RU2811990C2 - Animal feed - Google Patents

Abstract

FIELD: feed for farm animals.

SUBSTANCE: method of increasing the efficiency of production of livestock products for farm animals is proposed. The method involves the oral administration of cellulose butyryl ester with a number average molar mass from 2,000 to 1,000,000 g/mol, additionally containing acetyl groups of the ester.

EFFECT: invention provides a decrease in the feed conversion ratio, an increase in live weight and/or an increase in the average daily weight gain.

20 cl, 4 dwg, 6 ex

Description

TECHNICAL FIELD

The present invention relates to the field of feed for farm animals, in particular to feed material that is effective for the prevention and/or treatment of pathogenic infections in farm animals and/or for increasing the efficiency of production of animal products.

BACKGROUND OF THE ART

Increasing demand for livestock products around the world requires new strategies to improve the efficiency of livestock production. An integral element of such strategies is optimizing nutrient utilization. Consumer and government attention to food safety, reduced environmental impact, improved animal welfare, and stewardship of antimicrobials are creating additional demands on livestock products.

Particular attention in animal husbandry is paid to the destruction of pathogenic microorganisms such as Salmonella and other pathogenic bacteria. Salmonella is one of the most important causes of foodborne illnesses in humans, mainly due to the consumption of poultry meat or contaminated eggs. In the agricultural sector, attempts are being made to reduce the incidence of Salmonella infections through various measures such as vaccination, intensive sanitation procedures and/or the administration of antibiotics, probiotics, acidulants or short- and medium-chain fatty acids and their salts.

Recently, attention has been drawn to the bacteriostatic effects of short-chain volatile fatty acids on Gram-negative bacteria. The group of short-chain volatile fatty acids consists of biodegradable weak organic acids that can destroy pathogenic microorganisms without significantly affecting the intestinal microflora. It has been shown that short-chain volatile fatty acids can inhibit the growth of hemolytic strains of Escherichia coli by 50 percent. A number of short chain fatty acid compositions have been developed for use as feed additives to achieve a reduction in the presence of pathogens in animals.

EP1354520 describes a feed additive which is a microcapsule containing n-butyric acid in a matrix containing a lipid structure (wax) and is produced by spray cooling. According to EP1354520, microencapsulation of butyric acid is effective, in particular, to overcome the difficulties associated with the volatility and unpleasant odor of butyric acid, which pose difficulties in its use as a feed additive. EP1354520 also suggests that the drug is resistant to degradation in the stomach.

F. Van Immerseel et al. (2005 Poultry Science 84: 1851-1856) compared the effect of butyric acid with butyric acid embedded in a fat matrix when used as a feed additive for poultry. They reported that Salmonella colonization of the cecum and fecal shedding of Salmonella were significantly lower in the group given butyric acid embedded in the fat matrix.

WO2007/124949 describes the use of 3-hydroxybutyric acid and poly-3-hydroxybutyric acid compounds as feed additives.

BE1023491 describes a feed additive containing butyric acid in a wax matrix containing microcrystalline wax and its production by melt extrusion.

An object of the present invention is to provide animal feed additives that have improved effectiveness in preventing or treating pathogenic infections in farm animals and/or increasing the efficiency of production of animal products.

BRIEF DESCRIPTION OF THE INVENTION

The inventors of the present invention have surprisingly discovered that these objectives can be achieved using certain polysaccharide butyric acid esters.

It has been found that enteral administration of the butyryl esters of the polysaccharide of the present invention to farm animals results in an increase in the concentration of butyrate in the lower gastrointestinal tract. The present inventors believe that this correlates with the effectiveness of butyryl polysaccharide esters against the presence of pathogens in the lower gastrointestinal tract and feces, which was observed during in vivo testing. It was also unexpectedly found that administration of butyryl polysaccharide esters resulted in superior growth performance compared to other butyrate-based drugs or products. The polysaccharide butyryl esters of the present invention provide the highly sought after combination of improved average daily weight gain (ADG) and average daily feed intake (ADFI) coupled with reduced feed conversion ratio (FCR) and mortality over the period of supplementation without demonstrating adverse events during final fattening period.

It is assumed that butyric acid stimulates the growth of intestinal villi and/or modifies the development of microorganisms in the gastrointestinal tract. It is also believed that butyric acid can suppress the expression of genes involved in Salmonella invasion at low doses. Other beneficial effects that the inventors believe will be produced by the use of the polysaccharide butyric acid ester compositions of the present invention may include improved retention of intestinal contents in the small intestine, improved digestion/absorption of methionine, and/or more diverse microflora in the small intestine. lower gastrointestinal tract.

The reason for the increase in butyric acid concentration may be a prebiotic effect (for example, but not limited to, a prebiotic effect on butyric acid-producing bacteria), direct release of butyric acid as a result of decomposition of the butyryl ester of the polysaccharide, or any other mechanism leading to an increase in butyric acid concentration or a combination of them. Without being limited to any particular theory, the present inventors believe that the effects observed for the butyryl esters of the polysaccharide of the present invention may also correlate with the increased presence of microorganisms capable of fermenting polysaccharides in the lower gastrointestinal tract compared to the upper section of the gastrointestinal tract.

Therefore, the present invention relates to new butyryl polysaccharide esters, compositions such as feed additives and/or feeds containing butyryl polysaccharide esters, as well as uses of butyryl polysaccharide esters as a feed additive, for example, for the prevention or treatment of pathogenic infections in farm animals and/or to improve the efficiency of production of livestock products.

These and other aspects of the invention will become clearer from the following detailed description and the accompanying examples.

DETAILED DESCRIPTION OF THE INVENTION

The first aspect of the invention relates to a feed composition containing a butyryl ester polysaccharide.

Butyric acid is a short-chain volatile monocarboxylic acid with the molecular formula CH ₃ -CH ₂ -CH ₂ -COOH. The terms “butyric acid” and “butyrate” are used interchangeably as used herein and should be interpreted to mean the protonated (acid, butyrate) and deprotonated (conjugate base, butyrate) forms, respectively. One skilled in the art will appreciate that butyric acid, being a weak acid, is typically present in both protonated and deprotonated forms when dissolved in an aqueous medium, the concentration of each form depending on the pH of the medium. The acidic form can be absorbed by the intestinal walls and cell membranes of microorganisms.

As used herein, the term “butyryl polysaccharide ester” refers to compounds that generally contain a polysaccharide molecule as a central moiety, wherein said polysaccharide molecule is derivatized/replaced by a plurality of butyric acid molecules through the formation of ester bonds between the carboxylic acid group of the butyric acid molecule and hydroxyl group of the polysaccharide.

