AU748986B2

AU748986B2 - Application of phytase in feed having low content of phytate

Info

Publication number: AU748986B2
Application number: AU38128/99A
Authority: AU
Inventors: Robert Franciscus Beudeker; Jan Bart Hof; Arie Karst Kies
Original assignee: DSM NV
Current assignee: Koninklijke DSM NV
Priority date: 1998-04-01
Filing date: 1999-04-01
Publication date: 2002-06-13
Anticipated expiration: 2019-04-01
Also published as: EP1065942A1; CN1295443A; MXPA00009567A; AU3812899A; BR9909278A; CA2323581A1; WO1999049740A1; KR20010042395A; JP2002509704A

Description

WO 99/49740 PCT/EP99/02311 APPLICATION OF PHYTASE IN FEED HAVING LOW CONTENT OF

PHYTATE

Field of the invention The present invention relates to the application of the enzyme phytase in feed having a low content of phytate.

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, WO 99/49740 PCT/EP99/02311 -2- 2 vegetable 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 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 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 minerals.

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 -3non-ruminants) results in a marked improvement in daily gain and feed conversion ratio.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

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, ZnSO 4 and phytase; Group 2, only ZnSO 4 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, ZnSO 4 and phytase; Group 2, only ZnS0 4 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 o: 3o 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 Sanimal.

WO 99/49740 PCT/EP99/02311 -4- 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 average daily gain 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 animals.

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% 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.

WO 99/49740 PCT/EP99/02311 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% preferably from 0.01 to 0.15%, and more preferably from 0.01 to 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, T.G.Taylor and A.Williams, 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 piglets, 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 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 WO 99/49740 PCT/EP99/02311 -6products 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 WO 99/49740 PCT/EP99/02311 -7per 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 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 io 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 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 minerals. Some other feeds WO 99/49740 PCT/E P99/0231 1 -8or 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 more preferably less than 30%, more preferably less than more preferably less than 10%, and most preferably 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 most preferably at least 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 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.

WO 99/49740 PCTIEP99/02311 -9- 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 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 week fattening period: feed was either supplemented with microbial phytase (NATUPHOS® 5000, Gist-brocades, Delft, the Netherlands, obtainable from BASF, Ludwighshafen, Germany), ZnSO 4 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 77 760-764 (1994).

Based on the diet composition of Table 1 the contents of various nutrients have been calculated as follows for all diets: Nutrient Crude protein (185.2), Crude Fat (229.9), Crude Fiber Moisture Ash Nitrogen free extract (451.1), Lactose Starch Dairy protein non-dairy protein Lysine Methionine Met Cys Threonine Tryptophan Isoleucine Calcium Phosphorus Phytate-P 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.

WO 99/49740 WO 9949740PCT/EP99/0231

I

Table Diet composition for Groups 1 -4 during the fattening period Group 1 Group 2 Grou 1 Gow 2 Groupn 3 Raw material I1% 1-/ Whey powde 8i.

51.8 Soy protein isolate I I 51.8 12.5 12.5 12.5 Group 4 51.8 12.5 -f1 8 t I. I. Whleat gluten 11 Animal fat 8 8

I

Lard Coconut oil 5 5 5 Lecithin 1 1 11 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 formiate DL-methionine 0.05 0.05 0.05 0.05 Vitamin premix 1 1 1 1 Zinc sulphate 0.05 0.05 0 0 Phytase 500 0 500 0 (FT Table 2. Feed intake, growth and feed conversion ratio of Groups 1-4 during the fattening period 1 Group 2 Group 3 Group 4 Feed intake (kg) 327.5 328.5___ 335.1__323 Growth (kg) 1 64.4 168.4___ 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 ZnSO 4 WO 99/49740 PCT/EP99/02311 -11 Example 2 Effects of microbial phytase on zootechnical parameters up to 267 kq of live weight.

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 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 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 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 (60-70oC), mixed for 3-5 minutes, followed by addition of cold water while mixing until the required amount of milk with a temperature of 40-41oC was prepared. The animals were not allowed to drink extra water.

WO 99/49740 PCT/EP99/02311 12- Table 3: Diet composition for Groups 1-3 during the fattening period I GrouD 2 1 Grounn 3 Group 1 1 Group 1 Raw material Whey powder 41.7 41.7 41.7 Whey powder, delactosed 14 14 14 Whey protein 10 10 Soy protein (HP 100) 10 10 Pregelatinized starch 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 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.

