BE1030865B1 - FEED ADDITIVE FOR IMPROVING INTESTINAL HEALTH AND/OR DIGESTIBILITY OF FEED IN LIVESTOCK - Google Patents

Abstract

The present invention concerns a feed additive for improving intestinal health and/or the digestibility of feed in livestock, comprising 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, wherein the ratio between the first enzyme activity and the second enzyme activity is between 0.8/1 and 1/0.8. The invention also concerns a method for preparing said feed additive, as well as a feed composition comprising said feed additive and a use of said feed additive.

Description

1 BE2022/5716

FEED ADDITIVE TO IMPROVE 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 is applied and a use of the feed additive.

STATE OF THE ART

Due to the unbalanced amino acid content and resulting limited solubility of vegetable proteins, particularly those of wheat and corn 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 is not efficiently digested.

Soybeans contain many anti-nutritional factors that limit their widespread use. Protease inhibitors, trypsin inhibitors, or trypsin inhibitors (TIs) are among the most common anti-nutritional factors (ANFs) in soybeans and other grain feeds. TIs inhibit 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 enters the lymph and bloodstream through gaps between the intestinal epithelial cells.

These macromolecules have significant antigenic activity and stimulate the immune system, leading to specific antigen-antibody reactions and T-lymphoid cell-mediated delayed hypersensitivity. Soy antigens can therefore cause intestinal disorders, which reduces nutrient absorption and consequently lowers growth performance. Soy antigens are heat-stable, making heat treatment ineffective for removing them.

2 BE2022/5716

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

WO2004034776.

However, established techniques fail to consider the fact that in monogastric animals, feed is exposed to two pH regions: the acidic stomach (2-4) and the neutral intestinal tract (7-7.5). Serine proteases are neutral proteases that are virtually inactive in the acidic regions of the digestive system. As a result, efficient proteolysis in the digestive tract of monogastric animals remains suboptimal.

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

SUMMARY OF THE INVENTION

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

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

One object of the invention is to improve protein digestibility and protein utilisation throughout the gastrointestinal tract by, on the one hand, improving the digestibility of proteins produced by plants such as wheat, maize, soya, and Fabaceae as a reserve nitrogen source, and, on the other hand, improving the breakdown of protein-based anti-nutritional factors (ANFs) (glycinin and B-conglycinin, TI, etc.).

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

The invention aims to optimize proteolysis throughout the entire digestive tract of monogastric animals. To this end, the invention aims to achieve efficient proteolysis in both the acidic and neutral regions of the gastrointestinal tract.

3 BE2022/5716

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

It is an object 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 object of the invention is to improve the profitability and increase the valorisation 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 enhance or ensure food safety. The invention also improves litter quality.

DETAILED DESCRIPTION

The invention relates to 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 meaning as commonly understood by one skilled in the technical field of the invention.

To better understand the invention description, the following terms are explicitly defined. "A," "an," and "the" refer to both the singular and plural forms in this document unless the context clearly indicates otherwise. For example, "a segment" means one or more than one segment.

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

4 BE2022/5716 +/-0.1% or less than the quoted value, insofar as such variations are applicable to the invention described. However, this should 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”, “encompassing”, “containing”, “containing” are synonyms and are inclusive or open terms indicating the presence of what follows, and do not exclude or preclude the presence of other components, features, elements, members, steps known or described in the prior art.

The terms “consist of”, “comprising”, “comprising”, “comprising” are synonyms and are exclusive or closed terms indicating the presence of what follows, and excluding or preventing the presence of other components, features, elements, members, steps known or described in the prior art.

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

The terms "weight percent," "wt%," "m%," or "% wt" in this document refer to the relative weight of a component based on the total weight of the complete product being referenced. "IU" or "U" is the enzyme unit that represents enzymatic activity. 1 U or 1 IU (µmol/min) is defined as the amount of enzyme capable of catalyzing the conversion of 1 µmol of substrate in 1 minute.

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

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

According to one 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 of the first enzyme activity to the second enzyme activity is preferably between 0.8/1 and 1/0.8, expressed as 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 further preferred form, the feed additive comprises the serine protease and the acid endoprotease in a mass ratio of between 0.5/1 and 3.5/1, expressed in g serine protease to g acid endoprotease.