As used herein, the term "polysaccharide" refers to polymers containing a backbone containing monosaccharide repeat units and/or derivatized monosaccharide repeat units, typically cyclic pentoses, in particular C ₅ -aldoses or ketoses, or cyclic hexoses, in particular C ₆ -aldoses or ketoses. Non-limiting examples of C ₅ -C ₆ -aldoses include allose, altrose, glucose, mannose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose. Non-limiting examples of C ₅ -C ₆ -ketoses include ribulose, xylulose, fructose, sorbose and tagatose. As used herein, the term “monosaccharide derivatives” refers to any chemically or enzymatically modified monosaccharide unit.

The polysaccharide may be a homopolysaccharide or a heteropolysaccharide, preferably a homopolysaccharide.

The polysaccharide may be modified or unmodified. In a preferred embodiment of the invention, a butyryl ester of a polysaccharide is provided, wherein the polysaccharide is selected from the group consisting of starches, modified starches, amylopectin, modified amylopectin, amylose, modified amylose, chitosan, chitin, guar gum, modified guar gum, locust bean gum, tare, konjac gum, konjac flour, fenugreek gum, mesquite gum, aloe mannans, modified cellulose, oxidized polysaccharides, sulfated polysaccharides, cationic polysaccharides, acacia gum, karaya gum, xanthan, kappa-, iota- or lambda-carrageenans, agar- agar, alginates, callose, laminarin, chrysolaminarin, xylan, mannan, galactomannan, hemicellulose, pectin, arabinoxylan, xanthan gum, nigeran, isolichenan, laminaran, lichenan, glycogen, pullulan, dextran, pustulan, inulin, vegetable levans, carrageenan, galactocarolo PS, rodimenan, fucoidan, agarose, porphyran, alginic acid, keratosulfate, chondroitin, chrondroitin sulfates, heparin and cellulose, preferably from the group consisting of hemicellulose, starch and cellulose, most preferably the polysaccharide is cellulose.

In a preferred embodiment of the invention, a butyryl ester polysaccharide is provided, wherein the polysaccharide is selected from the group consisting of water-insoluble polysaccharides.

For the purposes of the present invention, the butyryl ester of a polysaccharide can be formed chemically, enzymatically, by the action of enzymes, by biosynthesis by a natural or genetically modified organism, etc. Generally, in accordance with the invention, the butyryl ester of a polysaccharide is obtained by esterification of a polysaccharide, e.g. , acid-catalyzed esterification.

The number of substituent groups on the (anhydro)monosaccharide units of polysaccharides can be expressed as a percentage by weight or as the average number of substituent groups attached to the ring, a concept known to polysaccharide chemists as the "degree of substitution" (DS). In the case of a _C6 monosaccharide, if all three available positions on each unit are substituted, the DS is 3; if an average of two positions on each ring are substituted, DS is 2, etc. By analogy, the number of substituent groups in polysaccharides can be expressed as a percentage of the available positions that are substituted.

In one embodiment, the butyryl ester polysaccharide has an average number of butyryl groups per monosaccharide unit (DS) of from 0.1 to 4. For example, according to this embodiment, the DS may be at least 0.25, at least 0 .5, at least 0.75, at least 1.0, at least 1.25, at least 1.5, at least 1.75, at least 2.0, at least 2, 25 or at least 2.5, and/or it may be less than 3.75, less than 3.5, less than 3.25, or less than 3. In illustrative embodiments, said DS is from 0.5 to 3.5, from 1.0 to 3.25, from 1.5 to 3 from 2.0 to 2.95.

In a particularly preferred embodiment of the invention, the butyryl ester of the polysaccharide is a cellulose butyryl ester having an average number of butyryl groups per monosaccharide unit (DS) of 0.1 to 4, preferably 1 to 3.5, preferably 1.75 to 3 .25, preferably from 2.25 to 3, preferably from 2.5 to 2.95.

In other embodiments, the butyryl ester polysaccharide has a butyryl content of at least 5 wt% based on the weight of the butyryl ester polysaccharide, preferably at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt.%, at least 30 wt.%, at least 35 wt.%, at least 40 wt.%, at least 45 wt.%, at least 50 wt.% or at least at least 55 wt.%. In embodiments of the invention, the butyryl ester polysaccharide is a butyryl ester of cellulose, characterized by a butyryl content of from 5 to 80 wt.% relative to the weight of the butyryl ester polysaccharide, preferably from 25 to 70 wt.%, from 40 to 60 wt.%, or from 50 to 56 wt.%.

In other embodiments, the butyryl ester polysaccharide has a butyryl content expressed as a percentage of available polysaccharide hydroxyl groups that are substituted at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%. In other embodiments, the butyryl ester polysaccharide has a butyryl content resulting in a degree of substitution, expressed as the percentage of available polysaccharide hydroxyl groups that are substituted, of 100% or less, e.g., less than 99%, less than 98%, less than 97% , less than 95%, less than 90%, less than 85%, less than 80% or less than 75%.

In embodiments of the invention, the polysaccharide butyryl ester is a cellulose butyryl ester characterized by a butyryl content, expressed as the percentage of available polysaccharide hydroxyl groups that are substituted, of 5 to 80%, preferably 25 to 70%, or 50 to 56%.

In other preferred embodiments, the butyryl esters of the polysaccharide of the invention further contain acetyl ester groups. In embodiments of the invention, the butyryl ester of the polysaccharide contains butyryl and acetyl groups in a molar ratio of at least 1/1, preferably at least 1.5/1, at least 2/1, at least 2.5/1, according to at least 3/1, at least 3.5/1, at least 4/1, at least 5/1, at least 6/1, at least 7/1, at least 8/1, at least 9/1 or at least 10/1. In embodiments of the invention, the butyryl ester of the polysaccharide contains butyryl and acetyl groups in a molar ratio of less than 100/1, preferably less than 75/1, less than 50/1, less than 40/1, less than 30/1, less than 25/1, less than 20/ 1 or less 15/1. In embodiments of the invention, the butyryl ester polysaccharide contains acetyl groups and is characterized by an acetyl group content of at least 1 wt.% relative to the weight of the butyryl ester polysaccharide, preferably at least 2 wt.%, preferably at least 3 wt.%. In embodiments of the invention, the butyryl ester polysaccharide contains acetyl groups and is characterized by an acetyl group content of no more than 10 wt.% relative to the weight of the butyryl ester polysaccharide, preferably no more than 8 wt.%, preferably no more than 5 wt.%.

