Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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APPLICATION OF PHYTASE IN FEED HAVING LOW CONTENT OF
PHYTATE
s Fieid of the invention
The present invention relates to the application of the enzyme
phytase in feed having a low content of phytate.
io
Background of the invention
The diets of ruminants vary considerably in different husbandry
systems, but also at different ages. In extensive husbandry systems,
~ s typically animals are fed with roughages, like grass, hay, grass silage
and/or maize silage. Some additional feeds or feedstuffs may be offered,
likewise a vitamin and/or mineral mixture. In such husbandry systems,
young animals typically suckle with their mother. In more intensive
husbandry systems, young animals, e.g. calves, are often offered a
zo milkreplacer, since the produced milk of the cows is used for human
consumption, or for processing to products for human consumption, like
butter and cheese. Veal calves are usually mainly offered a miikreplacer.
Some other feeds or feedstuffs, like hay, corn, or a special mixed
concentrate may be offered additionally. Often these feedstuffs require a
25 low level of available iron, when the aim is to produce white veal calf
meat. For calves aimed for rose meat production, the feeds preferably
contain some available iron. Older animals, like dairy cows, beef cattle,
sheep and deer, typically receive rations containing roughages, e.g. grass,
hay, grass silage and/or maize silage, and more concentrated feedstuffs,
3o e.g. mixed concentrates (usually produced in a feed mill), cereals,
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vegetable (by-) products and/or brewers grains. Additionally, a mineral
and/or vitamin mixture may be offered.
Phosphorus is an essential element for growth of all organisms. In
s livestock production, feed must be supplemented with inorganic
phosphorus in order to obtain a good growth performance of monogastric
animals (e.g. pigs, poultry and fish).
Phytate occurs as a storage phosphorus source in virtually all feed
substances originating from plants (for a review see: Phytic acid,
~o chemistry and applications, E. Graf (ed), Pilatus Press; Minneapolis, MN,
USA (1986). Phytate comprises 1-3% of all nuts, cereals, legumes, oil
seeds, spores and pollen. Complex salts of phytic acid are termed phytin.
Phytic acid is considered to be an anti-nutritional factor since it chelates
minerals such as calcium, zinc, magnesium, iron and may also react with
~ s proteins, thereby decreasing the bioavailability of protein and
nutritionally
important minerals.
The use of phytase in the nutrition of monogastric animals (e.g.
pigs, poultry and fish) has became widespread thanks to the availability of
microbial phytases at an affordable cost. Cloning and overexpression of
2o microbial phytase has resulted in a dramatic decrease in cost price of the
product enabling the commercialisation of this enzyme in the nutrition of
monogastric animals.
In contrast, typically no inorganic phosphate needs to be added to
the feed stuffs of older ruminants. Micro-organisms present in the rumen
is are thought to produce sufficient amounts of enzymes which catalyse the
conversion of phytate (myo-inositolhexakis-phosphate) to inositol and
inorganic phosphate.
In Europe and the USA, the feeds wherein phytase advantageously
is applied typically have a phytate-phosphorus content of 0.25 to 0.35%.
ao We now demonstrate that the addition of phytase to feed having a
low phytate content such as feed for young animals (ruminants or
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non-ruminants) results in a marked improvement in daily gain and feed
conversion ratio.
s Description of the Fi4ures
Figure 1. Contents of zinc in blood plasma as a function of time
during the fattening period, for each of the four groups with a different
diet composition: Group 1, ZnS04 and phytase; Group 2, only ZnS04;
~o Group 3, only phytase; Group 4, no additions.
Figure 2. Blood haemoglobin levels as a function of time during the
fattening period, for each of the four groups with a different diet
composition: Group 1, ZnS04 and phytase; Group 2, only ZnS04; Group
~s 3, only phytase; Group 4, no additions.
Description of the invention
2o The present invention relates to the application of the enzyme
phytase in feed with a low phytate content. In particular, the present
invention relates to the application of the enzyme phytase in the feeding
of animals which typically are fed low-phytate diets, such as poultry or
young animals.
25 According to an aspect of the invention there is provided a method
of feeding an animal, wherein the animal is fed a low-phytate diet
supplemented with a phytase. According to another aspect of the
invention there is provided a method of feeding a young animal a
low-phytate diet supplemented with a phytase which is applied as part of
so a method of breeding and/or keeping an animal.
