NL2035782A - FEED ADDITIVE FOR IMPROVING INTESTINAL HEALTH AND/OR DIGESTIBILITY OF FEED IN LIVESTOCK - Google Patents

Abstract

22 FEED ADDITIVE FOR IMPROVING INTESTINAL HEALTH AND/OR DIGESTIBILITY OF FEED IN CATTLE EXTRACT 5 The present invention concerns a feed additive for improving intestinal health and/or digestibility of feed in livestock, comprising a serine protease isolated from Bacillus licheniformis, an acid endoprotease isolated from Aspergil/us niger.

Description

FEED ADDITIVE FOR IMPROVING INTESTINAL HEALTH AND/OR

THE DIGESTIBILITY OF FEED IN LIVESTOCK

TECHNICAL DOMAIN

The invention relates to a feed additive for improving intestinal health and/or the digestibility of feed in livestock, as well as a feed in which such a feed additive has been applied and a use of the feed additive.

STATE OF THE TECHNOLOGY

Due to the unbalanced amino acid content and the resulting limited solubility of vegetable proteins, especially those of wheat and maize proteins, efficient proteolysis in the digestive tract of monogastric animals is suboptimal. For example, when soy or a soy derivative is included in the diet of monogastric animals such as pigs and poultry, a significant portion of the soy solids are not digested efficiently.

Soy contains many anti-nutritional factors that limit its extensive use. Protease inhibitor, trypsin inhibitor or trypsin inhibitor (TI) is one of the most common anti nutritional factors (ANFs) in soybeans and other grain feeds. TI inhibits the activity of enzymes that digest proteins in the digestive tract, such as trypsin and chymotrypsin.

TI is heat sensitive, but using heat treatment to reduce TI is not easy. Severe heat treatment completely eliminates TI, but can destroy the soy protein, reducing protein utilization in the animals.

Glycinin and B-conglycinin are soy antigens. When young animals are fed diets containing soy protein, a small portion of the undigested proteins enter the lymph and blood through gaps between the intestinal epithelial cells.

These macromolecules have significant antigenic activity and stimulate the immune system, leading to specific antigen-antibody responses and T-lymphoid cell-mediated delayed hypersensitivity. Soy antigens can therefore cause intestinal disorders, which reduces nutrient absorption and therefore growth performance. Soy antigens are heat stable, making heat treatment not very effective for removing soy antigens.

The use of serine proteases in animal feed is known, as shown for example

W02004034776.

However, known techniques do not take into account the fact that in monogastric animals the feed is exposed to 2 pH regions, the acidic stomach (2-4) and the neutral intestinal tract (7-7.5). Serine proteases are neutral proteases that have virtually no function in the acidic regions of the digestive system. As a result, efficient proteolysis in the digestive tract of monogastric animals is still suboptimal.

The present invention aims to find a solution for at least some of the above-mentioned problems.

SUMMARY OF THE INVENTION

The invention concerns a feed additive for improving intestinal health and/or the digestibility of feed in livestock according to claim 1. Preferred forms are shown in claims 2 to 11.

In a second aspect, the invention concerns a feed for livestock comprising a feed additive for improving intestinal health and/or the digestibility of feed in livestock according to claim 12. A preferred form is shown in claim 13. As well as a use according to claim 14.

An objective of the invention is to improve protein digestibility and protein utilization in the entire gastrointestinal tract by, on the one hand, improving the digestibility of the proteins that plants such as wheat, corn, soy, fabaceae, produce as a reserve nitrogen source, and on the other hand, improving the breakdown of the anti-nutritional factors (ANFs) on a protein basis (glycinin and B-conglycinin, TI, ...).

The common amino acid spectra of this complete group of protein-based ANF make them an ideal substrate for the Bacillus licheniformis serine protease.

It is an object of the invention to optimize proteolysis throughout the entire digestive tract of monogastric animals. To this end, the invention aims to provide efficient proteolysis in both the acidic and neutral regions of the gastrointestinal tract.

