Note: Descriptions are shown in the official language in which they were submitted.
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1
ANIMAL FEED ADDITIVES
TECHNICAL FIELD
The present invention relates to the combined use in animal feed of
one or more xylanolytic enzymes of Type I with one or more xylanolytic enzymes
of
Type II, as defined herein. It also relates to animal feed and animal feed
additives
comprising such combination of xylanolytic enzymes.
BACKGROUND ART
The types and amount of plant raw materials which can be used as
components in animal feed will often be limited by the ability of the animals
to digest
them. Feed enhancing enzymes are enzymes, usually of microbial origin, that by
improving feed digestibility are able to increase the efficiency of its
utilization.
When added to animal feed, feed enhancing enzymes improve the in
vivo break-down of plant cell wall material partly due to a reduction of the
intestinal
viscosity, whereby a better utilization of the plant nutrients by the animal
is achieved.
In this way the growth rate andlor feed conversion ratio (i.e. the weight of
ingested
2o feed relative to weight gain) of the animal becomes improved.
Xylanolytic enzymes (EC 3.2.1.8) are well known as feed enhancing
enzymes. According to one theory, their primary effect is to reduce the
viscosity of
the feed, whereas another theory focuses on the xylanases increasing the
availability of nutrients embedded in the plant cell wall. Thus WO 94/21785
2s describes xylanases derived from Aspergillus aculeatus and their use as
animal feed
additives, in particular in animal feed compositions containing high amounts
of
arabinoxylans and glucuronoxylans, e.g. feed containing cereals such as
barley,
wheat, rye or oats or maize.
WO 95123514 describes a process for reducing the viscosity of a plant
so material and for separating plant material into desirable components, which
process
comprises treating the plant material with a xylanase preparation having a
specific
activity on water-soluble/water in-soluble pentosans. However, WO 95/23514
does
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not relate to mixtures of xylanases.
SUMMARY OF THE INVENTION
s According to the present invention it has now been found that
xylanolytic enzymes can be distinguished by the way they degrade different
substrates. Moreover it has surprisingly been found that the combined action
of two
different types of xylanolytic enzymes is improved as compared to the action
of the
two separate enzymes, i.e. a synergistic effect occurs.
Therefore, one main object of the invention is the combined use of
these two types of xylanolytic enzymes in animal feed. In particular, they are
used
simultaneously and/or sequentially.
It is another main object of the present invention to provide an animal
feed additive which increases energy uptake from animal feed. Accordingly, in
one
~ s main aspect, the present invention provides an animal feed additive which
comprises
a mixture of one or more xylanolytic enzymes of Type I with one or more
xylanolytic
enzymes of Type II, Type I and Type II as defined herein.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is further illustrated by reference to the
accompanying drawing, in which:
Fig. 1 shows the action of six different xylanolytic enzymes [viz. a
multicomponent Trichoderma reesei xylanase preparation (A); a monocomponent
2s Aspergillus aculeatus xylanase preparation (B); a monocomponent Humicola
insolens xylanase preparation (C); a monocomponent Thermomyces lanuginosus
xylanase preparation (D); a multicomponent Humicola insolens xylanase
preparation
(E) and a multicomponent Trichoderma xylanase preparation (F)] on the
viscosity of
a wheat suspension determined after the so-called pepsin phase according to
3o Example 1, presented as relative viscosity (%) versus enzyme concentration
(FXU/g
wheat);
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Fig. 2 shows the action of the same six enzymes as in fig. 1 on the
viscosity of a wheat suspension, also presented as relative viscosity {%)
versus
enzyme concentration (FXU/g wheat), but here determined after the so-called
° pancreatin phase according to Example 1;
Fig. 3 shows the effect, determined after the pepsin phase, on the
viscosity of a wheat suspension of two individual Type I and Type II xylanase
preparations (viz. preparations D and A, respectively) compared to that of the
mixture of these two preparations as well as a theoretically calculated
additive effect;
the effect is presented as % relative viscosity; columns I the combined effect
of
preparations A and D; columns II theoretical additive effect of preparations A
and D;
columns ill the effect of preparation D alone; and columns IV the effect of
preparation A alone; and
Fig. 4 shows, as in fig. 3, the effect on the viscosity of a wheat
suspension of the same two individual Type I and Type II xylanase
preparations, but
here as determined after the pancreatin phase.
