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ANTIMICI~OBIAL ENZYMES IN ANIMAL FEED
Field of the invention
This invention relates to the use of antimicrobial enzymes, such as oxidases
and
lysozyme, and a polyunsaturated fatty acid (PUFA), in (monogastric and non-
ruminant)
animal feed to improve growth and feed conversion ratio of pigs, poultry, fish
and veal
calves.
Background of the invention,
Animals such as pigs, poultry, veal calves arid fish are grown intensively for
the
production of meat, fish and eggs. These animals are fed diets containing a
variety of raw
materials of animal and/or vegetable origin to supply energy and protein. Most
of the
feed that is consumed is produced commercially by the compound feed industry
but a
significant part is produced on the farm and fed directly. The feed is
supplemented with
vitamins and minerals to meet the requirements of animals for these essential
nutrients.
In the case of poultry, the feed is also supplemented with coccidiostats to
prevent
coccidiosis. The use of industrially produced enzymes as feed additives has
become
almost common practice. Examples of such enzymes comprise phytases, alpha-
amylases,
proteases and various plant cell wall degrading enzymes such as
f~-glucanases, endoxylanases and mannanases.
These enzymes are used to improve growth and feed conversion ratio and to
reduce the environmental pollution caused by manure from pigs, poultry and
fish.
However, feed costs are the most important cost factor in animal production.
During the 1950's it was realized that the addition of small amount of
antibiotics
to animal feed resulted in innproved zootechnical results in monogastric
animals.
Nowadays, antibiotics are used routinely as feed additives. The mode of action
of these
antibiotics on the improvennent of growth and feed conversion ratio is still
not fully
understood. The generic term for this class of feed additives is growth
promoters.
Examples of growth promoters include virginiamycin, tylosin, flavomycin and
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avoparcin.
The resistance of human pathogenic bacteria against antibiotics has been
increasing rapidly. This has made it more difficult to cure people from
bacterial
infections. The widespread use of antibiotics in animal feed has been blamed
by various
experts to accelerate the build-up of resistance to various antibiotics. This
has led to a ban
on the use of all antibiotics as growth promoters in animal feed in Sweden and
for
specific antibiotics, such as avoparcin, in Denmark. It is likely that other
countries will
follow these examples due to pressure from consumer and health care
organizations. The
feed industry therefore is very much interested in natural additives with
growth
promoting effects without any therapeutical use in humans.
Certain enzymes are known to be active as antimicrobial agents, and these may
be
used in the preservation of food. Glucose oxidase has also been suggested for
the
preservation of silage fodder and silage grain (WO-A-98/01694, Suomen Sokeri
Oy).
Plant cell wall degrading enzymes such as mannanase and b-glucanases are used
as
feed additives for diets containing high amounts of b-glucan and mannan to
reduce the
viscosity in the gastro-intestinal tract of monogastric animals caused by
these non-starch
polysaccharides. These enzymes also have some antifungal activity but do riot
exhibit any
antibacterial activity.
The antibacterial enzyme lysozyme has been added as a growth promoter to the
feed in monogastric animals (Latvietis, J., et al, In: Vitamine Zusatzstoffe
in der
Ernaehrung von Mensch and Tier. Symposium 5th (1995). Editor Rainer Schubert
et al.
Jena, September 28-29. ISBN 3.00.000361-4). These authors added lysozyme
prepared
from egg white to the feed ~of broilers and veal calves. Growth and feed
conversion were
allegedly improved. The concept however of using mixtures of antibacterial
enzymes in
combination with enzyme enhancers (eg. PUFAs) has not been published.
It is thus desirable for farmers and the compound feed industry to obtain an
optimum growth and feed conversion ratio, at minimum cost, in a sustainable
way,
respecting demands from both consumer and health care organisations alike.
Description of the Invention
The present invention provides an animal feed additive composition comprising
a
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mixture of antimicrobial enzymes which can show synergistic effects. The
effect of the
or each enzyme can be enhanced by the presence of one or more PUFAs. This may
allow the improvement of growth and feed conversion ratio of animals such as
pigs,
poultry, veal calves and aquatic or marine animals such as fish, and can allow
one to omit
an antibiotic as growth promoter.
A first aspect of the present invention relates to an animal feed additive
composition, suitable for a monogastric or non-ruminant animal, the
composition
comprising at least two antimicrobial enzymes and (as an enzyme enhancer) a
polyunsaturated fatty acid {PUFA).