An aim of the invention is to release extra available proteins, peptides and amino acids in as many regions of the gastrointestinal tract as possible, and thus also save on dietary costs by reducing the addition of extra required proteins and amino acids.

It is an objective of the invention to increase the digestibility of feeds with high levels of glutamine and asparagine in as many regions of the gastrointestinal tract as possible.

Another objective of the invention is to improve the profitability and increase the valorization of by-products in the entire gastrointestinal tract.

The invention also aims to reduce nitrogen excretion into the environment and water consumption. Furthermore, the invention aims to improve animal health and to improve or guarantee food safety. The invention is also responsible for improving the litter quality.

DETAILED DESCRIPTION

The invention concerns a feed additive for improving intestinal health and/or the digestibility of feed in livestock.

Unless otherwise defined, all terms used in the description of the invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art of the invention.

For a better appreciation of the description of the invention, the following terms are explicitly explained. “A”, “the” and “the” in this document refer to both the singular and the plural unless the context clearly dictates otherwise. For example, “a segment” means one or more than one segment.

When “about” or “around” is used in this document for a measurable quantity, a parameter, a period of time or moment, or the like, variations of +/-20% or less are meant, preferably +/-10% or less, more preferably +/- 5% or less, even more preferably +/- 1% or less, and even more preferably

+/-0.1% or less than and of the quoted value, to the extent that such variations apply in the described invention. However, this must be understood to mean that the value of the quantity for which the term “approximately” or “around” is used is itself specifically disclosed.

The terms “comprise”, “comprising”, “providing”, “involving”, “involving”, “containing”, “containing” are synonyms and are inclusive or open terms that indicate the presence of what follows, and that the presence does not exclude or preclude other components, features, elements, members, steps known or described in the prior art.

The terms “consist of”, “consisting of”, “containing”, “containing” are synonyms and are exclusive or closed terms which indicate the presence of what follows and which exclude or preclude the presence of other components, features, elements, members, steps, known from or described in the prior art.

Quoting numerical intervals through the endpoints includes all integers, fractions, and/or real numbers between the endpoints, including these endpoints.

The expression “% wt”, “wt%”, “m%” or “% wt” in this document refers to the relative weight of a component based on the total weight of the entire product being referred to. “IU” or “ U” is the enzyme unit that represents enzymatic activity. 1 U or 1 IU (umol/min) is defined as the amount of enzyme capable of catalyzing the conversion of 1 ymol of substrate in 1 minute.

In a first aspect, the invention concerns a feed additive for improving intestinal health and/or the digestibility of feed in livestock.

According to a preferred embodiment, the feed additive comprises a serine protease isolable from Bacillus licheniformis and an acid endoprotease isolable from Aspergillus niger.

According to an embodiment, the serine protease has a first enzyme activity measured at a pH between 7 and 8, preferably 7.5, and the acid endoprotease has a second enzyme activity measured at a pH between 2.5 and

3.5, preferably 3. The enzyme activity is measured in U/g protease. The ratio between the first enzyme activity and the second enzyme activity is preferably between 0.8/1 and 1/0.8, expressed in U/g serine protease to U/g acid endoprotease, more preferably between 0.85/1 and 1/0.85, even more preferably between 0.9/1 and 1/0.9, even more preferably between 0.95/1 and 1/0.95.

In an alternative or a further preferred form, the feed additive comprises the serine protease and the acid endoprotease, in a mass ratio between 0.5/1 and 3.5/1, expressed in g serine protease to g acid endoprotease.

It has been shown that the use of a combination of a serine protease and an acid endoprotease leads to synergistic protein hydrolysis. The serine protease ensures efficient proteolysis in the neutral regions of the gastrointestinal tract, while the acid endoprotease ensures efficient proteolysis in the acidic regions of the gastrointestinal tract.