DETAILED DISCLOSURE OF THE INVENTION
Xylanolytic Enzymes of Type I and Type II
2o According to the present invention it has now been found that
xylanolytic enzymes can be distinguished by the way they degrade different
substrates. Moreover it has surprisingly been found that the combined action
of two
different types of xylanolytic enzymes is increased as compared to the action
of the
two separate enzymes, i.e. a synergistic effect occurs.
2s The type of a xylanolytic enzyme may e.g. be determined by reference
to its action on the viscosity of a wheat suspension. As defined herein,
xylanolytic
enzymes of Type I increase initial viscosity of a wheat suspension, whereas
the
initial viscosity of a wheat suspension is unaffected or becomes decreased by
the
action of a xylanolytic enzyme of Type II. In particular, the increased
initial viscosity
3o referred to above refers to figs. 1 and 2 hereof, which show an initially
or at low
FXU-doses increased relative viscosity.
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The combined action of the two different types of xylanolytic enzymes
referred to above can be simultaneous, i.e. the two enzymes are active at the
same
time, or sequential, i.e. one type is acting first, the second type
subsequently. In the
sequential type of action the first type may be active or non-active, when the
second
s type excerts its effect, viz. a simultaneous and sequential, and a pure
sequential
action, respectively. In all these cases a synergistic effect is observed on
the
viscosity of the feed.
An assay for determining the type of xylanolytic enzyme is described in
more detail in Example 1.
Animal Feed and Animal Feed Additives
The present invention provides an animal feed as well as an animal
feed additive, which comprises a mixture of one or more xylanolytic enzymes of
Type I with one or more xylanolytic enzymes of Type li.
In the context of this invention, in particular, an animal feed means any
natural or artificial diet, meal or the like or components of such meals
intended or
suitable for being eaten, taken in, or digested by an animal.
In the context of this invention, an animal feed additive is an enzyme
preparation comprising one or more feed enhancing enzymes) and suitable
carriers
2o and/or excipients. In particular, the enzyme preparation is provided in a
form that is
suitable for being added to animal feed. The animal feed additive of the
invention
may be prepared in accordance with methods known in the art and may be
provided
in the form of a dry preparation or a liquid preparation. The enzyme to be
included in
the preparation, may optionally be stabilized in accordance with methods known
in
the art.
The animal feed additive of the invention may be a granulated enzyme
product which may readily be mixed with feed components, or more preferably,
form
a component of a pre-mix. The granulated enzyme product may be coated or
uncoated. The particle size of the enzyme granulates preferably is compatible
with
3o that of feed and pre-mix components. This provides a safe and convenient
mean of
incorporating enzymes into feeds.
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Also, the animal feed additive of the invention may be a stabilized
liquid composition, which may be an aqueous or oil-based slurry.
The animal feed additive of the invention may exert its effect either in
vitro or in vivo. The effect may be exerted by pre-treating an animal feed, if
desired
followed by an inactivation of the enzymes. But usually the xylanases exert
their
effect in vivo, i.e. they act directly in the digestive system of the animal,
during the
digestion. The feed additive of the invention is particularly suited for
addition to
animal feed compositions containing high amounts of arabinoxylans and
glucuronoxylans, e.g. feed containing cereals such as wheat, barley, rye,
oats, or
maize.
In a preferred embodiment an animal feed additive is provided in which
the ratio of xylanolytic enzymes of Type I to xylanolytic enzymes of Type 11
is in the
range of from about 119 to about 9/1, determined in terms of xylanolytic
activity, i.e.
10-90% of the total amount of xylanolytic activity, e.g. determined in terms
of FXU, is
~ 5 ascribable to a xylanolytic enzyme of Type I.
