MXPA98003507A - Food additives for anima - Google Patents

Abstract

The present invention relates to animal feed additives comprising galactase enzymes. More specifically, the invention relates to animal feed additives comprising an arabinogalactan endo-1,4-α-galactosidase and / or an arabinogalactan endo-1,3-α-galactoside

Description

Anra-VOS OF FOOD FOR ANTMAT.F-S TECHNICAL FIELD The present invention relates to animal feed additives comprising galactanase enzymes. More specifically, the invention relates to animal feed additives comprising an arabinogalactan endo-1, 4-β-galactosidase and / or an arabinogalactan endo-1,3-β-galactosidase.

Traditionally, animal feed diets, for example for pigs and pigs, are mainly based on cereals and soybean feed. However, in recent years there has been increasing interest in the use of alternative products such as peas, beans, soybean meal, rapeseed meal, lupines, cereal by-products and beet pulp. In some of these products, for example in sunflower meal, rapeseed meal, lupine, cereal byproducts and beet pulp, low digestibility often limits their inclusion in appreciable amounts in animal feed diets. This low REF: 27282 digestibility is associated with the composition of the carbohydrate fraction in these products, which consists mainly of polysaccharides other than starch. Polysaccharides other than starch are not degraded in the small intestine by the digestive enzymes of monogastric animals, and therefore do not offer all their energy potential to the animal. It is known that the hydrolysis of these polysaccharides would solve two problems, one regarding the welfare of the animal and the other related to an improvement in the economy of production. Enzymes that improve the diet are enzymes that improve the digestibility of the food and that are able to increase the efficiency of the food. Food enhancing enzymes work by improving the digestibility of food components. This improvement can be carried out, for example, by degradation of poly- and oligosaccharides in cereals and vegetable proteins. The established food enhancing enzymes include α-galactosidases, phytases, β-glucanases, proteases, cellulases and xylanases. However, the use of galactanases and β-galactosidases as food enhancing enzymes has never been suggested.

BRIEF DESCRIPTION OF THE INVENTION It has now been found that a certain group of enzymes designated galactanases are particularly beneficial for their incorporation into animal feed, particularly when incorporated together with one or more improved food enzymes. Accordingly, in its first aspect, the present invention provides feed additives for animals that comprise effective amounts of galactanase enzymes. In another aspect, the invention provides a method for improving the energy uptake of an animal diet, which method comprises supplementing the animal feed additive of the invention to monogastric animals. In still another aspect, the invention provides a process for the pretreatment of animal feed, a process by which the animal's food is subjected to the action of a galactanase enzyme.

BRIEF DESCRIPTION OF IOS DRAWINGS The present invention is further illustrated with reference to the accompanying drawing, in which: Figure 1 shows the degradation of galactana by a galactanase and a lactase.

