Note: Descriptions are shown in the official language in which they were submitted.
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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 al., 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
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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.
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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,
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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
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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.
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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)).
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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
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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.
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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 0.2ium to 0.4ium.
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)
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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 hydrolysing (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
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(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
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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 feedstuffs.
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 feedstuffs 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.
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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 LT94 , 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
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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
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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
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(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
(Hippog/ossus
hippog/ossus); 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
(ktalurus 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:
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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 mixturel 5
1
Per kg diet: vitamin A: 2500 IU; vitamin D3: 1500 IU; vitamin E: 200 mg;
vitamin K3: 10 mg; vitamin Bl: 15 mg; vitamin B2: 25 mg; vitamin B3: 75 mg;
vitamin B5: 30 mg; vitamin B6: 15 mg; vitamin B9: 5 mg; vitamin B12: 0.02 mg;
vitamin C: 125 mg; biotin: 0.25 mg; Ca: 1.1g; 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).
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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.1., 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 Ina 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):
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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.
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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 mixturel 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-containing diets were designed to contain enough SBM to induce
enteritis.
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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.