The serine protease is suitable for hydrolyzing peptide bonds located next to the amino acids aspartic acid (Asp) or glutamic acid (Glu). The acid endoprotease is capable of hydrolyzing glutamine-rich regions of protein chains in the stomach. The isoelectric point of glutamine, for example, is 5.7, which means that it will be protonated in the stomach and can therefore be more easily hydrolyzed by acid endoproteases.

The term "protease" is defined herein as an enzyme that hydrolyzes peptide bonds (protein bonds). The term "protease" includes any enzyme belonging to the

EC 3.4 enzyme group (including each of its thirteen subclasses). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego,

California.

The term “endoprotease” refers to a protease that hydrolyzes bonds internal to protein chains. Endoproteases exhibit activity on N- and C-terminally blocked peptide substrates that are relevant to the specificity of the protease in question. A serine protease is an endopeptidase that hydrolyzes the peptide bonds of proteins, with serine acting as a nucleophilic amino acid of the active site, i.e. serine is a part of the catalytic triad that acts as an active center. An acidic endoprotease is an endoprotease with optimal activity at acidic pH.

The term “feed”, as used in the text, refers to all compounds, preparations, mixtures or compositions suitable for or intended for consumption by an animal.

The term “improving the digestibility” of an animal feed means improving the availability of proteins, which leads to increased protein extraction, a higher protein yield, and/or improved protein utilization. The nutritional value of the feed is thereby increased and the growth rate and/or weight gain and/or feed conversion (i.e. the weight of the feed consumed in relation to the weight gain) of the animal is/are improved.

The term livestock includes all animals kept in agriculture and fisheries for economic reasons, usually as a source of food. Examples of animals are non-ruminants and ruminants. For example, ruminants include animals such as sheep, goats, horses and cattle, for example beef cattle, cows and young calves. In one embodiment, the animal is a non-ruminant animal. Non-ruminant animals include monogastric animals, e.g. pigs or pigs (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chickens (including but not limited to broilers, laying hens); young calves; and fish (including but not limited to salmon, trout, tilapia, catfish and carp; and crustaceans (including including, but not limited to shrimp and prawns).

In one embodiment, the feed additive further comprises a carrier material, preferably this carrier material is chalk.

In a preferred form, the feed additive comprises the serine protease, in an amount between 10 and 50 m%, expressed in relative weight of the serine protease on the total weight of the feed additive, preferably between 15 and 45 m%, more preferably between 20 and 40 m%, and most preferably between 25 and 35 m%.

In a preferred form, the feed additive comprises the acid endoprotease, in an amount between 1 and 40 m%, expressed in relative weight of the acid endoprotease on the total weight of the feed additive, preferably between 5 and 35 m%, even more preferably between 10 and 30 m%.

In a preferred form, the feed additive contains: - the serine protease and the acid endoprotease, in a mass ratio between 0.5/1 and 3.5/1, - additives and/or impurities, in an amount of maximum 1 m%, expressed in relative weight of the total amount of additives and/or impurities based on the total weight of the feed additive. - chalk, supplemented to 100%.

In a particular embodiment, the serine protease, in the form in which it is added to the feed, or when it is included in a feed additive, is well defined. Well defined means that the serine protease is at least 50% pure as determined by size exclusion chromatography. In other particular embodiments, the serine protease is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.

In a particular embodiment, the acid endoprotease, in the form in which it is added to the feed, or when it is included in a feed additive, is well defined. Well defined means that the acid endoprotease is at least 50% pure as determined by size exclusion chromatography. In other particular embodiments, the serine protease is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.

Serine proteases and acid endoproteases with purities of this order of magnitude can mainly be obtained using recombinant production methods.

A well-defined protease is advantageous. For example, it is much easier to correctly dose a protease that is essentially free of interfering or contaminating other proteases into the feed. The term correct dosing mainly refers to the objective of obtaining consistent and constant results, and to the possibility of optimizing the dosage based on the desired effect.

However, the proteases do not need to be so pure for use in animal feed; it can e.g. contain other enzymes.