Preferably one or two of the enzymes are antibacterial enzymes. These enzymes
may be of different types ancL~or may have different activity. One, eg. a
first, enzyme
may be able to disrupt the cell wall of bacteria. The enzyme may be one that
can attack
or degrade peptidoglycans. Far example, the enzyme may be able to cleave off
peptidoglycans. A preferred enzyme for this task is lysozyme. This (first)
enzyme may
I5 be present at a concentration to give from 50,000 to 150,000, such as from
70,000 to
130,000, optimally from 90,C>00 to 110,000 Shugar units per kilogram (or unit)
of animal
feed. In terms of weight, therefore, this first enzyme may be present at an
amount to
give a concentration in the animal feed of from 1 to 8 grams per kg of feed,
preferably
from 2 to 7 grams per kg of seed and optimally from 3 to 5 grams per kg of
feed.
The second enzyme rnay be able to generate a compound that is toxic to the
bacteria. This may be the same bacteria, of different, from the bacteria whose
cell walls
can be disrupted or degraded) by the first enzyme. The compound is preferably
a
peroxide, eg. hydrogen peroxide. Thus preferred enzyme are oxidases.
Particularly
preferred is glucose oxidase. This second enzyme may be present at a
concentration to
give from 500 to 1,500, preferably from 700 to 1,300, and optimally from 900
to 1,100
Sarett U per kilogram (or unit) of feed. Thus preferably this second enzyme
may be
present at an amount, by weight, to give a final concentration in the animal
feed of from
1 to 8 grams per kg of feed, ~~referably from 2 to 7 grams per kg of feed, and
optimally
from 3 to 5 grams per kg of feed.
Enzymes can function as antimicrobial agents in the following ways:
a) disruption of the cell wall;
b) generation of a toxic compound;
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c) removal of an essential nutrient; or
d) inactivation oi: enzymes essential for growth.
Each of these will be discussed in turn.
a) Microbial cell walls vary in structure for fungi, yeasts, gram negative and
gram positive bacteria. One c:an need different enzymes to disrupt the cell
wall of these
different types of microorganisms. The fungal and yeast cell wall, for
example, may be
disrupted by mannanases, chiainases and betaglucanases. The bacterial cell
wall,
however, is not sensitive to tlhese enzymes due to a different type of
structure. Gram
positive organisms have a peptidoglycan layer covered by some protein but
essentially
consists of peptidoglycan only. This substrate may be degraded by action of
lysozyme
(1,4-b-acetylmuramidase) which cleaves peptidoglycans between the C1 of N-
acetyl-
muramic acid and the C-4 of N-acetylglucosamine.
The peptidoglycan layer is covered by a tight lipopolysaccharide-protein-
divalent
cation-phospholipid layer in gram negative bacteria. This layer can hinder the
efficacy of
lysozyme in gram negative bacteria. Agents capable of disrupting this tight
lipopolysaccharide layer stimulate the action of lysozyme by giving the enzyme
access to
the peptidoglycan layer.
b) Oxidases are capable of producing hydrogen peroxide which is lethal to
most microorganisms. Glucose oxidase , for example, catalyses the conversion
of glucose
into gluconic acid and hydrogen peroxide. Xanthine oxidase, present in milk,
is also
capable of generating hydrogen peroxide.
Other antimicrobial compounds which may be enzymatically generated comprise
hypothiocyanate (produced b:y lactoperoxidase), chloramines (produced by
myeloperoxidase), free fatty acids (produced by lipase), poly-unsaturated
fatty acids,
lysophosphatidylcholine (produced by phospholipase A2) and
xylitol-5-phosphate {produced. by xylitol phosphorylase). This list is by no
means
exhaustive, however.
c) Oxygen may be removed from the media by means of oxidases such as e.g.
glucose oxidase. Complete removal of oxygen prevents the growth of aerobic
microorganisms.
d) Enzymes essential for growth of microorganisms may be inactivated by
means of other enzymes. Sulfhydryl oxidases, for example, are capable of
inactivating
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enzymes which depend on a<aive sulfhydryl groups for their activity.