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

Serine protease is suitable for hydrolyzing peptide bonds located next to the amino acids aspartic acid (Asp) or glutamic acid (Glu). Acidic endoprotease is suitable for hydrolyzing glutamine-rich regions on protein chains in the stomach. The isoelectric point of glutamine, for example, is 5.7, which means it will be protonated in the stomach and thus more easily hydrolyzed by acidic 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 by 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 relevant to the specificity of the protease in question. A serine protease is an endopeptidase that hydrolyzes peptide bonds in proteins, with serine acting as a nucleophilic amino acid of the active site—that is, serine is a member of the catalytic triad that functions as the active site. An acidic endoprotease is an endoprotease with optimal activity at acidic pH.

6 BE2022/5716

The term “feed” as used in the text refers to any compound, preparation, mixture or composition 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, higher protein yield, and/or improved protein utilization. This increases the feed's nutritional value and improves the animal's growth rate and/or weight gain and/or feed conversion (i.e., the weight of feed consumed relative to weight gain).

The term livestock encompasses all animals kept for economic reasons in agriculture and fisheries, usually as a food source. Examples of animals are non-ruminants and ruminants. Ruminants include, for example, animals such as sheep, goats, horses, and cattle, e.g., beef cattle, cows, and young calves. In one embodiment, an animal is a non-ruminant. Non-ruminant animals include monogastric animals, e.g., pigs or swine (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, 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 of 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 of 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%.

7 BE2022/5716

In a preferred form, the feed additive contains: - serine protease and acid endoprotease, in a mass ratio between 0.5/1 and 3.5/1; - additives and/or impurities, in a quantity of up to 1% by weight, expressed as a relative weight of the total quantity of additives and/or impurities based on the total weight of the feed additive; - chalk, made up to 100%.

In a particular embodiment, the serine protease, in the form in which it is added to the feed or when incorporated into 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 incorporated into 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.

The serine proteases and acid endoproteases with purities of this order of magnitude are mainly obtainable using recombinant production methods.

A well-defined protease is advantageous. For example, it is much easier to correctly dose a protease into the feed that is essentially free of interfering or contaminating other proteases. The term "correct dosing" refers primarily to the goal of achieving consistent and consistent results, and the ability to optimize the dosage based on the desired effect.

However, for use in animal feed, the proteases do not have to be as pure; they can, for example, contain other enzymes.

8 BE2022/5716

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 which 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. In a preferred form, the feed additive further comprises phytase, xylanase, amylase, or a combination of these.

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 grains and oilseeds) to release a usable form of inorganic phosphorus.

Xylanase (EC 3.2.1.8) is any enzyme capable of degrading the linear polysaccharide xylan to xylose, and thus degrading hemicellulose, one of the main constituents of plant cell walls.

Amylase is an enzyme that hydrolyzes starch during digestion. It is capable of targeting the linear α-1,4-glycosidic polymer bond between the glucose molecules of starch.

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

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

9 BE2022/5716

Proteins from plant seeds (corn, wheat, soybeans, rapeseed, peas, sunflowers, peanuts, etc.) are storage proteins and have a higher nitrogen content than animal storage protein sources (milk, egg white) to provide excess nitrogen (N) to the developing seedling. The higher nitrogen content is mainly due to the 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. These hydrogen bonds give the proteins a hydrophobic nature in 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, partially closely related protein components characterized by high glutamine and proline contents.

The insolubility of glutenins is due to the high concentration of nonpolar amino acid residues such as proline and leucine, and the highly polar but non-ionizable residue glutamine, as well as the low concentration of ionizable side chains such as lysine, arginine, glutamic acid, and aspartic acid. Hydrogen bonding between glutamine and asparagine plays a key role in promoting the association of gliadin and gluteninin molecules. The hydrophilic part of the protein is buried, and the hydrophobic part forms the exterior of the protein, making it water-insoluble.

Corn protein differs only partially from wheat proteins in that it 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 resulting limited solubility of vegetable proteins, particularly those of wheat and corn proteins, efficient proteolysis in the digestive tract of monogastric animals is suboptimal. The hydrophobic nature and non-uniform amino acid pattern require a highly specific proteolytic action.

10 BE2022/5716

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

In one embodiment, the serine protease in the feed additive is capable of solubilizing proteins. This is because the serine protease is able to hydrolyze the peptide bonds located next to the hydrophobic regions of the protein, thereby increasing solubility.

The protein may be an animal protein, such as meat and bone meal, and/or fish meal; or it may be a vegetable protein, preferably the feed includes 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 originating 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% (9/9).