PFflCffiTPCTOH PÜGTATITIADA DE A -MVEHCIQ Food Additives for Animals The present invention provides an animal feed additive comprising an effective amount of a galactanase enzyme. In a preferred embodiment, this galactanase enzyme is arabinogalactan endo-l, 4-β-galactosidase (EC 3.2.1.89). In another preferred embodiment, the galactanase enzyme is arabinogalactan endo-l, 3-β-galactosidase (EC 3.2.1.90). In a third embodiment, the animal feed additive comprises effective amounts of arabinogalactan endo-1,4-β-galactosidase and arabinogalactan endo-1,3-β-galactosidase. In the context of this invention, the animal feed additive is an enzyme preparation comprising a food enhancing enzyme (food enzyme) and suitable carriers and / or excipients, and enzyme preparation which is provided in a form that is suitable for administration to food. for animals. The animal feed additive of the invention can be prepared according to methods known in the art and can be in the form of a dry or liquid preparation. The enzyme to be included in the preparation can optionally be stabilized according to methods known in the art. The stabilized enzyme preparation is also known as protected or stabilized enzyme systems. In a specific embodiment, the animal feed additive of the invention is a granulated enzyme product which can be easily mixed with feed components, or more preferably, forms a premix component. The granulated enzyme product may be coated or uncoated. The particle size of the enzyme granulates are preferably compatible with those of the feed and premixing components. This provides a safe and convenient means of incorporating enzyme into foods. In another specific embodiment, the animal feed additive of the invention is a stabilized liquid composition, which may be an aqueous or oil-based suspension. Optionally, the liquid composition can be added to the animal feed composition after pelleting or pelleting this composition. In another preferred embodiment, the present invention provides an animal feed additive, which additive further comprises an effective amount of one or more food enhancing enzymes, in particular food enhancing enzymes selected from a group consisting of β-galactosidase, in particular lactase, α-galactosidase, phytase, β-glucanase, mannanase, xylanase, protease, cellulose or other hydrolases. In its most preferred embodiment, the present invention provides an animal feed additive comprising a galactanase and a lactase only. In an animal feed, polysaccharides such as galactane and arabino-galactane bind to rhamnogalacturan, a major constituent of the pectin matrix. Galactanase is capable of breaking those junctions that result in galactose monosaccharides, galactose dimer (gal-gal) and various polysaccharides. Only galactose is directly metabolizable. By adding lactase, the galactose dimer is hydrolyzed, resulting in more galactose monosaccharides and better utilization of the feed.

Microbial Sources The enzymes used according to the present invention can be obtained from any available source.

Preferably, the enzyme is of microbial origin, in particular of bacterial, or fungal or yeast origin. The enzyme can be derived from the source in question by the use of any suitable technique. In particular, the phytase enzyme can be obtained by fermentation of a microorganism in a suitable nutrient medium, followed by isolation of the enzyme in question by methods known in the art. Alternatively, the enzyme can be obtained by recombinant DNA techniques. In this manner, the enzyme can be obtained by general methods known in the art, for example by isolating a DNA fragment encoding the enzyme in question; combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector; introducing the plasmid vector into an appropriate host (i.e., an Escherichia coli or a member of the genus Bacillus, Aspergillus or Streptomyces) either as a replicating plasmid autonomously or integrated into the chromosome; cultivate the host organism under conditions that lead to the expression of the enzyme; and recovering the enzyme in question from the culture medium. The broth or medium used for culture may be any conventional means suitable for the growth of the host cell in question, and may be constituted according to the principles of the prior art.

Preferably, the medium contains carbon and nitrogen sources and other inorganic salts. Suitable media, for example minimal means or complex media are available from commercial suppliers, or can be prepared according to published recipes, for example, the catalog of strains of the American Type Culture Collection (ATCC). After cultivation, the enzyme is recovered by a conventional method for isolation and purification of proteins from a culture broth. Well-known purification procedures include separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components from the medium by means of salt such as ammonium sulfate and chromatographic methods such as, for example, ion exchange chromatography, gel filtration chromatography. , affinity chromatography, etc. The fermentation broth containing the enzyme is preferably treated by means of both filtration and ultrafiltration before being used according to the present invention. In addition, the enzymes in question can be incorporated as one or more single component preparations or as enzyme complex preparations. The galactanase enzyme contemplated in accordance with the present invention can be derived from any available source. In a preferred embodiment, the galactanase enzyme is derived from a filamentous fungus.