The serine protease and the acid endoprotease are present in a feed additive that is then added to livestock feed (or used in a treatment process).

Alternatively, the protease, i.e. the serine protease and the acid endoprotease, can be added directly to the feed (or used directly in a protein treatment process}, in this case the feed additive preferably consists mainly of the serine protease and the acid endoprotease.

Such proteases can of course be mixed with other enzymes. According to a preferred form, the feed additive further comprises phytase, xylanase, amylase, or a combination thereof.

A phytase is a phosphatase enzyme capable of catalyzing the hydrolysis of phytic acid (an indigestible, organic form of phosphorus found in many plant tissues, especially in grains and oilseeds) and releasing a useful form of inorganic phosphorus .

Xylanase (EC 3.2.1.8) is any enzyme capable of breaking down the linear polysaccharide xylan into xylose, thus breaking down hemicellulose, one of the main components of plant cell walls.

Amylase is an enzyme that hydrolyzes starch during digestion. An amylase is suitable for attacking the linear α-1,4-glycoside polymer bond between the glucose molecules of starch.

According to an embodiment, the feed has a crude protein content of 50-800 g/kg, and the feed furthermore comprises at least one serine protease isolable from Bacillus licheniformis.

The feed additive must of course be used in an effective amount, i.e. in an amount that is sufficient to improve the solubility, digestibility and/or nutritional value of the feed. In a preferred form, the total amount of protease, i.e. the serine protease and the acid endoprotease, is therefore added to the feed in a concentration of 50-1000 ppm. The term “ppm” is defined as mg protease per kg feed.

Proteins from plant seeds (corn, wheat, soy, rapeseed, pea, sunflower, groundnut, ...) are storage proteins and have a higher nitrogen content than animal storage protein sources (milk, egg protein) to prevent an excess of nitrogen (N} from accumulating in the developing seedling. The higher nitrogen content is mainly the result of a higher presence of amino acids with a secondary nitrogen content such as glutamine (GIn) and asparagine (Asn).

These amino acids have secondary amide groups that form hydrogen bonds with adjacent protein chains. Mutual hydrogen bonds ensure that the proteins have a hydrophobic nature in an aqueous solution.

Wheat protein contains approximately 80% gluten protein. Gluten proteins can be divided into two main fractions based on their solubility in aqueous alcohols: the soluble gliadins and the insoluble glutenins. Both fractions consist of numerous, partly closely related protein components characterized by high glutamine and proline contents.

The insolubility of glutenins is due to the high concentration of non-polar amino acid residues such as proline and leucine and of the very polar but non-ionizable residue glutamine, and to the low concentration of ionizable side chains such as lysine, arginine, glutamic acid and aspartic acid. The interactions between glutamine and asparagine through hydrogen bonds play an important role in promoting the association of gliadin and gluteninin molecules. The hydrophilic part of the protein is buried, and it and the hydrophobic part form the outside of the protein making it insoluble in water.

Corn protein differs only partially from wheat proteins and has an extensive (50%) fraction called zein. It also has a pronounced Gin and Asn content, which explains its limited water solubility and functionality.

Due to the unbalanced amino acid content and the resulting limited solubility of vegetable proteins, in particular those of wheat and maize proteins, efficient proteolysis in the digestive tract of monogastric animals is suboptimal. The hydrophobic character and non-uniform amino acid pattern require a very specific proteolytic action.

Proteases from Bacilius licheniformis have a high affinity for hydrophobic proteins and protein parts. According to a preferred form, the serine protease in the feed is suitable for hydrolyzing peptide bonds located next to the amino acids aspartic acid (Asp) or glutamic acid (Glu).

According to one embodiment, the serine protease in the feed additive is able to dissolve proteins. This is because the serine protease is able to hydrolyze the peptide bonds that are located next to the hydrophobic parts in the protein, thereby increasing the solubility.