According to the invention, the butyryl ester of the polysaccharide is a butyryl cellulose ester characterized by an acetyl content of from 0.1 to 60 wt.% relative to the weight of the butyryl cellulose ester, preferably from 0.5 to 45 wt.%, preferably from 1 to 30 wt. %, preferably from 2 to 15 wt.%, preferably from 3 to 10 wt.%, preferably from 4 to 6 wt.%.

In embodiments of the invention, the butyryl ester polysaccharide may have a number average molar mass (M _n ) from 2000 to 1,000,000 g/mol, preferably from 5,000 to 500,000 g/mol, from 7,000 to 250,000 g/mol, from 10,000 to 100,000 g/mol, 12,000 to 50,000 g/mol, 13,000 to 25,000 g/mol, or 15,000 to 17,000 g/mol. In embodiments of the invention, the butyryl ester polysaccharide may have a number average molar mass ( _Mn ) of at least 2000 g/mol, preferably at least 4000 g/mol, preferably at least 8000 g/mol, preferably at least 12,000 g /mol. Additionally, in embodiments of the invention, the butyryl ester polysaccharide may have a number average molar mass ( _Mn ) of less than 1,000,000 g/mol, preferably less than 500,000 g/mol, preferably less than 250,000 g/mol, preferably less than 100,000 g/ mol, preferably less than 70,000 g/mol, preferably less than 65,000 g/mol, preferably less than 30,000 g/mol, preferably less than 25,000 g/mol, preferably less than 17,000 g/mol. Number average molar mass ( _Mn ) can be determined by suitable methods known to one skilled in the art, such as osmosis, static light scattering, sedimentation equilibrium, gel permeation chromatography, viscometry, sedimentation rate, dynamic light scattering, end group analysis, etc. etc. The preferred method for determining number average molar mass ( _Mn ) is gel permeation chromatography.

According to the invention, the composition containing butyryl ester of a polysaccharide is a feed composition. As used herein, the term "feed" means suitable for consumption by an animal, in particular a farm animal. In an embodiment, it means that the composition has been determined to be safe, functional and suitable for its intended use in animal feed. For example, it is processed and labeled appropriately and/or satisfies the relevant regulations governing the use of the composition in animal feed in the relevant jurisdiction.

In embodiments of the invention, the feed composition contains at least one additional ingredient selected from the groups consisting of feed additives, feed formulation additives or excipients, and nutritional components.

It will be appreciated that the selection and (relative) amounts of such additional ingredients will depend on the precise form and purpose of the feed composition. Embodiments are provided where the feed composition is a feed additive or feed material. Embodiments are also provided where the feed composition is a feed premix or a ready-to-use feed or forage. Unless otherwise specified, the terms “feed additive” and “feed material” are used interchangeably herein and generally refer to compositions containing butyryl ester polysaccharide in high concentrations, which compositions are formulated and intended to be mixed with feed or forage for providing butyryl ester polysaccharide in acceptable quantities/dosages. It should be understood that these terms as used herein do not refer to legal definitions used in the context of animal feed regulations, which may vary between jurisdictions and/or change over time. Notwithstanding the above, it is clear that the feed compositions according to the invention can be presented in forms that satisfy the definition of a feed additive or feed material in a strict legal (ie regulatory) sense.

In embodiments of the invention, the feed composition is a feed material comprising a butyryl ester polysaccharide in combination with at least one other feed material. In embodiments of the invention, a feed material is provided containing from 0.1 to 80 wt.%, preferably from 0.5 to 60 wt.%, from 1 to 50 wt.%, from 2 to 40 wt.%, from 3 to 30 wt.%, from 4 to 20 wt.%, from 5 to 15 wt.% polysaccharide ester of butyric acid and one or more other feed materials.

Embodiments of the invention provide a feed material as defined herein, wherein said at least one other feed material is selected from the group of processing aids, flavoring agents, food additives, zootechnical additives, coccidiostats, and histomonostatics.

Examples of processing aids that may be suitably combined with the butyryl ester polysaccharide of the present invention include preservatives, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, binders, radionuclide control agents, anti-caking agents, acidity regulators, silage additives and denaturing agents.

Examples of flavoring agents that can be suitably combined with the butyryl ester polysaccharide of the present invention include colorants and flavoring compounds. Colorants are to be understood in a broad sense and may refer to substances that impart or restore color to feedstuffs, substances that, when fed to animals, impart color to food of animal origin, and/or substances that favorably affect the color of ornamental fish or birds.

Examples of food additives that can be suitably combined with the butyryl ester polysaccharide according to the present invention include vitamins, provitamins and substances with well-defined chemical properties having similar effects; trace element compounds; amino acids, their salts and analogues; urea and its derivatives.

Examples of zootechnical additives that can be suitably combined with the butyryl ester polysaccharide of the present invention include digestibility enhancers, intestinal flora stabilizers and substances that have a beneficial effect on the environment. In a preferred embodiment of the invention, at least one other feed material contained in the feed material is a binder, anti-caking agent, stabilizing agent, carrier and/or preservative. In a preferred embodiment of the invention, at least one other feed material is selected from the group of yeast product, clay, fatty acid salt, silica, sepiolite, bentonite, clinoptilolite, guar gum, xanthan gum, formic acid, sodium formate, calcium formate, acetic acid , calcium acetate, sodium propionate, calcium propionate, lactic acid, calcium lactate, tocopherol-rich vegetable oil extracts and wheat bran. As used herein, yeast product is to be understood in a broad sense and can refer to yeast and its derivative products, such as inactivated dry yeast, yeast cell walls, autolysates or nucleotides derived from, for example, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Kluyveromyces lactis, Kluyveromyces fragilis, Torulaspora delbrueckii, etc. Embodiments of the invention provide a feed material, as defined previously herein, containing a butyryl ester polysaccharide in an amount of up to 50 wt.% relative to the total weight of the composition, preferably up to 40 wt.%, up to 30 wt.%, up to 25 wt.%, up to 20 wt.%, up to 18 wt.%, up to 15 wt.%, up to 10 wt.% or up to 5 wt.%.

Embodiments of the invention provide a feed material, as defined herein, comprising one or more other feed materials, such as a feed material, as defined herein above, in a (cumulative) amount of at least 1% by weight, based on the total weight of the composition , preferably at least 2 wt.%, at least 3 wt.%, at least 5 wt.%, at least 10 wt.%, at least 20 wt.%, at least 30 wt.%, at least 40 wt.% or at least 50 wt.%.