Preferably, the filamentous fungus is an Ascomycotina (for example, the genus belonging to Loculorpycetes, Discomycetes, Plectcmycetes, Hemiascomycetes, Pyrenomycetes and Gymnoascales). In the most preferred embodiments, the fungus is an Ascomycete that belongs to the Plectomycetes, more specifically Eurotiales, Trichocomaceae or Aspergillus or an Ascomycete that belongs to Pyrenomycetes, more specifically Sordariales or Chaetomiaceae, or the filamentous fungus is an Ascomycete that belongs to Pirenomycetes mitosporica , more specifically Humicola or Myceliophthora. In other preferred embodiments, the filamentous fungus is a Basidiomycete, in particular a Basidi omycete belonging to Hymenomycetes (Dacrymycetales, Auriculariales, Cantharellal es, Tulasnellal es, Agaricales and Aphyllophorales), more specifically Aphyllophorales or Polyporaceae, more specifically Merypilus. In the most preferred embodiments, the galactanase enzyme is derived from a strain of Aspergillus, in particular Aspergillus aculeatus and Aspergillus niger, a strain of Bacillus, in particular Bacillus subtilis var. amylosacchari ticus, a strain of Humicola, in particular Humicola insolens, a strain of Merypilus, in particular Meripilus giganteus, a strain of Myceliophthora, in particular Myceliophthora thermophilum, a strain of Penicillium, in particular Penicillium citrium or a strain of hermomyces, in particular hermomyces lanuginosus . Bacterial galactosidases are available from E. coli strains and from Bacillus strains, in particular Bacillus stearothermophilus and Bacillus subtilis. Mycotic galactosidases are available from strains of Neusospora, Rhizopus and Aspergillus. Galactosidases are also available from yeasts, in particular strains of Saccharomyces cerevisiae and strains of Sacharomyces oleaginosus. In a preferred embodiment of the invention, the galactosidases are derived from a strain of Aspergillus oryzae, or a strain of Aspergillus ficuum, a strain of Aspergillus aculeatus or a strain of Aspergillus niger. The enzyme phytase can be derived from a fungal strain, in particular an Aspergillus strain, for example Aspergillus niger, Aspergillus oryzae, Aspergillus ficuum, Aspergillus awamori, Aspergillus nidulans and Aspergillus terreus. A phytase enzyme derived from a strain of Aspergillus niger or a strain of Aspergillus oryzae is more preferred. The phytase enzyme can also be derived from a bacterial strain, in particular a Bacillus strain or a Pseudomonas strain. Preferably, the phytase enzyme is derived from a strain of Bacillus subtilis. Finally, the enzyme phytase can be derived from a yeast, in particular a strain of Kluveromyces or a strain of Saccharomyces. Preferably, the phytase enzyme is derived from a strain of Saccharomyces cerevisiae. The enzyme β-glucanase can be derived from a strain of Aspergillus, in particular Aspergillus aculeatus, a strain of Humicola, in particular Humicola insolens, a strain of Thermomyces, in particular hermomyces lanuginosus or a strain of Trichoderma. The xylanolytic enzyme may be derived from a strain of Aspergillus, a strain of Bacillus, in particular Bacillus agaradherens or Bacillus pumilus, a strain of Dictyoglomus, a strain of Humicola, a strain of Rhodothermus, a strain of Thermotoga, a strain of Thermomyces in particular Thermomyces lanuginosus or a strain of Trichoderma. In the context of this invention "an enzyme derived from" encompasses an enzyme that is naturally produced by the particular strain, either recovered from that strain or encoded by a DNA sequence isolated from this strain and produced in a host organism transformed with the DNA sequence.

Method for Improving Energy Capture In another aspect, the invention relates to the use of a feed additive for animals of the invention, to improve the uptake of energy from the diet supplied to monogastric animals. In the context of this invention, monogastric animals include poultry, in particular tender chickens, laying hens and turkeys, pigs in particular young piglets and calves. According to this method, the animal feed additive of the invention is supplemented to the monogastric animal before or simultaneously with the diet. Preferably, the animal feed additive of the invention is supplemented to the monogastric animal simultaneously with the diet. In a more preferred embodiment, the animal feed additive is added to the diet in the form of a granulate or a stabilized liquid. In another preferred embodiment, the diet comprises substantial amounts of legumes, in particular soybeans, lupine, peas and / or beans and cruciferous, in particular rape seed. In another preferred embodiment, the diet additionally comprises additional quantities of cereals, mainly barley, wheat, rye, corn, rice and / or sorghum.