WO 99/49740 -13- Body weight (kg) at different times is shown in Table PCT/EP99/0231I Table Days experimental period Negative control group Group with 1,000 FTU/kg 140.8 180 0 140.8 28 176.9 Group with 10,000 FTU/kg 140.9 181.3 218 254.1 262.5 265.4 180 213.7 217.1 84 244 252 251 259.1 259zoL.

261t.4 ~b1 .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 1.83 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.

WO 99/49740 PCT/EP99/02311 -14- Example 3 Effects of microbial phytase 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 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 io 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 ZnSO 4 and phytase in the diet showed highest levels of zinc in blood plasma. Group 3, receiving phytase only and no added ZnSO 4 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 ZnSO 4 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 3) is another indication of a positive effect of phytase on the performance of the animals.

Claims

1. A method of feeding an animal a feed supplemented with a phytase, wherein said feed is a low-phytate feed having a content of phytate-P in a concentration ranging from 0.01 to 0.2%

2. A method according to claim 1, wherein said feed is a low-phytate feed having a content of phytate-P in a concentration ranging from 0.01 to 0.15%.

3. A method according to claim 1, wherein said feed is a low-phytate feed having a content of phytate-P in a concentration ranging from 0.01 to 0.1%.

4. A method according to claim 1, wherein said feed is a low-phytate feed having a content of phytate-P in a concentration ranging from 0.01 to 0.05%. A method of breeding and/or keeping an animal, comprising feeding the animal according to the method of any one of the preceding claims.

6. A method of improving the zootechnical results of an animal, comprising 20 feeding the animal according to the method of any one of claims 1 to 4.

7. A method of improving the mineral status of an animal, comprising feeding the animal according to the method of any one of claims 1 to 4.

8. A method according to any one of the preceding claims, wherein the feed .further comprises zinc.

9. A method according to any one of the preceding claims, wherein the phytase is supplemented at an activity level of at least 10 FTU per kg of feed.

10. A method according to any one of the preceding claims, wherein the animal is a young animal. W:\fioa\NK1\Spcies\38128.doc 16

11. The method according to claim 10, wherein the young animal is a young ruminant selected from the group consisting of calf, heifer, lamb, deer calf and goat.

12. The method according to claim 10, wherein the young animal is a piglet.

13. The method according to claim 10, wherein the young animal is a broiler or pullet.

14. A method according to any one of the preceding claims, wherein the feed is specifically adapted for a young animal. A method for preparing a low-phytate feed for animals having a content of phytate-P in a concentration ranging from 0.01 to 0.2% wherein the method comprises the step of mixing feed stuffs and a phytase.

16. A method according to claim 15, wherein the phytase is mixed with the feed stuffs as part of a premix. oo. 20 17. A method according to any one of the preceding claims, wherein the phytase is obtainable from a plant or micro-organism.

18. A method according to any one of the preceding claims, wherein the phytase is obtainable from a fungus.

19. A method according to claim 18 wherein the fungus is a fungus of the genus Aspergillus. A method according to claim 15 wherein the phytate-P content is 0.01 to 0.15%.

21. A method according to claim 15 wherein the phytate-P content is 0.01 to RJ?7 0.1%. W:ciskankd\species\38128-9g.doc 17

22. A method according to claim 15 wherein the phytate-P content is .01 to

23. A low-phytate feed for animals having a content of phytate-P in a concentration ranging from 0.01 to 0.2% comprising feed components specifically adapted for said animals and supplemented with a phytase which does not naturally occur in the feed components.

24. A low-phytate feed for animals according to claim 23 having a content of phytate-P in a concentration ranging from 0.01 to 0.15% A low-phytate feed for animals according to claim 23 having a content of phytate-P in a concentration ranging from 0.01 to 0.1%

26. A low-phytate feed for animals according to claim 23 having a content of phytate-P in a concentration ranging from 0.01 to 0.05% S' 27. A feed according to any one of claims 23 to 26, wherein the phytase is present at an activity level of at least 10 FTU per kg. of feed. i *20

28. A method according to claim 1, substantially as hereinbefore described with reference to any of the examples. 0 0

29. A feed according to claim 23, substantially as hereinbefore described. 0* o 0 DATED: 4 April, 2002 PHILLIPS ORMONDE FITZPATRICK Attorneys for: DSM NV W:\ciskaBnki\speciesl38128-99.doc