The protein may be an animal protein, such as meat and bone meal, and/or fish meal; or it can be a vegetable protein, preferably the feed comprises a vegetable protein.

The term “vegetable proteins,” as used herein, refers to any compound, composition, preparation or mixture containing at least one protein derived from or derived from a vegetable, including modified proteins and protein derivatives. In further embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60% (w/w).

Vegetable proteins can come from vegetable protein sources such as legumes and grains, for example materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soybean meal, lupine meal, and rapeseed meal. In one embodiment, the vegetable protein source is material from one or more plants of the Fabaceae family, for example soybean, lupine, pea, or bean. In another embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of plant-based protein sources are rapeseed, sunflower seed, cottonseed, and cabbage. Other examples of plant-based protein sources include grains such as barley, wheat, rye, oats, maize (corn), rice, triticale, and sorghum.

Soy is a preferred vegetable protein source in the feed. Soy can occur as, but not limited to, soy, soy meal, soy meal or a combination of these.

According to a preferred form, the feed is selected from the list of: wheat, maize, wheat bran, DDGS wheat, soya meal, rye, rapeseed, rapeseed meal, oats,

barley, peas, sunflower seed meal, rapeseed meal, sorghum or a combination of these.

These feeds all have a protein profile with high amounts of Gln and Asn, which are well hydrolyzed by both the serine protease and the acid endoprotease in the acidic and neutral regions of the gastrointestinal tract. The serine protease is suitable for hydrolyzing peptide bonds in neutral regions that are located next to the amino acids aspartic acid (Asp) or glutamic acid (Glu). The acid endoprotease is suitable for hydrolyzing glutamine-rich regions on protein chains in the stomach in acidic regions. This leads to a synergistic hydrolysis effect that is especially evident when used in feeds containing high levels of glutamine and asparagine.

The feed additive according to the invention results in an increased solubility of proteins, compared to the control. At least 101%, or 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, or at least at least 116% dissolved protein can be obtained using the serine protease of the invention, referring to the in vitro model of examples 1-9 herein. The term protein solubility basically means bringing protein(s) into solution.

Such solubility may result from protease-mediated release of protein from other components of the usually complex natural compositions such as animal feed. Solubility can be measured as an increase in the amount of soluble proteins, compared to a sample without serine protease treatment.

The feed additive according to the invention results in increased digestibility of proteins, compared to the control. At least 101%, or 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, or at least at least 116% digestible protein can be obtained using the feed additive comprising the serine protease.

In a preferred form the serine protease is isolable from Bacillus licheniformis, more preferably the serine protease is isolated from Bacillus licheniformis.

In a preferred form the acid endoprotease is isolable from Aspergillus niger, more preferably the acid endoprotease is isolated from Aspergillus niger.

According to one embodiment, the feed additive can be used for any livestock. In a preferred form, the feed additive is added to feed intended for monogastric cattle. More preferably, the livestock concerns pigs, poultry or aquaculture, even more preferably pigs and/or poultry.

According to one embodiment, the feed additive can be used for livestock of any age.

In a preferred form, the feed additive is added to feed intended for livestock between 0 and 1 year old, preferably between 0 and 6 months old, even more preferably between 0 and 3 months old.

In a preferred form the serine protease is suitable for hydrolyzing peptide bonds, more preferably the serine protease is suitable for hydrolyzing peptide bonds in trypsin inhibitors. Trypsin inhibitors include a high concentration of aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu) and glutamine (GIn). The serine protease is particularly suitable for hydrolyzing peptide bonds located next to the amino acids Asp or Glu. Asn and Gin are deamidated (in acidic conditions) in the stomach of cattle to Asp and

Glu. There are therefore a high number of peptide bonds in the protein that can be hydrolyzed by the serine protease.

According to a preferred form, the feed comprises soya, soya meal, or a combination thereof. Soy includes anti-nutritional factors such as glycinin, B-conglycinin and trypsin inhibitor (TI), against which the serine protease is preferentially effective.