In another preferred embodiment an animal feed additive is provided in
which the ratio of xylanofytic enzymes of Type I to xylanolytic enzymes of
Type II is
in the range of from about 2/8 to about 8/2, determined in terms of
xylanolytic
activity, i.e. 20-80% of the total amount of xylanolytic activity, e.g.
determined in
2o terms of FXU, is ascribable to a xylanolytic enzyme of Type I.
In a third preferred embodiment an animal feed additive is provided in
which the ratio of xylanolytic enzymes of Type I to xylanolytic enzymes of
Type II is
in the range of from about 317 to about 7/3, determined in terms of
xylanolytic
activity, i.e. 30-70% of the total amount of xylanolytic activity, e.g.
determined in
25 terms of FXU, is ascribable to a xylanolytic enzyme of Type I.
In a fourth preferred embodiment an animal feed additive is provided in
which the ratio of xylanoiytic enzymes of Type I to xylanolytic enzymes of
Type II is
in the range of from about 4/6 to about 6/4, determined in terms of
xylanolytic
activity, i.e. 40-60% of the total amount of xylanoiytic activity, e.g.
determined in
3o terms of FXU, is ascribable to a xylanolytic enzyme of Type I.
In a fifth preferred embodiment an animal feed additive is provided in
which the ratio of xylanolytic enzymes of Type I to xylanolytic enzymes of
Type II is
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about 515, determined in terms of xylanolytic activity, i.e. xylanolytic
enzymes of Type
I and Type II is present in approximately equal activities, e.g. determined in
terms of
FXU.
In a sixth preferred embodiment an animal feed additive is provided in
s which the ratio of xylanofytic enzymes of Type I to xylanolytic enzymes of
Type II is
in the range of from about 1/20 to about 20/1, determined in terms of
xylanolytic
activity, i.e. 5-95% of the total amount of xylanolytic activity, e.g.
determined in terms
of FXU, is ascribable to a xylanolytic enzyme of Type I. The particular
embodiment
of 1/20 of type Illtype I has turned up to be very satisfactory.
Other preferred ratios are 125:1, 100:1, 75:1, 50:1 and 25:1
(type I : type I I or vice versa).
Monocomponent Preparations
In a preferred embodiment, the present invention provides an animal
~ 5 feed additive, in which one or more of the xylanolytic enzymes are
provided in the
form of a monocomponent preparation. In the context of this invention, a
monocomponent preparation is an enzyme preparation, in which preparation
essentially all of the xylanolytic activity (i.e. the xylanolytic activity
detectable) is
owing to a single xylanase component. The monocomponent preparation may
2o preferably be obtained by way of recombinant DNA technology.
In a more specific embodiment all xylanolytic enzymes are provided in
the form of monocomponent preparations.
Feed Enhancing Enzymes
2s In a further preferred embodiment, the feed additive of the invention
comprises additional feed enhancing enzymes.
In the context of this invention feed enhancing enzymes comprise but
are not limited to other xyianases, a-galactosidases, ~i-galactosidases, in
particular
lactases, phytases, ~3-glucanases, in particular endo-(i-1,4-glucanases and
endo-~3-
30 1,3(4)-glucanases, xylosidases, galactanases, in particular arabinogalactan
endo-
1,4-~-galactosidases and arabinogalactan endo-1,3-~-galactosidases,
endoglucanases, in particular endo-1,2-(3-glucanase, endo-1,3-a-glucanase, and
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endo-1,3-~-giucanase, pectin degrading enzymes, in particular pectinases,
pectinesterases, pectin lyases, polygalacturonases, arabinanases,
rhamnogalacturonases, rhamnogalacturonan acetyl esterases, rhamnogalacturonan-
a-rhamnosidase, pectate lyases, and a-galacturonisidases, mannanases, ~3-
rnannosidases, mannan acetyl esterases, xylan acetyl esterases, proteases and
lipolytic enzymes such as lipases and cutinases.