Vegetable proteins can be derived from plant protein sources, such as legumes and grains, for example, materials from plants of the Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae families, 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, soybeans, lupines, peas, or beans. In another embodiment, the vegetable protein source is material from one or more plants of the Chenopodiaceae family, for example, beets, sugar beets, spinach, or quinoa. Other examples of vegetable protein sources include rapeseed, sunflower seeds, cottonseed, and cabbage. Other examples of vegetable protein sources include grains such as barley, wheat, rye, oats, maize (corn), rice, triticale, and sorghum.

Soy is a preferred vegetable protein source in feed. Soy can be found in, but not limited to, soybean, soybean meal, soybean meal, or a combination of these.

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

11 BE2022/5716 barley, peas, sunflower seed meal, rapeseed meal, sorghum or a combination of these.

These feeds all have a protein profile with high levels of Gin and Asn, which are effectively hydrolyzed by both serine protease and acid endoprotease in the acidic and neutral regions of the gastrointestinal tract. Serine protease is specifically designed to hydrolyze peptide bonds located next to the amino acids aspartic acid (Asp) or glutamic acid (Glu) in neutral regions. Acid endoprotease is also designed to hydrolyze glutamine-rich regions on protein chains in the stomach in acidic regions. This results in a synergistic hydrolysis effect, particularly evident when used in feeds containing high levels of glutamine and asparagine.

The feed additive according to the invention results in increased protein solubility 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 116% of solubilized 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 solubilization essentially means bringing protein(s) into solution.

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

The feed additive according to the invention results in increased protein digestibility 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 116% digestible protein can be obtained using the feed additive comprising the serine protease.

In a preferred embodiment, the serine protease is isolatable from Bacillus licheniformis, more preferably, the serine protease is isolated from Bacillus licheniformis.

In a preferred embodiment, the acid endoprotease is isolatable from Aspergillus niger, more preferably, the acid endoprotease is isolated from Aspergillus niger.

In one embodiment, the feed additive is suitable for any livestock. In a preferred form, the feed additive is added to feed intended for

12 BE2022/5716 monogastric livestock. Preferably, the livestock are pigs, poultry, or aquaculture, even more preferably pigs and/or poultry.

According to one embodiment, the feed additive is applicable to 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 embodiment, 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 contain 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 adjacent to the amino acids Asp or Glu. Asn and Gln are deamidated (in acidic conditions) in the stomach of cattle to Asp and

Glu. Consequently, the protein contains a high number of peptide bonds that can be hydrolyzed by serine protease.

In a preferred form, the feed comprises soy, soybean meal, or a combination of these. Soy contains anti-nutritional factors such as glycinin, β-conglycinin, and trypsin inhibitor (TI), against which the serine protease is preferably effective.

In a preferred embodiment, the serine protease is suitable for hydrolyzing peptide bonds in soy antigens, such as glycinin, β-conglycinin, or glycinin and β-conglycinin. Glycinin and β-conglycinin contain high concentrations of aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu), and glutamine (Gin). The serine protease is particularly suitable for hydrolyzing peptide bonds adjacent to the amino acids Asp or Glu. Asn and Gln are deamidated (under acidic conditions) in the stomach of cattle to Asp and Glu. Consequently, a high number of peptide bonds are present 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 colourings, for example carotenoids such as beta-carotene, astaxanthin and lutein; flavouring compounds;

13 BE2022/5716 stabilizers; antimicrobial peptides; polyunsaturated fatty acids; reactive oxygen generating species.

Examples of antimicrobial peptides (AMPs) are CAP18, Leucocine 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) include Aspergillus giganteus and

Aspergillus niger peptides, as well as variants and fragments thereof retaining 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 oxidase, oxygenase, or syntethase.

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

In a preferred embodiment, the method comprises applying a serine protease isolatable from Bacillus licheniformis having a first enzyme activity measured at a pH between 7 and 8, and an acidic endoprotease isolatable 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, whereby a ratio between the first enzyme activity and the second enzyme activity in the feed additive of between 0.8/1 and 1/0.8 is achieved.

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

14 BE2022/5716

In a third aspect, the invention relates to a feed composition for livestock comprising a feed additive according to the first aspect and a feed, the feed comprising ingredients 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 thereof.

In a preferred form, the feed consists at least partly of soy, soybean meal, soybean flour, or a combination of these.

In a fourth aspect, the invention relates to a use of a feed additive according to the first aspect for improving intestinal health and/or feed digestibility 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 the following, the invention is described by means of non-limiting examples which illustrate the invention, and which 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 adding the proteases to the animal feed.