In a preferred embodiment, the serine protease is suitable for hydrolyzing peptide bonds in soy antigens, such as glycinin, B-conglycinin, or glycinin and B-conglycinin. Glycinin and B-conglycinin include a high concentration of aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu) and glutamine (Gin). The serine protease is particularly suitable for hydrolyzing peptide bonds located next to the amino acids Asp or Glu. Asn and GIn are deamidated (in acidic conditions) in the stomach of cattle to Asp and Glu. There are therefore a high number of peptide bonds in the protein that can be hydrolyzed by the serine protease.

In one embodiment, macrominerals are also included in the feed additive.

Furthermore, possible feed additive components are colorants, for example carotenoids such as beta-carotene, astaxanthin and lutein; aroma compounds;

stabilizers; antimicrobial peptides; polyunsaturated fatty acids; reactive oxygen generating species.

Examples of antimicrobial peptides (AMPs) are CAP18, Leucocin A, Tritrpticine,

Protegrin-1, Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as

Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides described in WO 03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFPs) are Aspergillus giganteus and

Aspergillus niger peptides, as well as variants and fragments thereof that retain antifungal activity, as described in WO 94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are polyunsaturated

C18, C20 and C22 fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating compounds are chemicals such as perborate, persulfate or percarbonate; and enzymes such as an oxidase, an oxygenase or a synthethase.

In a particularly preferred form, the feed additive further comprises a deaminase. The term “deaminase” refers to an enzyme that performs a deamination, which means that they can catalyze the removal of amino groups (-NHz) from amino acids. The inventors experienced a synergy between the deaminase enzyme and the proteases, serine protease and the acid endoprotease. This finding was established after extensive laboratory testing and in vitro studies.

The deaminase enzyme appeared to play a role in the modification of specific amino acids in proteins. This modification not only resulted in a significant improvement in protein digestibility, but also reduced the presence of anti-nutritional factors, such as glycinin and B-conglycinin. This discovery of the synergistic effect between deaminase and proteases in the feed additive has led to a breakthrough in the optimization of protein processing in the gastrointestinal tract.

Examples of deaminases are Arginase; Glutaminase; Asparaginase, Adenosine deaminase (ADA); Cytidine deaminase; Tryptophan deaminase; Phenylalanine

deaminase. Most preferred is asparaginase. Asparaginase has asparagine as a substrate, which is beneficial in breaking down anti-nutritional factors, such as glycinin and B-conglycinin.

In a further preferred form, the feed additive for improving intestinal health and/or feed digestibility in livestock comprises a serine protease isolated from Bacillus licheniformis, an acid endoprotease isolated from Aspergillus niger, and a deaminase, preferably an asparaginase, wherein the feed additive serine protease and the acid endoprotease in a mass ratio between 0.5/1 and 3.5/1.

In a preferred form, the feed additive comprises the deaminase, in an amount between 0 and 50 m%, expressed in relative weight of the deaminase on the total weight of the feed additive, preferably between 1 and 50 m%, more preferably between 5 and 45 m%, even more preferably between 10 and 40 m%, and most preferably between 15 and 35 m%.

In a preferred form the feed additive comprises the deaminase and a total amount of serine protease and acid endoprotease in a mass ratio between 0.1/1 and 1/1.

In a further preferred form, the feed additive comprises the serine protease and the acid endoprotease in a mass ratio between 0.5/1 and 3.5/1, and the feed additive comprises the deaminase and a total amount of serine protease and acid endoprotease in a mass ratio between 0.1/1 and 1/1.

In a further preferred form, the feed additive consists of: - a serine protease, an acid endoprotease and a deaminase, wherein the feed additive comprises the serine protease and the acid endoprotease in a mass ratio between 0.5/1 and 3.5/1, - additives and/ or impurities, in an amount of up to 1 m%, expressed in relative weight of the total amount of additives and/or impurities based on the total weight of the feed additive. - chalk, supplemented to 100%.