The composition can comprise or enzyme enhancer, such as a compound that can
significantly improve the activity of the or each antimicrobial (eg.
antibacterial) enzyme,
preferably in a synergistic manner. The enzyme enhancer can comprise one or
more
polyunsaturated fatty acids, otherwise known as PUFAs. The or each PUFA can be
of
the n-3 or n-6 family. Preferably it is a C18, C20 or C22 PUFA. The PUFA can
be
provided in the form of a free fatty acid, as a fatty acid ester (e.g. methyl
or ethyl ester),
as a phospholipid and/or in t:he form of a triglyceride.
Preferred PUFAs incllude arachidonic acid (ARA), docosohexaenoic acid (DHA),
eicosapentaenoic acid (EPA) and/or y-linoleic acid (GLA). Of these, ARA is
preferred.
The PUFAs rnay be from a natural (e.g. vegetable or marine) source or may be
derived from a single cell or :microbial source. In particular, the PUFA may
be produced
by a bacteria, fungus or yeast:. Fungi are preferred, preferably of the order
Mucorales, for
example Mortierella,1'~thium: or Entomophora. The preferred source of ARA is
from
Mortierella alpina or I'~thium: insidosum.
The PUFA may be present as an oil. Suitable oils that include ARA are
available
from DSM N.V., Wateringseweg 1, P.O. Box 1, 2600 MA Delft, The Netherlands,
under
the trade mark VEVODARz~. Another commercially available oil is ARASCO~ from
Martek Corporation, 6480 bobbin Road, Columbia, MD 21045, United States of
America. Other PUFAs are available, for example DHA as a DHA oil (DHASCO~
from Martek Corporation or DHA from Pronova, Norway, under the trade mark
EPAX~).
The PUFA is preferably at a concentration that it allows it to be added to the
animal feed to give a final concentration of from 0.1 or 1 to 1000, such as
from 0.5 to 50
or 1 to 100, and preferably from 1 to 10 grams per kilogram (or unit) of feed.
All the antimicrobial enzymes can be produced on industrial scale and/or may
be
recombinant. Lysozyme is commercially available, isolated from egg white, or
may be
recombinant. The or each enzyme may be naturally occurring or may be an (eg.
recombinant) variant or mutant thereof.
The or each antibacterial enzyme is preferably recombinantly produced such as
by expression of a heterologous gene or cDNA in a suitable organism, or
alternatively by
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homologous (over)expression. of a suitable endogenous gene. The glucose
oxidase gene,
for example, has been overex~pressed in recombinant systems
(WO-A- 89/12675, Chiron). ~:.ysozyme (from egg white) can be recombinantly
expressed
by expression of the gene in Aspergillus niger (Archer, D.B. et al.,
Bio/Technology 8:
741-745 (1990). Lysozyme mutants (produced by protein engineering) can also be
used
which have better heat stability and stronger antimicrobial action.
The antimicrobial enzymes used in the invention will usually be either ones
which are not a natural constituent of the animal feed or are present in the
feed at a
concentration different from its natural concentrations.
A second aspect of the present invention relates to an animal feed composition
comprising at least two antim.icrobial enzymes and a PUFA. As with the
additive
composition, a first enzyme may be able to disrupt the cell wall of bacteria,
and a second
enzyme may be capable of generating a compound toxic to the bacteria.
A third aspect of the invention relates to a process for the preparation of an
animal feed composition, the process comprising adding to one or more edible
feed
substances) or ingredients) two or more antirnicrobial enzymes and a PUFA, or
an
additive of the first aspect.
The enzymes and/or F'UFA can be added to the animal feed composition
separately from the feed substances) or ingredient(s), individually or in
combination
with other feed additives. Alternatively, or in addition, the or each enzyme
can be an
integral part of one of the feed substances. This aspect includes both
preparing a feed
composition with the two en~:ymes and PUFA or supplementing an existing feed
composition with these compounds.
A particularly preferred method for the (exogenous) addition of the
antimicrobial
enzyme to animal feed is to add the or each enzyme as transgenic plant
material and/or
(e.g. transgenic) seed. The enzyme may thus have been synthesized through
heterologous
gene expression, for example t:he gene encoding the desired enzyme may be
cloned in to a
plant expression vector, under control of the appropriate plant expression
signals, e.g. a
tissue specific promoter, such as a seed specific promoter. The expression
vector
containing the gene encoding the enzyme can be subsequently transformed into
plant
cells, and transformed cells can be selected for regeneration into whole
plants. The thus
obtained transgenic plants can be grown and harvested, and those parts of the
plants
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containing the heterologous (to the plant) enzyme can be included in one of
the
compositions, either as such ~or after further processing. Reference here is
made to
WO-A-91/14772 which discloses general methods for the (heterologous)
expression of
enzymes in (transgenic) plants, including methods for seed-specific expression
of
enzymes. The heterologous enzyme may be contained in the seed of the
transgenic plants
or it may be contained in other plant parts such as roots, stems, leaves,
wood, flowers,
bark and/or fruit.