In embodiments of the invention, the feed composition is a livestock feed, also referred to as forage, comprising a butyryl ester polysaccharide as described herein and one or more animal feed ingredients. As understood by those skilled in the art, the term "feed ingredient" as used herein refers to feed components that provide universal nutrients essential for normal growth and development of an animal, the key nutrients being amino acids, carbohydrates, lipids, vitamins and minerals . Generally, feed ingredients are roughly classified into protein sources, energy sources, fat sources and mineral sources.

Thus, in embodiments of the invention there is provided a feed for farm animals, as defined herein, wherein one or more feed ingredients are selected from the group of protein sources, energy sources, fat sources and mineral sources. Examples of suitable protein sources include soybean meal, rapeseed meal, palm meal, sunflower meal, peas, beans, lupins, fish meal, poultry meal and blood plasma. Suitable examples of energy sources include corn, wheat, barley and rice. Suitable examples of fat sources include fish oil, tall oil, corn oil, soybean oil, rice bran oil, palm oil and canola oil. Suitable examples of mineral sources include calcium, magnesium, phosphorus, potassium, sodium, copper, selenium, zinc, iron, manganese, iodine, cobalt.

In embodiments of the invention, there is provided a food for farm animals, as defined herein, containing a butyryl ester polysaccharide in an amount of at least 0.0001 wt.% relative to the total weight of the composition, preferably at least 0.001 wt.%, at least 0.005 wt.%, at least 0.01 wt.%, at least 0.025 wt.%, at least 0.05 wt.% or at least 0.1 wt.%. In embodiments of the invention, there is provided a feed for farm animals, as defined herein, containing a butyryl ester polysaccharide in an amount of up to 10 wt.% relative to the total weight of the composition, preferably up to 5 wt.%, up to 2 wt.%, up to 1 wt. %, up to 0.5 wt.% or up to 0.1 wt.%.

In embodiments of the invention, the feed composition has a free acid content of less than 5 wt.% relative to the weight of the composition, preferably less than 2 wt.%, preferably less than 1 wt.%, preferably less than 0.5 wt.%, preferably less than 0.1 wt.% , preferably less than 0.05 wt.%, preferably less than 0.01 wt.%. The free acid content can be determined by suitable methods known to one skilled in the art, such as Karl Fischer titration.

In embodiments of the invention, the feed composition has a water content of less than 5 wt.% relative to the weight of the composition, preferably less than 2 wt.%, preferably less than 1 wt.%, preferably less than 0.5 wt.%, preferably less than 0.1 wt.%, preferably less than 0.05 wt.%, preferably less than 0.01 wt.%. Water content can be determined by suitable methods known to one skilled in the art, such as Karl Fischer titration.

Feed compositions containing butyryl ester polysaccharide, as described above herein, may be provided in powder, compressed, granular or briquetted form. In preferred embodiments, the butyryl ester polysaccharide is uniformly distributed throughout the feed composition.

In preferred embodiments, the feed composition is provided in compressed, granular or briquetted form, and the butyryl ester polysaccharide is distributed among the main particles constituting the feed composition.

In highly preferred embodiments, the feed composition is provided in compressed, granular or briquetted form, where the main particles constituting the feed composition do not contain distinguishable layers or phases. In highly preferred embodiments, the feed composition is provided in compressed, granular or briquetted form, wherein the main particles constituting the feed composition containing butyryl polysaccharide ester do not contain a coating consisting of or containing butyric acid ester in amounts greater than 70% by weight. , more than 50 wt.% or more than 20 wt.% relative to the total mass of the coating.

A second aspect of the present invention relates to a method of treating an animal by administering a butyryl ester polysaccharide as defined herein. Typically, as will be clear from the foregoing, such methods involve enteral, in particular oral, administration to an animal of a butyryl ester polysaccharide, preferably in admixture with the animal's food, for the purpose of influencing the physiological state of the animal, in particular for the purpose of improving intestinal health , condition and/or productivity of the animal. Thus, the methods of the present invention may have a prophylactic or therapeutic effect. The methods according to the present invention can also have a purely economic purpose.

According to the invention, the animal is preferably a farm animal, including bird species, aquatic species and mammal species. Examples of bird species include poultry species such as turkey, duck and chicken. Examples of aquatic species include fish species such as salmon, trout, tilapia, catfish and carp, and crustacean species including shrimp and shrimp. Examples of mammalian species include ruminant species such as sheep, goats and cattle, and non-ruminant species such as horses, piglets and pigs. In a preferred embodiment of the invention, the animal is selected from the group consisting of monogastric animals with lower intestinal fermentation. In other preferred embodiments, the animal is selected from the group consisting of chickens, piglets, horses, calves, goats, sheep, rabbits, dogs, cats and fish. In more preferred embodiments, the animal is selected from the group consisting of chickens, piglets, horses, calves, goats, sheep and rabbits, more preferably from the group consisting of chickens, piglets, horses and calves, more preferably from the group consisting of chickens and piglets, most preferably chickens.

In embodiments of the invention, the animal to be treated may be an animal that is in the weaning period, an animal that is in the starter period, an animal that is in the growing period, or an animal that is in the finishing period. In preferred embodiments, the animal is an animal that is in the starter period.

In embodiments of the invention, the method of treatment comprises feeding the animal a food or forage, typically a food or forage as defined elsewhere herein, containing a butyryl ester polysaccharide. The optimal treatment regimen may depend on the species being treated and/or the desired effect, and, based on the information herein, determination of appropriate treatment regimens will be within the skill of those skilled in the art. In embodiments of the invention, treatment will comprise enteral, eg oral, administration of butyryl ester polysaccharide at a dose of at least 0.0001 wt.% based on the total weight of animal feed consumed by the animal within 24 hours, preferably at least 0.001 wt.%. %, at least 0.005 wt%, at least 0.01 wt%, at least 0.025 wt%, at least 0.05 wt% or at least 0.1 wt%. Embodiments of the invention provide a treatment as defined herein comprising enteral, e.g. oral, administration of butyryl ester polysaccharide at a dose of up to 10% by weight based on the total weight of animal feed consumed by the animal over a 24 hour period, preferably up to 5% by weight. .%, up to 2 wt.%, up to 1 wt.%, up to 0.5 wt.% or up to 0.1 wt.%.

In embodiments of the invention, the butyryl ester polysaccharide is administered in such dosages at least once a week, preferably at least once every three days, more preferably at least once every two days, most preferably once a day.

The methods of the invention may be carried out for a number of reasons, as will be apparent from the foregoing, in particular to improve and/or maintain the health of the animal and/or to improve the performance of the animal.

Therefore, an embodiment of the invention provides a method as defined herein, wherein the method is non-therapeutic. Therefore, a method is provided, as defined herein, wherein the animal to be treated is an animal that is in good or normal health. In a preferred embodiment of the invention, this method is aimed at reducing the feed conversion ratio, increasing live weight and/or increasing average daily weight gain.