In a second aspect, the invention concerns a method for manufacturing a feed additive according to the first aspect.

In a preferred embodiment, the method comprises applying a serine protease isolable from Bacillus licheniformis with a first enzyme activity measured at a pH between 7 and 8, and an acid endoprotease isolable from

Aspergillus niger with a second enzyme activity measured at a pH between 2.5 and 3.5, on a carrier material, for example by spraying, wherein the feed additive has a ratio between the first enzyme activity and the second enzyme activity between 0.8/1 and 1/0.8 is reached.

In one embodiment, the proteases can be applied in liquid state to the carrier material by spraying. In another embodiment, the proteases can be applied to the carrier material in powder form by means of mixing.

In a third aspect, the invention concerns a feed composition for livestock comprising a feed additive according to the first aspect and a feed, wherein the feed comprises ingredients selected from the list of: wheat, maize, wheat bran, DDGS wheat, soybean meal, rye, rapeseed, rapeseed meal, oats, barley, peas, sunflower seed meal, rapeseed meal, sorghum or a combination of these.

In a preferred form, the feed consists at least partly of soya, soya meal, soya meal, or a combination thereof.

In a fourth aspect, the invention concerns a use of a feed additive according to the first aspect for improving intestinal health and/or the digestibility of feed in livestock, wherein this feed additive is dosed to the feed of monogastric animals, such as poultry and/or pigs. .

When used according to the invention, the protease can be fed to the animal before, after, or simultaneously with feeding. The latter is preferred.

In what follows, the invention is described by means of: non-limiting examples that illustrate the invention and are not intended or should be construed to limit the scope of the invention.

EXAMPLES

Example 1-9: Improved digestibility

Table 1 shows the in vitro improved digestibility by administering the proteases to the animal feed.

TABLE 1

Digestibility | Digestibility | Improved proteins proteins with protease digestibility (*) (300 ppm)

Rapeseed meal 55.3 61.5 11.21% wn

Sunflower seed- 63.2 77.2 22.15% soins * improved digestibility (%) = digestibility The proteins '406

The digestibility has been measured and calculated using the CVB protocol (central animal feed agency).

Example 10-13: Improved digestibility

Table 2 shows the in vivo improved digestibility by administering the protease to the cattle feed.

TABLE 2

Digestibility | Digestibility | Improved proteins d proteins with protease digestibility (*)

Rapeseed meal * improved digestibility (%) — ER protein digestibility * 100

Example 14-16: amino acid composition anti nutritional factors

TABLE 3 [TIGR 38) [Gein 15) | Becongtveinine ee 16

WG) | [4%] aAl ww] wm awe © | [244] | Rev. oe

REG) | SILER | ows ae

MO | ola] =| seem] am

Ew

MO | [0%] G| 00% ol 00%

Sec) | o|0O%| ol B] 9 00%

The three anti-nutritional factors, glycinin, B-conglycinin and TI, each have a high concentration of Asp, Asn, Glu and Gin:

TI: 5.2% (Asn) + 8.9% (Asp) + 2.6% (Glu) + 7.3% (Gin) = 24%

Glycinine: 7.8% (Asn) + 3.6% (Asp) + 10.1% (Glu) + 8.6% (Gln) = 30.1%

B-conglycinin: 7.8% (Asn) + 3.5% (Asp) + 10.7% (Glu) + 8.3% (Gin) = 30.3%

Examples 17-19: protease activity assay

A protease activity assay was performed on different enzyme extracts. The extracts were prepared as follows: - 0.5 g enzyme in 5.0 ml buffer (buffer pH 7.5 /pH 3.0), - extract for 30 min at room temperature, - centrifugation and dilution.

The protease activity measurement was performed at 37°C and buffer pH 7.5/pH 3.0.

The results, shown in Table 4, show that the serine protease/acid endoprotease mixture (ex. 19) has a higher activity than the average of the activity of the preparations alone (ex. 17 and 18).