The addition of the enzyme in the form of transgenic plant material, e.g.
transgenic seed containing the antimicrobial enzymes, may require the
processing of the
plant material so as to make the enzyme available, or at least improve its
availability.
Such processing techniques may include various mechanical (eg. milling and/or
grinding)
techniques or thermomechanical treatments such as extrusion or expansion.
The antibacterial enz~rmes may be added to the feed composition at a
concentration which varies a;s a function of diet composition, type of enzyme
and target
animal species.
Preferably the compositions of the invention do not contain any antibiotics.
The
compositions) of the invention may also be free of a mineral component (such
as zinc
and/or iodine) and/or an im;munomodulating agent (such as ascorbic acid).
Although
each of the two antimicrobial enzymes and the PUFA can all be produced by a
micro-
organism, it is preferred that the composition is free of microorganisms that
produced
any of these compounds (or microorganisms from Streptomyces). Furthermore, the
composition may be devoid of microorganisms that produce lactic acid inside
the animal
(eg. those of the genus Lactobacillus or Enterococcus).
A fourth aspect of the present invention relates to a process for promoting
growth and/or feed conversion in a monogastric or non-ruminant animal, the
process
comprising feeding the animal at least two antimicrobial enzymes and a PUFA or
a feed
composition of the first or second aspect or preparable by the third aspect.
Suitable animals include farm, monogastric and/or non-ruminant animals such as
pigs (or piglets), poultry (suc:h as chickens, turkeys), calves or veal or
aquatic (e.g. marine)
animals (for example fish).
A fifth aspect relates t:o the use of a composition of the first aspect as an
additive
for a monogastric or non-ruminant animal feed composition.
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Preferred features and characteristics of one aspect of the present invention
are
applicable to another aspect ~~reutatis mutandis.
The present invention will now be described by way of example with reference
to
the following Examples which are provided by way of illustration and are not
intended
to limit its scope.
Comparative Examples 1 to 4 and Example 5
Characterization of antibacterial enz me products
Glucose oxidase (EC 1.1.3.4), an oxidase capable of generating hydrogen
peroxide,
was obtained as a commercial) product under the trade mark FERMIZYME GO~ from
DSM/Royal Gist-brocades, Bakery Ingredients Division, PO Box 1, 2600 MA DELFT,
The Netherlands. This enzyme preparation exhibits an activity of 500 Sarett
Units per
gram. One Sarett unit is the amount of enzyme that will cause an uptake of
lOmm3 of
oxygen per minute in a Warburg manometer at 30°C in the presence of
excess oxygen
and 3.3% glucose monohydrate in a phosphate buffer pH 5.9. The enzyme was
produced
by the fungus Aspergillus nige;r.
Lysozyme obtained from chicken egg-white was obtained as a commercial
product under the trade mark DELVOZYME~ from DSM/Royal Gist-brocades, Dairy
Ingredients Group, PO Box 1, 2600 MA DELFT, The Netherlands. The product
contains 5.1 x 106 Shugar units/ml product. One Shugar unit is defined as the
amount of
enzyme which causes a decrease of absorbance of 0.001 per minute at 450 nm and
pH 6.2
at 25°C in a suspension of Micrococcus lysodeikticus (0.25 mg/ml)
obtainable from Sigma
Chemicals.
Characterization of arachidonic acid
Arachidonic acid (ARA) was also obtained from DSM/Royal Gist-brocades under
the trade mark VEVODART"~. This is in the form of a microbial oil (ARA content
at
least 35%) obtained by culturing the fungus Mortierella alpina.
Application of antibacterial en.zymes in animal feed for poultry
Trials we carried out with broilers to test the efficacy of glucose oxidase
and
lysozyme alone and the combination of both. Male broilers (Ross) we kept from
day 1
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to day 5 on a standard diet. .At day S, animals we selected from this group
and are
divided into cages. The weight of the animals and their variation were
measured. The
average weight and its deviation were equal per cage. Fifteen animals were
kept in one
cage. The cages were situated in an artificially heated, ventilated and
illuminated broiler
house. The floor space of each cage was 0.98 mz, with wire floors. The broiler
house was
illuminated for 24 hours per day. During the experimental period, light
intensity was
gradually reduced. The temperature was gradually reduced from 28°C
during the first
week to 23°C during the last week of the experiment. Humidity in the
broiler unit was
approximately 60% during the experimental period. The animals had been
vaccinated
against New Castle disease (u:>ing the spray method) at an age of one and
fourteen days.