Microbial Sources
The xylanolytic enzymes of Type I and Type II according to the
invention may originate from any source of xylanolytic enzymes. Preferably the
xylanolytic enzymes are originally derived from microbial sources, and may in
particular be of fungal origin, of bacterial origin, or derived from a yeast
strain. In
case of recombinant xylanoiytic enzymes, any host cell can be employed for
their
production such as fungi, bacteria, yeast, transgenic plants or animal cell
lines.
Preferred xylanolytic enzymes of fungal origin are xylanases originally
derived from a strain of Aspergillus, in particular Aspergillus aculeatus,
Aspergillus
awamori, Aspergillus niger, Aspergillus tubigensis, a strain of Cochliobolus,
in
particular Cochliobolus carbonum, a strain of Disporotrichum, in particular
Disporotrichum dimorphosporum, a strain of Humicola, in particular Humicola
2o insolens, a strain of Neocallimastix, in particular Neocallimastix
patriciarum, a strain
of Thermomyces, in particular Thermomyces lanuginosus (syn. Humicola
lanuginosa), or a strain of Trichoderma, in particular Trichoderma
Iongibrachiatum
and Trichoderma reesei.
Preferred xylanolytic enzymes of bacterial origin are xylanases derived
from a strain of Bacillus, in particular Bacillus pumilus, Bacillus
stearothermophilus, a
strain of Dictyoglomus, in particular Dicfyoglomus thermophilum, a strain of
Microtetraspora, in particular Microtefraspora flexuosa, a strain of
Streptomyces, a
strain of Thermotoga, in particular a strain of Thermotoga neapolitana,
Thermotoga
maritima and Thermotoga thermarum, or a strain of Rhodothermus.
3o In a preferred embodiment the animal feed additive of the invention
comprises a xylanolytic enzyme of Type I derived from a strain of Thermomyces
lanuginosus which is provided in the form of a monocomponent preparation.
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In another preferred embodiment the animal feed additive of the
invention comprises a xylanolytic enzyme of Type II derived from a strain of
Trichoderma which is provided in the form of a monocomponent preparation.
s Xylanolytic Activity
The xylanolytic activity can be expressed in FXU-units, determined at
pH 6.0 with remazol-xylan (4-O-methyl-D-glucurono-D-xylan dyed with Remazol
Brilliant Blue R, Fluka) as substrate.
A xylanase sample is incubated with the remazol-xylan substrate. The
background of non-degraded dyed substrate is precipitated by ethanol as a stop
reagent. The remaining blue color in the supernatant (as determined
spectrophotometricaily at 585 nm) is proportional to the xylanase activity,
and the
xylanase units are then determined relatively to an enzyme standard at
standard
reaction conditions, i.e. at 50.0°C and 30 minutes reaction time in a
0.1 M phosphate
15 buffer pH 6Ø
EXAMPLES
The invention is further illustrated with reference to the following
2o examples which are not intended to be in any way limiting to the scope of
the
invention as claimed.
Example 1
Determination of Type of Xylanolytic Enzyme
25 This example demonstrates how xylanolytic enzymes can be
distinguished by the way they affect the viscosity of a wheat suspension, and
how
they can be classified xylanolytic enzymes of Type I and Type II,
respectively.
Xylanolytic enzymes of Type I increase the initial viscosity of a wheat
suspension,
whereas the initial viscosity of a wheat suspension is unaffected or becomes
so decreased by the action of a xylanolytic enzyme of Type II.
Foregut digesta viscosity has been identified as a major nutritional
constraint affecting digestibility of wheat and barley based broiler diets. A
close
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correlation between the reduction of digesta viscosity results and
improvements in
chicken feed conversion efficiency have been found.