TABLE 4 a | Activity (U/g) Average activity (U/g)

Serine protease 113.6 (ex. 17)

Acid endoprotease 121.3 (ex. 18) 110.7 erine protease acid endoprotease 145.4 (ex. 19) 3.0 | 180.1

Claims (15)

CONCLUSIONS A feed additive for improving intestinal health and/or feed digestibility in livestock, comprising a serine protease isolated from Bacillus licheniformis, an acid endoprotease isolated from Aspergillus niger, and a deaminase, wherein the feed additive comprises the serine protease and the acid endoprotease in a mass ratio between 0.5/1 and 3.5/1. 2. Feed additive according to claim 1, wherein the deaminase is an asparaginase. Feed additive according to any one of the preceding claims, wherein the feed additive comprises the deaminase and a total amount of serine protease and acid endoprotease in a mass ratio between 0.1/1 and 1/1. 4. Feed additive according to any one of the preceding claims, wherein the feed additive comprises the deaminase, in an amount between 0 and 50 m%, expressed in relative weight of the deaminase on the total weight of the feed additive. 5. Feed additive according to any one of the preceding claims, wherein the feed additive further comprises a carrier material, preferably chalk. 6. Feed additive according to any one of the preceding claims, wherein the feed additive is a serine protease isolated from Bacillus licheniformis with a first enzyme activity measured at a pH between 7 and 8, and an acid endoprotease isolated from Aspergillus niger with a second enzyme activity measured at a pH between 2.5 and 3.5, wherein the ratio between the first enzyme activity and the second enzyme activity is between 0.8/1 and 1/0.8, expressed in U/g serine protease to U/g acid endoprotease. 7. Feed additive according to any one of the preceding claims, wherein the feed additive comprises the serine protease, in an amount between 25 and 35 m%, expressed in relative weight of the serine protease on the total weight of the feed additive. 8. Feed additive according to any one of the preceding claims, wherein the feed additive comprises the acid endoprotease, in an amount between 10 and 30 m%, expressed in relative weight of the acid endoprotease on the total weight of the feed additive. 9. Feed additive according to any one of the preceding claims, wherein the feed additive contains the serine protease and the acid endoprotease, in a mass ratio between 0.5/1 and 3.5/1, and chalk. 10. Feed additive according to any one of the preceding claims, wherein the feed additive comprises the serine protease and the acid endoprotease in a mass ratio between 0.5/1 and 3.5/1, and the feed additive comprises the deaminase and a total amount of serine protease and acid endoprotease in a mass ratio between G,1/1 and 1/1. 11. Feed additive according to any one of the preceding claims, wherein the livestock is monogastric livestock, preferably the livestock is pigs, poultry or aquaculture, and wherein the livestock is between 0 and 1 year old, preferably between 0 and 6 months. 12. Feed additive according to any one of the preceding claims, wherein the feed additive further comprises phytase, xylanase, amylase, or a combination thereof. 13. A method for manufacturing a feed additive according to any one of claims 1-12, comprising applying a serine protease isolable from Bacillus licheniformis with a first enzyme activity measured at a pH between 7 and 8, and an acid endoprotease isolable from Aspergillus niger with a second enzyme activity measured at a pH between 2.5 and 3.5, on a carrier material, for example by spraying, wherein the feed additive has a ratio between the first enzyme activity and the second enzyme activity between 0.8/1 and 1 /0.8 is reached. 14. A feed composition for livestock comprising a feed additive according to any one of claims 1-12 and a feed, wherein the feed comprises ingredients selected from the list of: wheat, maize, wheat bran, DDGS wheat, soybean meal, rye, rapeseed, rapeseed meal, oats , barley, peas, sunflower seed meal, rapeseed meal, sorghum or a combination of these. 15. A use of a feed additive according to any one of claims 1-12, for improving intestinal health and/or the digestibility of feed in livestock, wherein this feed additive is dosed to the feed of monogastric animals, such as poultry and/or pigs .