In embodiments of the invention, the method may be aimed at increasing live weight at a certain point in time, such as slaughter or day 35, preferably day 35, by more than 1%, preferably by more than 2%, preferably by more than 4%, preferably by more than 6%. In a preferred embodiment, the method aims to increase live weight at a given point in time by 1-12%, preferably by 2-6%. One skilled in the art can determine the increase in body weight at a given point in time through routine experiments, such as an in vivo experiment using a control group.

In embodiments of the invention, the method may be aimed at reducing the feed conversion ratio calculated over a certain period, such as the period of supplementation or life expectancy, preferably the period of supplementation, by more than 1%, preferably by more than 2%, preferably by more than 4%, preferably more than 6%. In a preferred embodiment, the method is aimed at reducing the feed conversion ratio calculated over a certain period by 1-12%, preferably by 2-6%. One skilled in the art can determine the decline in feed conversion ratio calculated over a period by routine experimentation, such as an in vivo experiment using a control group.

In embodiments of the invention, the method may be aimed at increasing average daily weight gain, calculated over a certain period, such as the period of supplementation or life expectancy, preferably the period of supplementation, by more than 1%, preferably by more than 2%, preferably by more than 4%, preferably more than 6%. In a preferred embodiment, the method is aimed at increasing the average daily weight gain, calculated over a certain period, by 1-12%, preferably by 2-6%. One skilled in the art can determine the increase in average daily weight gain calculated over a period through routine experiments, such as an in vivo experiment using a control group.

Embodiments of the invention provide a method as defined herein, wherein the method is carried out for the purpose of improving or maintaining the health of an animal.

Accordingly, embodiments of the invention provide methods, as defined herein, wherein the animal to be treated is an animal suffering from or at risk of developing a disorder or pathology. Additionally, embodiments of the invention provide methods, as defined herein, for therapeutically and/or prophylactically treating a disorder or pathology in an animal.

In particular, embodiments of the invention provide methods, as defined herein, wherein the animal to be treated is an animal suffering from or at risk of developing a pathogenic infection, preferably a pathogenic intestinal infection, more preferably a pathogenic cecal infection and/or colon. In embodiments of the invention, a method, as defined herein, is provided for treating and/or preventing a pathogenic infection, preferably a pathogenic intestinal infection, more preferably a pathogenic infection of the cecum and/or colon. According to the invention, said pathogen may be selected from bacteria, Eimeria, viruses and fungi, more preferably, said pathogen is selected from bacteria, most preferably Clostridium acetobutylicum, Escherichia coli, Streptococcus cremoris, Lactococcus lactis, Lactococcus cremoris, Clostridium perfringens, Campylobacter jejuni, Campylobacter coli , Lawsonia intracellullaris, Brachyspira hyodysenteriae, Enterococcus caecorum, Streptococcus suis, Salmonella enteritidis and combinations thereof, preferably Clostridium perfringens, Campylobacter jejuni, Campylobacter coli, Lawsonia intracellullaris, Brachyspira hyodysenteriae, Enterococcus caecorum, Streptococcus suis, Salmonella enteriti dis and their combinations.

In an embodiment, a method is provided, as defined herein, wherein the animal to be treated is an animal suffering from, or at risk of developing, a disorder of the intestinal flora, in particular a disorder of the microflora of the cecum and/or colon. In embodiments of the invention, a method, as defined herein, is provided for improving the intestinal microflora of an animal and/or for maintaining the health of the intestinal microflora in an animal, in particular for improving the microflora of the cecum and/or colon, and/or for maintaining the health of the intestinal microflora cecum and/or colon. Embodiments of the invention provide methods, as defined herein, that result in and/or aim to increase the microbial count of bacteria of the genus Lactobacillus or Bifidobacterium in the gastrointestinal tract, preferably in the lower gastrointestinal tract, preferably in the cecum or colon gut. Embodiments of the invention provide methods, as defined herein, that result in and/or aim to increase the ratio of microbial counts of bacteria of the genus Lactobacillus or Bifidobacterium relative to the microbial counts of bacteria of the family Enterobacteriaceae in the gastrointestinal tract, preferably the lower gastrointestinal tract , preferably the cecum or colon. Embodiments of the invention provide methods, as defined herein, that result in and/or aim to increase the ratio of microbial counts of bacteria of the genus Lactobacillus or Bifidobacterium relative to the microbial counts of bacteria of the genus Salmonella in the gastrointestinal tract, preferably in the lower gastrointestinal tract. intestinal tract, preferably in the cecum or colon. In embodiments of the invention, methods are provided, as defined herein, that result in and/or are aimed at improving intestinal microflora, including increasing the ratio of microbial counts of bacteria of the genus Lactobacillus or Bifidobacterium compared to microbial counts of bacteria of the genus Salmonella in the gastrointestinal tract, preferably in the lower gastrointestinal tract, preferably the cecum or colon, where the ratio is approximated by determining the microbial count of one bacterial species representative of each genus and calculating the ratio using the microbial count of the representative bacteria. In accordance with such embodiments, a decrease or increase in microbial number or microbial number ratio should be interpreted broadly and, for example, can be understood to mean one or more of the following:

- a decrease or increase in the number of colony forming units (CFU), determined using a suitable method known to one skilled in the art, measured 1-10, preferably 1-5, preferably 3 days after the start of treatment, and compared with a group of animals not exposed treatment,

- decrease or increase in the number of colony forming units (CFU), determined using a suitable method known to one skilled in the art, measured in the same animal(s) before treatment and 1-10, preferably 1-5, preferably 3 days after initiation treatment, or

- any other method known to one skilled in the art for determining the effect of introducing into the feed the polysaccharide butyric acid ester additive according to the invention.

A further aspect of the present invention is directed to the use of a butyryl ester polysaccharide, as defined herein, and/or a feed composition containing said butyryl ester polysaccharide, as defined herein, in methods of treatment, as defined above herein.

A further aspect of the present invention is directed to the use of a butyryl ester polysaccharide, as defined herein, and/or a feed composition containing said butyryl ester polysaccharide, as defined herein, for the manufacture of a composition for use in methods of treatment, as defined above in this document.

A further aspect of the present invention is directed to a butyryl ester polysaccharide, as defined herein, and/or a feed composition containing said butyryl ester polysaccharide, as defined herein, for use in methods of treatment, as defined above herein.

A further aspect of the invention is directed to the use of a butyryl ester polysaccharide, as defined herein, as a component or ingredient of animal feed.