In this example six different xylanolytic enzymes are examined with
a respect to their activity on initial viscosity of a wheat suspension:
s A) A multicomponent Trichoderma reesei {previously identified as
Trichoderma longibrachiatum) xylanase preparation (enzyme complex, NovozymTM
431, available from Novo Nordisk A/S, Denmark);
B) A monocomponent Aspergillus aculeatus xylanase preparation
obtained according to Example 3 of WO 94/21785;
C) A monocomponent Humicola insolens xylanase preparation
obtained according to Example 1 of WO 92/17573;
D) A monocomponent Thermomyces lanuginosus xylanase
preparation obtained according to Examples 1-3 of WO 96/23062;
E) A multicomponent Humicola insolens xylanase preparation (enzyme
complex, Bio-Feed Plus CT, available from Novo Nordisk A/S, Denmark); and
F) A multicomponent Trichoderma xylanase preparation (enzyme
complex, AvizymeTM 1300, available from Finnfeeds, Finland).
The determination is carried out under conditions that mimic the
conditions of the gastro-intestinal tract of poultries. Therefore two
determinations are
2o made, one after incubation for 45 minutes in presence of pepsin (the pepsin
phase,
which mimics the conditions of the gizzard), and one after additional 120
minutes in
presence of pancreatin (the pancreatin phase, which mimics the conditions of
the
small intestine).
Therefore two samples and two reference solutions (blank sample,
2s control) are prepared, each containing 8.00 g of ground wheat in 12 ml of a
solution
of 1.042 g of pepsin in 250 ml of 0.1 N HCI. After stirring for 2 minutes, a
solution of
the xylanase sample in question is added to the two samples, and this point is
accorded the time zero (t = 0). The controls are added the same volume of a 1
M
NaHC03.
3o After 45 minutes (t = 45) of incubation under stirring at 40°C, 4 ml
of a
1 M NaHC03 solution is a added to one of the samples, and this sample is now
being
cooled to 0°C. This sample represents the pepsin phase. Also at t = 45,
4 ml 1 M
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NaHC03 is added to one of the controls.
At the same time (t = 45) 4 ml of the supernatant of a solution
containing 200 mg pancreatin in 50 ml of 1 M NaHC03 solution is added to the
second sample. After additional 120 minutes (t = 165) of incubation under
stirring at
5 40°C the sample is cooled to 0°C. This sample represents the
pancreatin phase.
Also at t = 165, 4 ml 1 M NaHC03 is added to the the second control.
After centrifugation, the supernatants of the two samples are subjected
to viscosity determination on a Brookfield DV-III viscosimeter at 40°C,
using spindle
no. 18. The viscosity is determined relative to the viscosity of the blank
sample.
The results of these determinations are presented as dose/response
shown in the appended Figs. 1-2. From these figures it is noticed that the
xylanolytic
enzymes designated above as C, D and E represent xylanolytic enzymes of Type I
that increase the initial viscosity of the wheat suspension, and the
xylanolytic
enzymes designated above as A, B and F represent xylanolytic enzymes of Type
II
that decrease the initial viscosity of the wheat suspension.
Example 2
Effect of Mixtures of Xylanolytic Enzymes of Type 1 and Type II
This example demonstrates the effect of mixtures of xylanolytic
2o enzymes of Type I and Type If on viscosity reduction of a wheat suspension.
Using the procedure described in example 1, above, combinations of
the Type I enzymes C, D and E with the Type II xylanases A and B was examined.
The activity of the Type I xylanases was varied as shown in Table 1
(as in example 1 ), while the activity of the Type II xylanases was kept
constant at the
dose that gave 30 % (preparations A and B) or 60 % (preparation B) viscosity
reduction after the pancreatin phase; i.e. for preparation B an activity of
0.01 FXU/g
wheat, which resulted in a relative viscosity of 0.7, and an activity of 0.12
FXUIg,
which resulted in a relative viscosity of 0.4; and for preparation A an
activity of
0.20 FXU/g wheat, which resulted in a relative viscosity of approximately 0.7
(fig. 2).
3o Table 1 shows the relative viscosity of the in vitro test system after
addition of different xylanase combinations and also the single Type I
enzymes. The
viscosity is relative to the viscosity of a parallel incubation without added
enzyme.
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Using only a xylanase of type I, rather high doses have to be applied
with a view to reducing the viscosity. However, when admixing with a xylanase
of
type II, a reduced viscosity results even in those cases in which the overall
concentration of xylanase I + xylanase II is smaller than that concentration
of
s xylanase I alone, which had to be applied for observing any effect on the
viscosity.