TABLE 1

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

15 BE2022/5716

Rapeseed meal 55.3 61.5 11.21% and

Zonnebloemzaad- 63,2 77,2 22,15% mame LEE * improved digestibility (%) = digestibility proteins with supplement Vertbaarheld proteins chien mer supplenen vertsorbaameid ever , 100

Digestibility was measured and calculated using the CVB protocol (Central Animal Feed Bureau).

Example 10-13: Improved digestibility

Table 2 shows the in vivo improved digestibility by adding the protease to the animal feed.

TABLE 2

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

Rape seed meal * improved digestibility (%) = digestibility proteins with supplement Vertbaarheld proteins chien mer supplenen vertsorbaameid siwitten , 100

Example 14-16: Amino acid composition anti-nutritional factors

TABLE 3 [Free [Gee (ex. 15) | B-congiycmine (ex. 26) mem | sJéM]| | sw] 5] +

16 BE2022/5716

CEO

HS | shi] 6] | 5] mem | >|] | #8] 4

MO | oy0mm| 6y Go] 6] 00% eur | 0 [00% [0] 00e] 5y 00%

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

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

Glycinin: 7.8% (Asn) + 3.6% (Asp) + 10.1% (Glu) + 8.6% (GIn) = 30.1%

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

Examples 17-19: Protease activity determination

A protease activity assay was performed on various 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.

17 BE2022/5716

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

TABLE 4

Activity (U/g) Average activity (U/g)

Serine protease 113.6 (e.g. 17)

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

Claims (14)

18 BE2022/5716 CONCLUSIONS 1. A feed additive for improving intestinal health and/or feed digestibility in livestock, comprising a serine protease isolatable from Bacillus licheniformis having a first enzyme activity measured at a pH between 7 and 8, and an acid endoprotease isolatable from Aspergillus niger having a second enzyme activity measured at a pH between 2.5 and 3.5, wherein the ratio of the first enzyme activity to the second enzyme activity is between 0.8/1 and 1/0.8, expressed in U/g serine protease to U/g acid endoprotease, the feed additive comprising the serine protease and the acid endoprotease in a mass ratio between 0.5/1 and 3.5/1, and chalk. 2. A feed additive according to claim 1, wherein the feed additive comprises the serine protease and the acid endoprotease in a mass ratio of between 0.5/1 and 3.5/1, expressed in g serine protease to g acid endoprotease. 3. Feed additive according to any one of the preceding claims, wherein the feed additive further comprises a carrier material, preferably chalk. 4. A feed additive according to any preceding claim, wherein the feed additive comprises the serine protease in an amount of between 25 and 35 m%, expressed in relative weight of the serine protease on the total weight of the feed additive. 5. A feed additive according to any preceding claim, wherein the feed additive comprises the acid endoprotease in an amount of between 10 and 30 m%, expressed in relative weight of the acid endoprotease on the total weight of the feed additive. 6. A feed additive according to any preceding claim, wherein the serine protease in the feed is suitable for hydrolysing peptide bonds located next to the amino acids aspartic acid (Asp) or glutamic acid (Glu). 7. A feed additive according to any preceding claim, wherein the serine protease is isolated from Bacillus licheniformis. 19 BE2022/5716 8. A feed additive according to any preceding claim, wherein the acid endoprotease is isolated from Aspergillus niger. 9. Feed additive according to any one of the preceding claims, wherein the livestock is monogastric livestock, preferably the livestock is pigs, poultry or aquaculture. 10. Feed additive according to any one of the preceding claims, wherein the cattle are between 0 and 1 year old, preferably between 0 and 6 months old. 11. A feed additive according to any preceding claim, wherein the feed additive further comprises phytase, xylanase, amylase, or a combination thereof. 12. A method for manufacturing a feed additive according to any one of claims 1 to 11 comprising applying a serine protease isolatable from Bacillus licheniformis having a first enzyme activity measured at a pH between 7 and 8, and an acid endoprotease isolatable from Aspergillus niger having a second enzyme activity measured at a pH between 2.5 and 3.5, to a carrier material, for example by spraying, wherein a ratio between the first enzyme activity and the second enzyme activity in the feed additive is achieved between 0.8/1 and 1/0.8. 13. A feed composition for livestock comprising a feed additive according to any one of claims 1 to 11 and a feed, wherein the feed comprises ingredients 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 thereof. 14. A use of a feed additive according to any one of claims 1-11 for improving intestinal health and/or feed digestibility in livestock, wherein this feed additive is dosed to the feed of monogastric animals, such as poultry and/or pigs.