A further aspect of the invention relates to a method for preparing an animal feed composition as described herein, comprising:

- providing the first component of animal feed,

- providing a butyrate polysaccharide ester, and

- mixing the first component of the animal feed with the butyrate ester of the polysaccharide until a homogeneous mixture is obtained.

In an embodiment, the method includes:

- providing the first component of animal feed,

- providing butyrate polysaccharide ester,

- mixing the first animal feed component with the butyrate polysaccharide ester until a homogeneous mixture is obtained, and

- granulation of said homogeneous mixture.

Thus, the invention has been described with reference to the specific embodiments discussed above. It should be understood that these embodiments are subject to various modifications and alternative forms well known to those skilled in the art. Many modifications in addition to those described above may be made to the structures and methods described herein without departing from the spirit and scope of the invention. Accordingly, although specific embodiments have been described, they are examples only and are not intended to be limiting on the scope of the invention. In addition, for a proper understanding of this document and the claims set forth herein, it should be understood that the verb “comprise” and its conjugations are used in its non-limiting sense to mean that the elements following the word are included, but elements not specifically mentioned are not excluded . In addition, referring to an element in the singular does not exclude the possibility that more than one element is present unless the context clearly requires the presence of one and only one of the elements. Thus, the singular usually means "at least one." The following examples are offered for illustrative purposes only and are in no way intended to limit the scope of the present invention.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: Butyrate concentrations measured in Example 1 - ileal inoculum.

Fig. 2: butyrate concentrations measured in example 1 - colon inoculum.

Fig. 3: butyrate concentrations measured in example 1 - cecal inoculum.

Fig. 4: butyrate concentrations measured in example 2 - colon.

Examples

Example 1: In Vitro Fermentation Assay

To evaluate the ability of the butyryl polysaccharide esters of the present invention to increase the concentration of butyrate in the gastrointestinal (GIT) tract of animals, an in vitro fermentation assay was conducted where bacteria from the GIT of broiler chickens were inoculated together with the product of the invention, a positive control and a negative control.

Wednesday

1 liter of medium consists of 0.6 g KCL, 0.6 g NaCl, 0.2 g CaCl2⋅2H ₂ O, 0.5 g MgSO ₄ ⋅7H ₂ O, 1.5 g Pipes buffer, 0.54 g NH ₄ Cl, 1.0 g trypticase, 1 ml resazurin solution (0.2 g resazurin per 200 ml distilled water), 10 ml “microelements solution”, 12 ml vitamin/phosphate solution, 10 ml hemin solution (0.1 g hemin /1 l of distilled water), 4 mg/ml NaHCO ₃ and 1 mg/ml cysteine HCl.

- Solution of microelements

Solution 0.025 g CuCl⋅2H ₂ O, 0.020 g FeSO ₄ ⋅7H ₂ O, 0.025 ZnCl ₂ , 0.025 g CuCl ₂ ⋅H2O, 0.050 g CoCl ₂ ⋅6H ₂ O, 0.050 g SeO ₂ , 0.250 g NiCl ₂ ⋅6H ₂ O, 0.250 g Na ₂ MoO ₄ ⋅2H ₂ O, 0.0314 g NaVO ₃ , 0.250 H ₃ BO ₃ /1 l 0.02 M HCI

- Vitamin/phosphate solution

0.0204 g biotin, 0.0205 g folic acid, 0.1640 g calcium D-pantothenate, 0.1640 g nicotinamide, 0.1640 riboflavin, 0.1640 g thiamine HCl, 0.1640 g pyridoxine HCl, 0.0204 g para-aminobenzoic acid, 0.0205 cyanocobalamin (vitamin B12) per 1 liter of distilled water containing 54.7 g KH ₂ PO ₄ .

Inoculum

An intestinal sample was collected from the ileum, colon, and cecum of the gastrointestinal tract of a 4-week-old Ross 408 broiler. The inoculum was prepared identically for each segment. Immediately after preparation, specimens were placed under anaerobic conditions at 37°C (anaerobic chamber, Ruskinn Technology, Bridgend, UK). The contents were weighed for each segment and diluted 1:9 with prewarmed (37°C) anaerobic sterile sodium phosphate buffer. After homogenization, the test products were added and the diluted material was inoculated at a ratio of 1:10 into the above medium.

Material under study

As shown in the table below, three different materials were tested for their ability to increase butyrate concentrations in the gastrointestinal tract. All tests were performed at a final concentration of 0.5% w/v.

Cellulose acetobutyrate was used as a representative butyryl ester polysaccharide according to the invention. Xylooligosaccharides were included as a positive control due to their known prebiotic effects on butyrate-producing bacteria. To evaluate the effect of the cellulose backbone on butyrate concentration, a negative control consisting of cellulose was also tested.

Test Product Provider Characteristics 1 Cellulose acetobutyrate (CAB) Acros Organics® Butyryl content 50-54%, acetyl content less than 4% 2 Xylooligosaccharides (XOS) Longlive® 35% XOS 3 Cellulose Sigma-Aldrich®

Measuring butyrate concentration

Liquid samples were collected at time points 0 h, 4 h, 6 h, and 24 h after inoculation for analysis of butyrate concentration by HPLC-UV. The extraction protocol and equipment were used as described by De Baere et al. (Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 107-115.

results

In fig. Figure 1 shows butyrate concentrations measured from an ileal inoculum.

In fig. Figure 2 shows butyrate concentrations measured from a colonic inoculum.

In fig. Figure 3 shows butyrate concentrations measured from a cecal inoculum.

The results clearly show that CAB is able to significantly increase butyrate concentration relative to the butyrate concentration measured for the negative control.

Example 2: Determination of butyrate concentration in the colon of poultry in vivo

To evaluate the ability of the butyryl polysaccharide esters of the present invention to increase butyrate concentrations in the lower gastrointestinal tract (GIT) of animals, an in vivo experiment was conducted. The product according to the invention was added to poultry feed, the concentration of butyrate in the colon was determined and compared with the results obtained for other drugs or delayed release products.

Animals

Ross 308 broiler chicks were used in this feed trial. 1-day-old chicks were obtained from a commercial hatchery and kept in isolation. All experimental groups were housed in the same room in separate cages with bedding on the floor. 60 chickens were divided into eight groups of 7 or 8 chickens each (including a control group).

Tested feed compositions

Seven different test feed compositions were prepared by mixing the test products listed in the table below with a commercially available broiler mash (Versele-Laga, Belgium) at a concentration of 3 g sodium butyrate per kg feed. The test feed was subsequently pelletized (without steam) to avoid selective feed intake. The briquetting technique, briquette size, and commercially available broiler feed were the same for all products tested.