The experiment lasted 33 days, comprising a pre-test period of 5 days and a
test period of
28 days.
The experimental diets were offered ad lib. to the animals. Water was freely
available. The feed was cold pelleted (temperatures were kept below
65°C) at a diameter
of 3 millimeter.
The experiment comprised the following treatments:
1) basal diet (nega.tive control)
2) basal diet + glucose oxidase (1000 Sarett U/kg feed)
3) basal diet + lysozyme (100.000 Shugar units/kg of feed)
4) basal diet + glucose oxidase (1000 Sarett U/kg of feed) + lysozyme
(100.000 Shugar units/kg of feed)
5) basal diet + glucose oxidase (1000 Sarett U/kg of feed) + lysozyme
(100,000 Shugar units/kg of feed) + arachidonic acid (ARA) to a final
concentration of 1 g/kg of feed.
Each treatment was replicated six times (90 birds per treatment in total).
Gain and
feed conversion were measured. The composition of the feed (basal diets) used
was:
Ingredients Content (%)
Rye 10
Wheat 40
Soy oil 1
Animal fat 6
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Manioc 4.3
Soya bean meal (45.4,~o crude protein) 22
Full fat toasted Soya beans 10
Meat meal tankage (58% crude protein) 3
Vitamins/premix 1
Limestone 0.9
Monocalciumphosphate 1.2
Salt (NaCI) 0.3
D,L-methionine 0.2
ME broilers {KCaI/kg;) 2850
Crude protein (%) 21.4
Crude fat (%) 10.5
Lysine (available, %) 1.23 (1.04)
Methionine + cystein~e (available, %) 0.90 {0.79)
The enzymes and arachidonic acid were added to this basal diet by mixing it
first
with a carrier.
The effects of the antibacterial enzymes and arachidonic acid on growth and
feed
conversion ratio in broilers between 5 and 33 days of age are shown below in
Table 1.
TABLE 1
Example Diet Feed Growth Feed Improvement
Intake (g) conversionin feed
(g) ratio conversion
ratio
1 Basal diet 2,760 1,540 1.79 -
2 Basal diet + 2,750 1,554 1.77 -0.02
glucose oxidase
3 Basal diet + 2,748 1,553 1.77 -0.02
lysozyme
4 Basal diet + 2,731 1,589 1.72 -0.05
glucose oxidase
~-
lysozyme
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B
l di
asa 2,710 1,595 I.70 -0.09
et +
oxidase +
lysozyme +
arachidonic
acid
The addition of one type of antibacterial enzyme or a combination of different
types of antibacterial enzymes both improved the growth and feed conversion
ratio in
broilers. However, more importantly a synergistic effect was found for the
combination
S of glucose oxidase and lysozyme on the feed conversion ratio and the
inclusion of
arachidonic acid in diets contaiining antibacterial enzymes resulted in an
even further
improvement.
Comparative Examples 6 to 9 and Example 10
Application of antibacterial enzymes in animal feed for ~iQs
Crossbred pigs (equal barrows and gifts; n =100) of a similar age and weight
were
used in this trial. They were housed in environmentally controlled rooms, and
had ad
lib. access to feed and water at all times. The room temperature was set
initially at 29°C
and was lowered about 2°C peer week after the second week. The pigs
were allotted to
one of five treatments. There were two pigs in each pen with 10 replications
(weight
1! 5 blocks) per treatment.
Body weight and pen feed consumption were measured weekly.
The basal diet was a typical American diet, of the composition:
Raw Material Content%r)
Corn 63.6
Soyabean meal 30.9
Vitamin premix 0.25
Trace mineral premix 0.1
Selenium premix 0.05
Dicalcium phosphate 1.2
Salt (NaCI) 0.3
Limestone 3.6
No antibiotic was added to the feed.
