Background of the invention The diets of ruminants vary considerably in different husbandry systems, but also at different ages. In extensive husbandry systems, 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 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 milkreplacer.

Some other feeds or feedstuffs, like hay, corn, or a special mixed concentrate may be offered additionally. Often these feedstuffs require a 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, e. g. mixed concentrates (usually produced in a feed mill), cereals,

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 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, 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 proteins, thereby decreasing the bioavailability of protein and nutritionally important mineras.

The use of phytase in the nutrition of monogastric animals (e. g. pigs, poultry and fish) has become widespread thanks to the availability of microbial phytases at an affordable cost. Cloning and overexpression of 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 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%.

We now demonstrate that the addition of phytase to feed having a low phytate content such as feed for young animals (ruminants or

non-ruminants) results in a marked improvement in daily gain and feed conversion ratio.

Description of the Figures 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; 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 3, only phytase; Group 4, no additions.

Description of the invention 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.

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 a method of breeding and/or keeping an animal.

One would not expect any beneficiai 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 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 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 animas.

Contents of zinc in blood plasma and haemoglobin levels are also 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 only slightly improves the zootechnical results such as growth rate and feed conversion ratio.

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 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 amount of feed (kg) required per kg of growth of the animal.

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 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, <BR> <BR> <BR> <BR> Biochemical Journal 92,42-46). Phytate-P is calculated as phytic acid * 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, especially young ruminants, as defined hereinafter. Young animals can be young ruminants such as calves, heifers, lamb, deer calves or goats.

Young animals can also be young non-ruminants, such as pilets, broilers or pullets.

A phytase is herein defined as an enzyme which is a phosphatase 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 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 phytase is obtainable and/or the structure and/or biochemical properties of the phytase molecule. Thus, the phytase applied in the methods and

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 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.

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 6.0. 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 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 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 animal feed. The activity 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 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

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, 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 stabilise 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 such as other enzymes, vitamins, mineras. 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 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 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%.

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 vegetable origin such as soybean isolates, soybean concentrates and wheat as well as animal fat and vitamins and mineras. Some other feeds

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 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 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 animais, especially for pilets.

Thus, according to one aspect of the invention, there is provided a 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 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.

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 mineral status of said animal.

The foliowing 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 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 (NATUPHOSQ'5000, Gist-brocades, Delft, the Netherlands, obtainable from BASF, Ludwighshafen, 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 (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 (0.6), Moisture (35.9), Ash (65.3), Nitrogen free extract (451.1), 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 ratio (FCR) calculated therefrom for each of the four groups.

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 11 11 11 11 Animal fat 8 8 8 8 Lard 7. 5 7. 5 7. 5 7.5 Coconutoil 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 Vitamin premix 1 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 1 Group 2 Group 3 Group 4 Feed intake (kg) 327.5 328.5 335.1 323 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 supplemental phytase and no additional ZnS04.

Example 2 Effects of microbial phytase on zootechnical parameters up to 267 kg of live weight.

A similar experiment as described in Example 1 was conducted with older animas. 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 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 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 was as described in Table 3.

Microbial phytase was supplemented as NATUPHOSO 5000 (Gist-brocades, Delft, the Netherlands, obtainable from BASF, Ludwigshafen, Germany) in the diets.

During the experiment, the animals were fed according to the schedule shown in Table 4. Animals were fed in the morning and in the afternoon. The milk replacer including the phytase was dissolve 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 water.

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, delactosed 14 14 14 Wheyprotein 10 10 10 Soy protein (HP 100) 10 10 Pregeiatinizedstarch 2. 5 2. 5 2.5 Fat20. 15 20.15 20. 15 Methionine 0. 35 0. 35 0.35 Lysine 0. 75 0. 75 0.75 Zincsulphate 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 and 3% emulsifier.

The above diet contained 0.04% phytate-P.

Table 4: Feeding schedule

Day Kg milk per calf Concentration (g Intake powder (g per feeding 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 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. Body weight (kg) at different times is shown in Table 5.

Table 5

Days Negative control Group 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.

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 control Group with Group with experimental group 1,000 FTU/kg 10,000 FTU/kg period 0-28 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.

Example 3 Effects of microbial phvtase on mineral status Animals were grown as detailed in Example 1. Blood samples were taken at the start of the fattening period and 2,4,8,12,16 and 20 weeks thereafter. Blood was analyse 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 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 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 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 mineras (Group 3) is another indication of a positive effect of phytase on the performance of the animas.