Food-enhancing enzymes should be applied in amounts suitable for the degradation of non-digestible polysaccharides. It is now contemplated that the enzyme is administered in an amount corresponding to an activity in the range of from about 0.1 to about 10 mg of enzyme protein per kg of animal feed, preferably from about 0.1 to about 5 mg of enzyme protein per kg of protein. animal food Food pretreatment for animals In another aspect, the invention provides a process for the pretreatment of the animal feed, by which process the animal feed is subjected to the action of a galactanase enzyme. Preferably, the enzyme galactanase is an arabinogalactan endo-l, 4-β-galactosidase or arabinogalactan endo-1,3-β-galactosidase. In a preferred embodiment of the invention, the process further comprises treating the animal feed with an effective amount of one or more enzymes that are selected from the group consisting of β-galactosidase, in particular lactase, α-galactosidase, phytase, β -glucanase and / or xylonasa.

EXAMPLES The invention is further illustrated with reference to the following examples, which are not intended to be in any way limiting the scope of the invention as claimed.

Example 1 Degradation of galactan hydrolysis products by lactase Soy galactane is isolated, according to the procedure described by Labavitch et al. \ J. Biol. Chem. 1976 251 5904-5910]. To a 1% solution of the soy galactana isolated in 0.1M sodium acetate buffer, pH 5.0, a galactanase (endo-1,4-β-galactosidase, obtained from Aspergillus aculeatus in accordance with 92/13945) is added. , see in particular example 3), and incubated overnight at 30 ° C. After activation of the galactanase by heat, 10 ml of a 1% solution of sumilactase (Sumilact ^, Lot number 40303-01, available from Shinihon, Japan) are added to 1 ml of galactane degradation products, and incubation It is carried out overnight at 30 ° C.

The degradation is verified by high resolution chromatography by size exclusion, as described by Christgau et al. I "Curr. Genet, 1995, 27 135-141], figure 1. This figure shows that the hydrolysis products resulting from the action of galactanase are further degraded by the action of lactase, therefore, lactase increases the amount of galactose, which is digestible, and significantly decreases the amount of galactobiose and higher oligomers and polymers, which are not digestible.

Ejepplo 2 Real Metabolizable Energy Using a bioassay for real metabolizable energy (TME), in food, described by Sibbald [Sibbald I R; PQ ltry Science 197655303-308], and modified by Dale & Fuller (Dale N and Fuller HL, Poultry Science 1984 63 1008-1012), examined the effects of a galactanase (endo-1,4-β-galactosidase, obtained according to 29 92/13945, see in particular example 3 ), a lactase enzyme Sumilact ", batch number 40303-01, available from Shinihon, Japan), and a mixture containing both enzymes, with respect to the uptake of energy from an animal feed composition. The food composition of the basal diet is soy bean flour, galactanase is included at a dosage of 0.2 g / kg (experiment A) or 1.0 g / kg, experiment B) of soybean meal, and the lactase at a dosage of 0.1 g / kg (experiment A) or 0.5 g / kg (experiment B) of soybean meal In experiment A, a total of 34 adult cocks are used and experiment B uses a total of 42 adult roosters Before the experiments, the birds are fasted for 21 hours for aciar your digestive tracts. At the beginning of the experiment, the roosters are weighed individually and then forced feeding is provided by the appropriate amount of feed. After feeding, the birds are returned to their cages and the excreta is collected. Exactly 48 hours after the forced feeding, the birds are weighed again and the excreta are emptied quantitatively. The excreta is frozen, lyophilized, allowed to reach equilibrium with atmospheric humidity, weighed and crushed. Tests of crushed food samples and excreta are made, respectively, for coarse determination of energy using a calorimeter. Tests of dry matter food samples are carried out. Table 1 and 2 below show the results, determined as the difference between the energy of the food supplied and the energy of the excreta emptied.