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One would not expect any beneficial effect of phytase when
applied in feeds with a low content of phytate. The present invention
surprisingly shows that the addition of exogenous phytase to low-phytate
feeds results in a marked improvement in zootechnical results, such as
s average daily gain (i.e. growth rate) and feed conversion, in animals fed
such phytase-containing low-phytate feeds.
Accordingly, one aspect of the invention provides a method of
improving the zootechnical results of an animal, wherein the method
comprises feeding the animal a low-phytate feed supplemented with a
~o phytase. This improvement of the zootechnical results of animals fed with
a low-phytate diet supplemented with a phytase may be apparent in any
animal fed with a low-phytate diet. In particular, an improvement of the
zootechnical results was measured in young animals.
Contents of zinc in blood plasma and haemoglobin levels are also
is significantly increased as a consequence of phytase addition to
low-phytate feed whereas levels of calcium, iron, magnesium and copper
remain unaffected. The mere addition of a mineral such as zinc to the feed
does improve the mineral status of the animals but anly slightly improves
the zootechnical results such as growth rate and feed conversion ratio.
2o Accordingly, yet another aspect of the invention provides a method
of improving the mineral status of animals, wherein the method comprises
feeding the animal a low-phytate diet supplemented with a phytase. In a
preferred method according to the invention, the low-phytate diet fed to
the animal further comprises zinc in addition to a phytase. Preferably
zs 0.005 - 0.2% (w/w) zinc is included in the diet, more preferably 0.01 -
0.03% is included in the diet.
The term "zootechnical results" is herein understood to mean
animal performance in terms of daily gain (growth rate), feed conversion
ratio (FCR), daily milk production and the like. FCR is defined as the
ao amount of feed (kg) required per kg of growth of the animal.
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Throughout this invention, a low-phytate feed or diet is defined as a
feed or diet comprising phytate-phosphorus (phytate-P) in a concentration
ranging from 0.01 to 0.2% (w/w), preferably from 0.01 to 0.15%, and
more preferably from 0.01 to 0.1 %. Most preferably, low-phytate feeds
s have a phytate-P content of 0.01 to 0.05 %, which is up to ten times
lower than the feed of an older animal.
Phytate can be determined as phytic acid, according to the method
of e.g. Oshima et al., 1964 (Oshima, M., T.G.Taylor and A.Williams,
Biochemical Journal 92, 42-46). Phytate-P is calculated as phytic acid *'
~0 0.29, since phytic acid contains approximately 29% phosphorus.
For the purpose of this invention, young animals are defined as
farmed animals which are in their growing and/or fattening period and/or
which would still suckle with their mother in natural conditions and/or
which receive a feed product specifically adapted for young animals,
~ s especially young ruminants, as defined hereinafter. Young animals can be
young ruminants such as calves, heifers, Iamb, deer calves or goats.
Young animals can also be young non-ruminants, such as piglets, broilers
or pullets.
A phytase is herein defined as an enzyme which is a phosphatase
2o capable of liberating at least one inorganic phosphate from a myo-inositol
phosphate. A typical example of a phytase is a
myo-inositol-hexakisphophate-3- phosphohydrolase (E.C. 3.1.3.8).
The phytase to be applied in the methods and products according
to the invention is not present as a natural constituent of any of the feed
is stuffs in the animal diet but rather is a supplement to feed stuffs or the
diet in general. This means that the phytase applied in the methods and
products of the invention differs from any of the natural phytases that
might be present in the feed stuffs with respect to the amount of phytase
activity and/or the nature of the phytase, i.e. the organism from which the
ao phytase is obtainable and/or the structure and/or biochemical properties of
the phytase molecule. Thus, the phytase applied in the methods and
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products of the invention is a supplement to the low-phytate feed of the
animal. The phytase supplement can be an exogenously added phytase,
which can be produced by fermentation of microbial host cells expressing
the phytase. Cloning and overexpression of microbial phytases has been
s described in detail in EP-A-0 420 358. Another possibility of
supplementing a low phytate feed with a phytase is to add
phytase-containing transgenic plant material, preferable transgenic seed,
which has been genetically engineered so as to express (or overexpress) a
phytase as described in detail in EP-A-0 449 375.
