris - - 200

Fish oil 135 135 150

Gelatine 75 75 75

Potato starch 75 75 75

Vitamin, mineral, marker

and astaxanthin mixture¹ 5 5 5 1 as Example 2

Example 5 - Feeding study

This experiment was designed to evaluate the effectiveness of a Chlorella vulgaris (Chlorella) algae-derived meal at a higher level of inclusion in protecting Atlantic salmon against enteritis caused by plant materials in the feed. Soybean meal was used as the enteritis inducing plant material in this experiment. The Chlorella-contammg diets were designed to contain enough SBM to induce enteritis. Materials and methods

Three diets (compositions shown in Table 4 above) were prepared using the method described in Example 3.

Triplicate tanks with Atlantic salmon of about 100 g initial weight were fed one of the three diets for four weeks. After completion of the trial, the intestines of the salmon were evaluated for signs of enteritis as described in Example 3.

Results

The results of the experiment are shown in Figure 1 , which clearly shows the effect of diet on the level of enteritis in the distal intestine of the Atlantic salmon. In Figure 1, the level of confidence in the results is shown by the "+" or "*" symbols above the corresponding bar. "+++" denotes a confidence level of p<0.001 relative to the corresponding value from diet 2, whereas "++" denotes a p<0.01 confidence level. "***" denotes a confidence level of p<0.001 relative to the corresponding value from diet 1, whereas "**" denotes a p<0.01 confidence level.

The results of this experiment clearly show that inclusion of Chlorella (microalgae) at a level of 20% by weight of feed in the diet of Atlantic salmon abolished the intestinal changes signifying plant-induced enteritis (SBM positive control). In the fish fed the algae, all of the investigated parameters are significantly different from the positive, soybean meal control while none differ significantly from the negative, fish meal control.

Claims

Claims:

1. Algae-derived material (e.g. intact or ruptured cells, and/or a cell fraction) for the prevention or amelioration of plant-induced enteritis in fish.

2. A fish feed for prevention or amelioration of plant- induced enteritis in fish, said fish feed comprising algae-derived material (e.g. intact or ruptured cells and/or a cell fraction).

3. The algae-derived material of claim 1 or the fish feed of claim 2, wherein the plant-induced enteritis is soybean-induced enteritis.

4. A fish feed (preferably in a dry pelleted, expanded or extruded form, or in a moist or semi-moist form) comprising algae-derived material (e.g. intact or ruptured cells and/or a cell fraction) in combination with an enteritis-causing plant material.

5. A fish feed having a reduced fishmeal content, characterised in that some or all of the fishmeal is replaced by an enteritis-causing plant material and an algae- derived material (e.g. intact or ruptured cells and/or a cell fraction).

6. A feed component comprising (e.g. consisting essentially of) algae-derived material (e.g. intact or ruptured cells and/or a cell fraction) in combination with an enteritis-causing plant material.

7. The fish feed of claim 4 or claim 5 or the feed component of claim 6, wherein the enteritis-causing plant material is soybean meal.

8. The fish feed or feed component of any one of claims 4 to 7, wherein the ratio of said plant material to said algae-derived material is less than 8: 1, preferably between 4: 1 and 1 : 1, especially about 2: 1.

9. The algae-derived material, fish feed or feed component of any one of claims 1 to 8, wherein said material comprises intact or ruptured cells and/or a cell fraction of algae of the genus Chlorella, especially one or more of Chlorella minutissima, Chlorella pyrenoidosa, Chlorella variabilis and Chlorella vulgaris.

10. The algae-derived material, fish feed or feed component of claim 9, wherein at least 75% of the algal cells in the algae-derived material are ruptured cells.

11. The algae-derived material, fish feed or feed component of any one of claims 1 to 10, which is derived by one or more of the following processes fermentation, centrifugation, filtration (e.g. ultrafiltration), solvent extraction, homogenisation, hydrolysis, autolysis and drying (e.g. spray drying).

12. The algae-derived material, fish feed or feed component of any one of claims 1 to 11, for feeding to salmonid fish, e.g. salmon or trout.

13. A method of preventing or ameliorating plant- induced enteritis in fish, said method comprising feeding to said fish an effective amount of an algae-derived material or fish feed as defined in any one of claims 1-5 and 7-12.

14. Use of an algae-derived material as defined in any one of claims 1 to 12 in the manufacture of an agent for the prevention or amelioration of plant-induced enteritis in fish.

15. A process for preparing the fish feed or feed component of any one of claims 4 to 12, which process comprises admixing an algae-derived (e.g. as defined in any one of claims 1 to 12) and an enteritis-causing plant material.