When mixing for instance 0.12 FXU of xylanase B with 0.25 FXU of xylanase C
(i.e.
overall activity 0.37 FXU) a viscosity reduction of approximately 50% is
obtained in
the pepsin phase - as compared with a much smaller viscosity reduction of 11
when as much as 1.2 FXU of xylanase C is used, without admixed type II
xylanase.
Usually the enzyme costs are set on an activity basis, and accordingly, when
using a
mixture of the type I and the type II enzymes, the enzyme costs can be
reduced.
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12
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13
Moreover, the effect on viscosity of a wheat suspension of two
individual Type I and Type II xylanase preparations (preparations D and A,
respectively} has been compared to that of the mixture of these two
preparation as
well as a theoretically calculated additive effect. For preparation D three
activity
s levels were chosen, and for preparation A one activity level (that which
resulted in a
30% viscosity reduction, i.e. a relative viscosity of 0.7) was chosen. The
results of
these experiments are presented in Figs. 3 and 4.
Fig. 3 shows the effect after the pepsin phase (after incubation for 45
minutes, cf. Example 1 ), and Fig. 4 shows the effect after the pancreatin
phase (after
incubation for 165 minutes, cf. Example 1 ). In these figures, the effect is
presented
as % relative viscosity.
The first columns (I) of each cluster represent the actual relative
viscosity determined using a mixture of preparations D and A. Thus, in the
first
cluster of Figs. 3 and 4, columns (I), a mixture of 0.22 FXUIg wheat of
preparation D
and 0.2 FXUIg wheat of preparation A was used, in the second cluster, a
mixture of
0.56 FXUIg wheat of preparation D and 0.2 FXU/g wheat of preparation A was
used,
and in the third cluster, a mixture of 1.12 FXUIg wheat of preparation D and
0.2
FXU/g wheat of preparation A was used.
The second columns (II) of each cluster of each of fig. 3 and fig. 4
2o represent the theoretical viscosity of the mixture, calculated using a
multiplicative
statistical model. Thus, referring for example to fig. 3, column (II) of the
first cluster
represents the theoretically additive effect of using 0.22 FXUIg wheat of
preparation
D and 0.2 FXU/g wheat of preparation A, column {II) of the second cluster
represents the theoretically additive effect of using 0.56 FXU/g wheat of
preparation
25 D and 0.2 FXU/g wheat of preparation A, and column (II) of the third
cluster
represents the theoretically additive effect of using 1.12 FXUIg wheat of
preparation
D and 0.2 FXUIg wheat of preparation A.
The third and fourth columns (III and IV) of each cluster represent the
actual relative viscosity determined using each of preparations D and A atone,
3o respectively. Thus, e.g. in the first cluster of Fig. 3, columns III and IV
represent the
effect of using 0.42 FXUIg wheat of preparation D and 0.42 FXUIg wheat of
preparation A, respectively.
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It is apparent from these figures, that the actual viscosity determined
using a mixture of preparations A and D is lower than the calculated additive
value,
and this is owing to a synergy between the enzymes.
It is presently contemplated that xylanases of Type I, such as
preparation D, are acting preferably or initially (at low doses) on insoluble
xylans
(constituting approximately 80-90% of the total amount of xylans), thus
liberating
pentosans which are soluble, and therefore the viscosity is increased. These
pentosans, however, are preferred substrates for the Type II xylanases, such
as
preparation A, so when acting in combination xylanases I and II degrade more
xylans and the viscosity is adequately reduced. Still further, also other
nutrients,
which were originally embedded by the xylans, are made nutritionally
available.
In general the synergistic effect is most pronounced when the total
activity is low. This is probably because at higher activities of Type I
xylanases, they
will also start degrading the soluble pentosans, thus reducing the synergistic
effect.
~5 In addition, statistical analysis showed that the synergistic effect is
highest during the pepsin phase, probably because the viscosity reduction here
is
smaller than in the pancreatin phase, thus leaving more room for improvement.