~o The phytase to be applied in the methods of the invention is
preferably a phytase with an acidic pH optimum, i.e. with an optimum at
a pH less than 7.0, preferably less than 6Ø Preferably the phytase is
obtainable from plants or micro-organisms. The microbial phytase is
preferably obtainable from a fungus, more preferably from a fungus of the
i s genera Aspergillus and Thermomyces. Most preferably the microbial
phytase is obtainable from a black Aspergillus that belongs to the
Aspergillus niger Group as defined by Raper and Fennell (1965, In: The
Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pp
234-344), such as Aspergillus niger, Aspergillus ficuum and Aspergillus
Zo awamori.
In the methods and products according to the invention the phytase
is preferably added to the low-phytate feed of the animal in an amount
exceeding the amount of phytase activity naturally present in any of the
feed stuffs ordinarily used in the preparation of anima! feed. The activity
Zs level of the phytase supplemented to the low-phytate feed is preferably at
least 10 FTU (for definition see Example 1 ) per kg of low-phytate feed,
more preferably at least 20 FTU per kg of low-phytate feed, more
preferably at least 50 FTU per kg of low-phytate feed, more preferably at
least 100 FTU per kg of low-phytate feed, more preferably at least 200
so FTU per kg of low-phytate feed. Usually the activity level will be less
than
10,000 FTU per kg of low-phytate feed, preferably less than 5000 FTU
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per' kg of low-phytate feed, more preferable less than 2500 FTU per kg of
low-phytate feed.
According to another aspect of the invention there is provided a
method for preparing a phytase-containing low-phytate feed for animals,
s wherein feed stuffs are mixed with a phytase, and optionally zinc. The
phytase may be mixed with the feed stuffs in dry form, e.g. as an enzyme
containing granulate, or in liquid form, e.g. in the form of a stabilised
liquid concentrate. In a preferred method the phytase is mixed with the
feed stuffs as part of a premix which may contain other feed additives
~o such as other enzymes, vitamins, minerals. For animals receiving (mainly)
a milkreplacer, this feed would be an optional way to offer phytase to the
animal.
A product according to the invention provides a low-phytate feed
comprising feed components specifically adapted for the said animal
is supplemented with a phytase. In one embodiment of the invention, a
low-phytate feed may be provided by mixing feed components with a high
phytate content with feed components with a low phytate content. In
another embodiment of the invention, a low-phytate feed may be provided
by including a plant source in the feed which is genetically modified
Zo and/or obtained by classical selection techniques to contain a lower
amount of phytate than the amount which is present in the unmodified
and/or parental plant. For instance, the use of low-phytate corn or soy
variants for feed preparation may provide feeds with a phytate-P content
of about 0.2%.
25 Another product according to the invention provides a low-phytate
feed specifically adapted for young animals, especially young ruminants,
supplemented with a phytase. For instance, a feed for veal calves in the
growing and fattening period will usually consist of mixtures of skim milk
powder (and other milk products) and milk substitutes (milk replacers) of
ao vegetable origin such as soybean isolates, soybean concentrates and
wheat as well as animal fat and vitamins and minerals. Some other feeds
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or feedstuffs, like hay, corn, or a special mixed concentrate may be
offered additionally.
Accordingly, a low-phytate feed specifically adapted for young
animals, especially young ruminants, in the growing and fattening period
s is a milk replacer which, in addition to phytase, preferably comprises less
than 60% (w/w solid matter) skim milk powder, more preferably less than
40%, more preferably less than 30%, more preferably less than 20%,
more preferably less than 10%, and most preferably 0%. Preferably the
feed for young animals in the growing and fattening period comprises at
~o least 5% of milk substitutes of a vegetable origin, more preferably at
least
25%, most preferably at least 50%.
Some milk replacer produced also is used for monogastric animals,
especially for piglets.
Thus, according to one aspect of the invention, there is provided a
i s method for feeding an animal, such as poultry, a low-phytate feed
supplemented with a phytase. Preferably, said low-phytate feed comprises
a genetically modified or classically obtained low-phytate corn or soy
source, and/or alternatively, another low-phytate plant source.
According to a preferred aspect of the invention there is provided a
2o method of feeding a young animal in the growing and/or fattening period,
such as a young ruminant, a low-phytate diet supplemented with a
phytase. The diet comprises milk products, of which the amount of skim
milk powder preferably is as low as possible, and milk substitutes as
described hereinabove and is supplemented with a phytase.
is The invention is also directed to the non-therapeutic use of a
phytase in any of the methods according to the invention such e.g. a
method of feeding an animal a low phytate feed, the use of a phytase for
the improvement of zootechnical results in breeding and/or keeping of said
animal, as well as to the use of a phytase for the improvement of the
so mineral status of said animal.