Test Product Provider Characteristics 1 Control - no additive ND (No data available) ND 2 Admix 30 Coated® Nutri-Ad® 30% sodium butyrate coated with palm oil 3 Butyryl ester polysaccharide: cellulose acetobutyrate (CAB) Acros Organics® Butyryl content 50-54%, acetyl content less than 4% 4 Polyhydroxybutyrate (Ralstonia) Metabolix® 100% hydroxybutyrate 5 Wax matrix with 10% starch Prepared by yourself 30% sodium butyrate in Lunacera M® crystalline matrix and starch. 6 Tributyrin Prepared by yourself 60% tributyrin (Proviron) on silica (Caldic, Belgium) 7 Unprotected butyrate (sodium butyrate) Nutri-Ad® 100% sodium butyrate

Experimental technique

At 19, 20 and 21 days of life, average daily feed intake per pen was measured. Male and female chicks at 22 days of age were randomly assigned to one of the test feed compositions described above and the chicks were fed a restrictive diet (95% of previously measured average intake) for a period of 22–27 days.

At 28 days of age, birds were sacrificed and colons were collected.

Measuring butyrate concentrations

One gram of intestinal contents of the colon of each chicken was weighed and diluted with 1 ml of distilled water. After dilution, samples were homogenized and centrifuged at 13,000 rpm for 20 minutes and stored at −20°C until extraction for HPLC-UV analysis. The extraction protocol and equipment were used as described by De Baere et al. (Journal of Pharmaceutical and Biomedical Analysis 80 (2013) 107-115). For test 4, polyhydroxybutyrate, the hydroxybutyrate concentration shown in FIG. 4.

results

The results are shown in Fig. 4 as butyrate concentration in millimoles (mM)/gram of intestinal contents plotted for each treatment group. If intestinal contents were insufficient or extraction failed, data for this measurement were not included.

The results clearly show that CAB is able to significantly increase intestinal butyrate concentrations relative to butyrate concentrations measured for control or other delayed-release drugs or products.

Example 3 Determination of the post-infectious presence of Salmonella in the cecum in vivo

To evaluate the ability of the polysaccharide butyric acid esters of the present invention to reduce Salmonella colonization in the intestines of animals infected with Salmonella Enteritidis, an in vivo experiment was conducted. The product of the invention was added to poultry feed, the presence of Salmonella Enteritidis in the cecum was determined and compared with results obtained for other butyrate-based formulations or delayed release products.

Animals

Ross 308 broiler chicks used in this trial. 1-day old chicks were obtained from a commercial hatchery. All experimental groups were housed under the same conditions in separate cages with bedding on the floor. 160 chickens were divided into eight groups of 20 chickens each (including a control group). The four test feed compositions were tested in two parallel assays. All chickens had free access to water and feed.

Tested feed compositions

Four different test feed compositions were prepared by mixing the test products listed in the table below with a commercially available broiler mash (Versele-Laga, Belgium) at a concentration of 3 g sodium butyrate per kg feed. The test feed was subsequently pelletized (without steam) to avoid selective feed intake. The briquetting technique, briquette size, and commercially available broiler feed were the same for all products tested.

Test Product Provider Characteristics 1 Control - no additive ND ND 2 Admix 30 Coated® Nutri-Ad® 30% sodium butyrate coated with palm oil 3 Wax matrix with 10% starch Prepared by yourself 30% sodium butyrate in Lunacera M® crystalline matrix and starch 4 Butyryl ester polysaccharide: cellulose acetobutyrate (CAB) Acros Organics® Butyryl content 50-54%, acetyl content less than 4%

Salmonella strain

The strain used in this experiment was Salmonella Enteritidis SE147, a well-characterized streptomycin-resistant strain isolated from a poultry farm (Methner et al., 1995). The strain was grown for 6 h in Luria-Bertani medium (LB, Sigma, St. Louis, MO), after which the number of CFU per milligram was determined by plating 10-fold dilutions of the bacterial suspension on xylose-lysine deoxycholate agar (XLD, Oxoid, Basingstoke, UK) containing 100 μg/ml streptomycin (Sigma, St. Louis, MO). The bacterial suspension was kept at 4°C during plate counting and diluted in PBS to obtain the desired infective dose.

Experimental technique

17 days after hatching, all chicks were orally inoculated with 10 ⁵ CFU of Enteritidis per bird. On day 4 postinfection, all birds were sacrificed and cecal samples were collected for bacteriological analysis. The experiment was approved by the ethics committee of the Faculty of Veterinary Medicine of Ghent University.

Bacteriological analysis

Cecal samples were homogenized in a volume of buffered peptone water (BPW, Oxoid, Basingstoke, UK) and 10-fold dilutions were prepared in PBS. For each dilution, 6 x 20 μl was inoculated onto XLD plates containing 100 μg/ml streptomycin. After overnight incubation at 37°C, the number of CFU per gram of tissue was determined. Samples that were negative after direct culture were prefortified overnight at 37°C in buffered peptone water (BPW, Oxoid, Basingstoke, UK), after which all samples were fortified by adding 1 ml of this suspension to 9 ml of brilliant green tetrathionate broth (Oxoid, Basingstoke, UK). Samples that were Salmonella negative after titration but positive after enrichment were considered to contain 1 x 10 ¹ CFU/g tissue. Samples that remained negative after enrichment were considered to contain 0 CFU/g tissue.

results

As shown in the table below, administration of a feed containing a polysaccharide butyryl ester, such as CAB, provides significantly improved results against Salmonella colonization of the cecum compared to other formulations or delayed release products.

Presence of Salmonella Control 2 3 4 Negative 0 0 0 0 Positive after enrichment 6 14 13 27 x more than 10 ² , less than 10 ³ CFU/g 12 8 7 4 x more than 10 ³ , less than 10 ⁴ CFU/g 6 14 5 8 More than 10 ⁴ CFU/g 15 4 15 1

Example 4: Determination of the post-infectious presence of Salmonella in cloacal smears in vivo

To evaluate the ability of the butyryl polysaccharide esters of the present invention to reduce fecal shedding of Salmonella in animals infected with Salmonella Enteritidis, an in vivo experiment was conducted. The product of the invention was added to poultry feed, the presence of Salmonella Enteritidis in cloacal swabs was determined and compared with results obtained for other formulations or delayed release butyrate products.

Animals

Ross 308 broiler chicks used in this trial. 1-day old chicks were obtained from a commercial hatchery. All experimental groups were housed under the same conditions in separate cages with bedding on the floor. 80 chickens were divided into four groups of 20 chickens each (including a control group). Four test feed compositions were tested, listed below. All chickens had free access to water and feed.