Table 1 Real M tabolizable Energy (TME) Values with different subscripts are significantly different (P <0.05) Table 2 Real Metabolizable Energy (TME) Values with different subscripts are significantly different (P <0.05) Example 3 Apparent Metabolizable Energy The effects of the enzymes on the nutritional value of the basal diet are determined using a classic apparent metabolizable energy test (AME) to estimate the amount of energy in the diet available to the bird. The AME study is carried out with an experimental basal diet containing sorghum (64%) and soybean meal (30%). Commercial broiler chickens (Ingham ™ IM98) are reared from incubation until 24 days of age in a pen on the floor in a temperature-controlled shed. The birds are provided with commercial starter feed for 21 days and then commercial finished feed. The chickens are weighed in groups of five and transferred to 48 metabolic cages located in another room in the same shed. Experimental diets are provided for 7 days (days 1-7). The first three days (days 1-3) allow chickens to adapt to cages and food. The ingestion of the food during this period is measured. During the next four days (days 4-7) the ingestion of food is measured and all the excreta are collected and dried. The moisture content of the excreta collected on day 5 is determined by drying overnight at 90 ° C. Each diet is provided to 25 birds. The dry matter (DM) content of the samples of sorghum, settled foods and ground foods is determined by overnight drying at 105 ° C. The coarse energy (GE) values of excreta and crushed food are measured with a Parr isoperibol calorimeter. The nitrogen content of the food and excreta samples is measured by Kjeltec digestion, distillation and titration methods. In this experiment, galactanase is included at a dosage of 6.7 ml / kg of feed, and lactase is included at a dosage of 3.3 ml / kg of feed. Table 3 below presents the results, determined as the difference between the energy of the food supplied and the energy of the excreta emptied.

Table 3 Apparent Metabolizable Energy (AMEn) The values with different sub-indices are significantly different (P <0.05) It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to that it refers. Having described the invention as above, property is claimed as contained in the following:

Claims (16)

raiVOOICACIQNES

1. An animal feed additive, characterized in that it comprises an effective amount of an isolated endo-galactanase enzyme.

2. The animal feed additive according to claim 1, characterized in that endo-galactanase is obtained by recombinant DNA techniques.

3. The animal feed additive according to any of claims 1 or 2, characterized in that the endo-galactanase enzyme is an arabinogalactan endo-1,4-β-galactosidase.

4. The animal feed additive according to any of claims 1 or 2, characterized in that the endo-galactanase enzyme is an arabinogalactan endo-1,3-β-galactosidase.

5. The animal feed additive according to any of claims 1 to 4, characterized in that it further comprises an effective amount of one or more enzymes that are selected from the group consisting of β-galactosidase, α-galactosidase, phytase, β-glucanase and xylanase.

6. The animal feed additive according to claim 5, characterized in that the β-galactosidase is a lactase.

7. A premix for animal feed, characterized in that it comprises an animal feed additive according to any of claims 1 to 6.

8. The method for preparing food for animals, characterized in that the additive according to any of claims 1 to 6 or the premix according to claim 7 is mixed with the components before the granulation.

9. The method for preparing feed for animals, characterized in that the additive according to any of claims 1 to 6 is in the form of a liquid composition which is added to the animal feed composition after the granulation.

10. An animal feed, characterized in that it comprises an effective amount of an isolated endo-galactanase enzyme.

11. The animal feed according to claim 10, characterized in that it further comprises an effective amount of one or more enzymes that are selected from the group consisting of β-galactosidase, α-galactosidase, phytase, β-glucanase and xylanase.

12. The use of an isolated endo-galactanase characterized because it is used in animal feeds.

13. The use of an isolated endo-galactanase and one or more enzymes that are selected from the group consisting of β-galactosidase α-galactosidase, phytase, β-glucanase and xylanase, characterized in that they are used in animal feed.

14. A method for improving the energy uptake of an animal diet, which method is characterized in that it comprises the supplementation of the animal feed additive according to any of claims 1 to 6 or of the animal feed according to any of the claims 10 or 11, to monogastric animals.

15. A process for the pretreatment of animal feed, the process is characterized in that the animal feed is subjected to the action of an isolated endo-galactanase enzyme.

16. The process according to claim 15, which process is characterized in that it comprises treating the animal feed with an effective amount of one or more enzymes that are selected from the group consisting of β-galactosidase α-galactosidase, phytase, β- glucanase and xylanase.