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The following Examples serve to illustrate the invention and are by
no means intended to limit the invention in any way.
Example 1
Example 1
Effects of microbial phytase on Growth and feed conversion ratio
up to 177 kg of live weight
io
Trials are carried out with 4 groups of 19 piebald bull calves. After
a starting period of 6 weeks animals received the following diets during a
20 week fattening period: feed was either supplemented with microbial
phytase (NATUPHOS° 5000, Gist-brocades, Delft, the Netherlands,
~s obtainable from BASF, l.udwighshafen, Germany), ZnS04 or both. Group
4 served as a negative control. Microbial phytase was supplemented at a
dose of 500 FTU (phytase units) per kg of feed. The analytical method for
determining microbial phytase activity and the definition of a phytase unit
has been published by Engelen et al. (Journal of AOAC International 77
Zo (3): 760-764 (1994).
Based on the diet composition of Table 1 the contents of various
nutrients have been calculated as follows for all diets:
Nutrient (g/kg): Crude protein (185.2), Crude Fat (229.9), Crude
Fiber (O.6), Moisture (35.9), Ash (65.3), Nitrogen free extract (451.1 ),
25 Lactose (35.1 ), Starch (83.7), Dairy protein (51.8), non-dairy protein
(13.5), Lysine (17.9), Methionine (7.5), Met + Cys (10.4), Threonine
(8.4), Tryptophan (2.2), Isoleucine (8.4), Calcium (8), Phosphorus (5.7),
Phytate-P (0.5), Iron (23 mg/kg). ME = 4471.1 kcal/kg.
Table 2 shows the average feed intake, growth and feed conversion
3o ratio (FCR) calculated therefrom for each of the four groups.
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Table 1. Diet composition for Groups 1-4 during the fattening period
Group 1 Group 2 Group 3 Group
4
Raw material
Whey powder 51.8 51.8 51.8 51.8
Soy protein 12.5 12.5 12.5 12.5
isolate
Wheat gluten 1 1 1 1 1 i 1 1
Animal fat 8 8 g g
Lard 7.5 7.5 7.5 7.5
Coconut oil 5 5 5 5
Lecithin 1 1 1 1
Emulsifier 0.65 0.65 0.65 0.65
(Berol)
Dicalcium 0.2 0.2 0.2 0.2
phosphate
Calcium 1.25 1.25 1.25 1.25
formiate
DL-methionine 0.05 0.05 0.05 0.05
Vitamin premix1 1 1 1
Zinc sulphate 0.05 0.05 0 0
Phytase 500 0 500 0
(FTU/kg)
Table 2. Feed intake, growth and feed conversion ratio of Groups 1-4
during the fattening period
Group Group Group 3 Group 4
1 2
Feed intake 327.5 328.5 335.1 323
(kg)
Growth (kg) 164.4 168.4 177.2 161.2
FCR (kg/kg) 1.99 1.95 - 1.89 2
Best results were obtained for group 3 grown on feed containing
1o supplemental phytase and no additional ZnS04.
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Example 2
Effects of microbial phytase on zootechnical parameters
up to 267 ka of live weiaht
A similar experiment as described in Example 1 was conducted
with older animals. This is relevant for practice since results obtained may
vary as a function of body weight and age.
The study was conducted with veal calves with a live weight of
130-150 kg at the start of the experimental period. In total 24 male
~o black-and-white veal calves (Holstein-Friesian) were used for the
experiment.
On day 0 all animals were weighed and divided among three
experimental groups in such a way, that the groups were as
homogenuous as possible. Allocation was based on body weight and
5 haemoglobin content of the blood.
The animals were housed individually under conventional conditions
in wooden boxes (75 *' 175 cm) with slatted floors.
After allocation, the animals were changed from a commercial milk
replacer to the experimental diet plus premix. The composition of the diet
Zo was as described in Table 3.
Microbial phytase was supplemented as NATUPHOS~ 5000
(Gist-brocades, Delft, the Netherlands, obtainable from BASF,
Ludwigshafen, Germany) in the diets.