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The experiment comprised the following treatments (Examples 6 to 10):
a) basal diet (nel;ative control);
b) basal diet + glucose oxidase (1000 Sarett U/kg feed);
c) basal diet + lysozyme (100,000 Shugar units/kg of feed);
d) basal diet + glucose oxidase (1000 Sarett U/kg of feed) + lysozyme
(100,000 Shuga~r units/kg of feed);
e) basal diet + ghucose oxidase (1000 Sarett U/kg of feed) + lysozyme
(100,000 Shugar units/kg of feed) + arachidonic acid to a final
concentration of 1 g/kg of feed.
The results obtained in terms of feed intake, growth and feed conversion ratio
are
shown below in Table 2.
TABLE 2
Effects of antibacterial enzymes and ARA on growth and feed conversion ratio
in
growing pigs (23 to 54 kg body weight).
Exam Di
le
p et Daily Daily Feed Improvement
Feed gain conversionin feed
Intake (kg) ratio conversion
( ) ratio
6 Basal diet 2.20 0.90 2.44 -
7 Basal diet + 2.15 0.90 2.39 -0.05
glucose oxidase
8 Basal diet + 2.14 0.89 2.40 -0.04
lysozyme
9 Basal diet + 2.10 0.94 2.23 -0.21
glucose oxidase
+
lysozyme
10 Basal diet + 2.05 0.95 2.16 -0.28
oxidase +
lysozyme +
arachidonic
acid
The addition of one type of antibacterial enzyme or combinations of different
types of antibacterial enzymes __<;howed a favourable effect on daily gain and
feed
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conversion ratio. However, the combination of two different types of
antibacterial
enzymes (i.e. glucose oxidase and lysozyme) resulted in a surprising
synergistic effect on
feed conversion ratio and the addition of arachidonic acid to feed containing
antibacteria
enzymes resulted in a further improvement.
Comparative Examples 11 to '14 and Example 15
The use of antibacterial enz,~ie in fish nutrition
Effects of supplemental antibacterial enzymes on growth and feed conversion
ratio were studied with trout (Uncorhynchus mykiss).
The diet composition used was as follows:
a0 Raw material Content
Soyabean meal 43
Soya beans, pressure cooked 20
Wheat gluten 20.5
Fish oil 12
1.5 L-lysine-HCl 0, g
D, L-methionine 0,2
Vitamin/mineral premix 3.5
No growth promoting antibiotic was added to the feed.
Experiments were conducted with 200 trout with a mean initial body weight of
20 8.8 g/trout which were allottecL to 5 equal groups. Diets were fed to these
5 groups over a
period of 53 days. The water temperature was kept constant at 15°C. The
diets were fed
twice daily to satiation avoiding feed losses. Weight gain and feed conversion
ratio were
determined.
The experiment comprised the following treatments (Examples 11 to 15):
a) basal diet (negatiive control);
b) basal diet + glucose oxidase (1000 Sarett U/kg feed);
c) basal diet + lyso:zyme (100,000 Shugar units/kg of feed);
d) basal diet + glucose oxidase (1000 Sarett U/kg of feed) + lysozyme
(100,000 Shugar units/kg of feed); and
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e) basal diet + glucose oxidase (1000 Sarett LJ/kg of feed) + lysozyme
{100,CC0 Shugar units/kg of feed) + arachidonic acid to a final
concentration of 1 g/kg of feed.
The results obtained, in terms of growth and feed conversion, are shown below
in
Table 3.
TABLE 3
Gain, feed intake and feed conversion ratio in trout fed for 53 days on diets
supplemented with antibacterial enzymes + /- arachidonic acid.
Example Diet Feed Gain Feed Improvement
Intake (g/trout)conversionin feed
(g/trout) ratio conversion
ratio
11 Basal diet 18.5 12.5 1.48 -
12 Basal diet 20.6 14.1 1.46 -0.02
+
glucose oxidase
13 Basal diet 20.4 14.1 1.45 -0.03
+
lysozyme
14 Basal diet 21.5 16.4 1.31 -0.17
+
glucose oxidase
+
lysozyme
Basal diet 22.9 18.4 1.24 -0.24
+
oxidase +
lysozyme +
arachidonic
acid
15 The results obtained demonstrate the favourable effects of one type of
antibacterial enzyme or a combination of antibacterial enzymes on growth and
feed
conversion ratio in trout. The combination of different types of antibacterial
enzymes
showed a synergistic effect on feed conversion ratio and the addition of
arachidonic acid
to diets containing antibacterial enzymes gave a further improvement.