During the experiment, the animals were fed according to the
is schedule shown in Table 4. Animals were fed in the morning and in the
afternoon. The milk replacer including the phytase was dissolved in hot
water (60-70°C), mixed for 3-5 minutes, followed by addition of cold
water while mixing until the required amount of milk with a temperature
of 40-41 °C was prepared. The animals were not allowed to drink extra
so water.
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Table 3: Diet composition for Groups 1-3 during the fattening period
_" Group 1 Group 2 Group 3
Raw material % %
Whey powder 41.7 41.7 41.7
Whey powder, delactosed14 14 14
Whey protein 10 10 10
Soy protein (HP 100) 10 10 10
Pregelatinized starch 2.5 2.5 2.5
Fat' 20.15 20.15 20.15
Methionine 0.35 0.35 0.35
Lysine 0.75 0.75 0.75
Zinc sulphate 0.02 0.02 0.02
Premix (%) 0.5 0.5 0.5
Phytase (FTU/kg) < 20 1,000 10,000
'~ Fat composition: 37% tallow, 40% lard, 15% coconut fat, 5% lecithin
s and 3% emulsifier.
The above diet contained 0.04% phytate-P.
Table 4: Feeding schedule
Day Kg milk per calfConcentration Intake powder
per feeding (g (g
powder per kg per animal per
milk) feeding)
0-7 8.5 130 1,105
7-14 8.5 135 1,148
14-21 8.5 140 1,190
21-28 8.5 145 1,233
28-35 8.5 150 1,275
35-42 8.5 155 1,318
42-49 8.5 160 1,360
49-56 8.5 165 1,403
56-63 8.5 170 1,445
63-77 8.5 175 1,488
77-90 8.5 180 1,530
91 8.5 130 1,105
~o
The animals were weighed before allocation and at 4,8,12 and 13
weeks after allocation with a precision of 0.2 kg. Feed intake was
registered per animal. With these figures and weight gain results, feed
conversion ratio was calculated as kg feed per kg weight gain.
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Body weight (kg) at different times is shown in Table 5.
Table 5
Days Negative controlGroup with Group with
experimental group 1,000 FTU/kg 10,000 FTU/kg
period
0 140.8 140.8 140.9
28 176.9 180 181.3
56 213.7 217.1 218
84 244 252 254.1
90 251 259.1 262.5
91 252.9 261.4 265.4
Experimental groups receiving diets comprising phytase show a
substantially higher daily gain in bodyweight than the negative control
group.
~o Cumulative feed conversion ratio (kg feed/kg body weight gain)
during the different periods for the three different groups is shown in
Table 6.
Table 6
Days Negative controlGroup with Group with
experimental group 1,000 FTU/kg 10,000 FTU/kg
period
0-28 1.83 1.69 1.64
0-56 1.93 1.85 1.85
0-84 2.16 1.99 1.99
0-90 2.2 2.04 2.02
0-91 2.19 2.02 1.99
Experimental groups receiving diets containing supplemented
microbial phytase show a considerable improvement in feed conversion
ratio.
Zo
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Example 3
Effects of microbial phytase on mineral status
Animals were grown as detailed in Example 1. Blood samples were
s taken at the start of the fattening period and 2, 4, 8, 12, 16 and 20
weeks thereafter. Blood was analysed for the content of haemoglobin,
iron, calcium, magnesium, copper, and zinc following methods known to
people skilled in the art. Contents of calcium, magnesium, copper and iron
were the same for all test groups. The content of iron in blood plasma
~o decreased markedly during this period but did not differ significantly
between the groups. There appeared to be a significant difference in zinc
contents in blood plasma between the groups as shown in Figure 1.
Group 1 receiving elevated levels of ZnS04 and phytase in the diet
showed highest levels of zinc in blood plasma. Group 3, receiving phytase
~ s only and no added ZnS04, showed intermediate zinc levels in blood
plasma. The same is true for group 2 receiving supplemental zinc in the
diet and no phytase. The negative control group showed very low levels
of zinc in blood plasma.
It is concluded that it is possible to improve the mineral status of
Zo the animal by either supplementing the diet with ZnS04 , by the addition
of phytase or by the combination of both.
The effects of age and diet composition on blood haemoglobin
levels are shown in Figure 2. The increased levels of haemoglobin in
animals receiving diets containing phytase and no added minerals (Group
25 3) is another indication of a positive effect of phytase on the performance
of the animals.