Tested feed compositions

Four different test feed compositions were prepared by mixing the test products listed in the table below with a commercially available broiler mash (Versele-Laga, Belgium) at a concentration of 3 g sodium butyrate per kg feed. The test feed was subsequently pelletized (without steam) to avoid selective feed intake. The briquetting technique, briquette size, and commercially available broiler feed were the same for all products tested.

Test Product Provider Characteristics 1 Control - no additive ND ND 2 Admix 30 Coated® Nutri-Ad® 30% sodium butyrate coated with palm oil 3 Wax matrix with 10% starch Prepared by yourself 30% sodium butyrate in Lunacera M® crystalline matrix and starch. 4 Polysaccharide butyryl ester: cellulose acetobutyrate (CAB) Acros Organics® Butyryl content 50-54%, acetyl content less than 4%

Salmonella strain

The strain used in this experiment was Salmonella Enteritidis SE147, a well-characterized streptomycin-resistant strain isolated from a poultry farm (Methner et al., 1995). The strain was grown for 6 h in Luria-Bertani medium (LB, Sigma, St. Louis, MO), after which the number of CFU per milligram was determined by plating 10-fold dilutions of the bacterial suspension on xylose-lysine deoxycholate agar (XLD, Oxoid, Basingstoke, UK) containing 100 μg/ml streptomycin (Sigma, St. Louis, MO). The bacterial suspension was kept at 4°C during plate counting and diluted in PBS to obtain the desired infective dose.

Experimental technique

17 days after hatching, all chicks were orally inoculated with 10 ⁵ CFU of Enteritidis per bird. Cloacal smears were taken for bacteriological analyzes 1 day before infection and on days 1 and 3 after infection. The experiment was approved by the ethics committee of the Faculty of Veterinary Medicine of Ghent University.

Bacteriological analysis

Cloacal swabs were plated on XLD plates containing 100 μg/ml streptomycin and incubated overnight at 37°C. Samples that were negative after direct culture were prefortified overnight at 37°C in buffered peptone water (BPW, Oxoid, Basingstoke, UK), after which the samples were fortified by adding 1 ml of this suspension to 9 ml of brilliant green tetrathionate broth ( Oxoid, Basingstoke, UK). This suspension was again incubated overnight at 37°C and then plated on XLD containing 100 μg/ml streptomycin. After overnight incubation at 37°C, the number of CFU per cloacal swab was determined. Samples that were Salmonella negative after titration but positive after enrichment were designated as Salmonella positive. Samples that remained negative after enrichment were designated as Salmonella negative.

results

As shown in the table below, administration of a feed containing a polysaccharide butyryl ester, such as CAB, provides significantly improved results against fecal shedding of Salmonella compared to other formulations or delayed release products.

Days after infection Presence of Salmonella Control 2 3 4 -1 Negative 20 20 20 20 Positive 0 0 0 0 1 Negative 5 14 17 16 Positive 15 6 3 4 3 Negative 5 14 13 17 Positive 15 6 7 3

Example 5 Determination of in vivo growth parameters

To evaluate the ability of the butyryl polysaccharide esters of the present invention to influence growth parameters under suboptimal agricultural practices, an in vivo experiment was conducted. The product of the invention was added to a nutritional diet of rapeseed meal (RSM), the effect on growth performance was determined and compared with results obtained with other butyrate-based delayed release formulations or products.

Experimental plan

The experimental design was complete block randomization. The experimental groups were allocated as a 3 × 2 factorial plus control with 3 delayed-release butyrate-based drugs or products and two supplementation levels (0.25 or 1 g butyrate/kg feed consumed). Each experimental group was presented six times, resulting in a total of six blocks. The diets were based on the RSM-corn-wheat starter, grower and finisher diets detailed in the tables below. Experimental starter and grower diets were derived from the base recipe by adding feed additives instead of soybean oil.

Product Provider Characteristics 1 Control - no additive ND ND 2 Admix 30 Coated® Nutri-Ad® 30% sodium butyrate coated with palm oil 3 Wax matrix Prepared by yourself 30% sodium butyrate in Lunacera M® crystalline matrix. 4 Butyryl ester polysaccharide: cellulose acetobutyrate (CAB) Acros Organics® Butyryl content 50-54%, acetyl content less than 4%

Starter composition

Product 1-CTR 2-H 2-L 3-H 3-L 4-H 4-L Ingredient Corn 295.96 295.96 295.96 295.96 295.96 295.96 295.96 Wheat 200 200 200 200 200 200 200 Rapeseed meal flour 350 350 350 350 350 350 350 Fish flour 75 75 75 75 75 75 75 Soybean oil 55.29 51.96 54.46 51.96 54.46 53.29 54.79 Premix 5 5 5 5 5 5 5 Crushed lime 9 9 9 9 9 9 9 Monocalcium phosphate 5.5 5.5 5.5 5.5 5.5 5.5 5.5 Salt 0.9 0.9 0.9 0.9 0.9 0.9 0.9 NaHCO3 2.8 2.8 2.8 2.8 2.8 2.8 2.8 L-lysine HCl 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Natuphos 1000G 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Admix 30 Coated® 0 3.33 0.83 0 0 0 0 Wax matrix 0 0 0 3.33 0.83 0 0 CAB 0 0 0 0 0 2 0.5 TOTAL 1000 1000 1000 1000 1000 1000 1000

Growth composition

Product 1-CTR 2-H 2-L 3-H 3-L 4-H 4-L Ingredient Corn 215.35 215.35 215.35 215.35 215.35 215.35 215.35 Wheat 300 300 300 300 300 300 300 Rapeseed meal 350 350 350 350 350 350 350 Fish flour 55 55 55 55 55 55 55 Soybean oil 63.00 59.67 62.17 59.67 62.17 61.00 62.50 Premix 5 5 5 5 5 5 5 Crushed lime 8 8 8 8 8 8 8 Salt 1.2 1.2 1.2 1.2 1.2 1.2 1.2 NaHCO3 1.4 1.4 1.4 1.4 1.4 1.4 1.4 L-lysine HCl 1 1 1 1 1 1 1 Natuphos 1000G 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Admix 30 Coated® 0 3.33 0.83 0 0 0 0 Wax matrix 0 0 0 3.33 0.83 0 0 CAB 0 0 0 0 0 2 0.5 TOTAL 1000 1000 1000 1000 1000 1000 1000

Final composition

Product 1-CTR Ingredient Corn 229.35 Wheat 300 Rapeseed meal flour 350 Fish flour 40 Soybean oil 64.00 Premix 5 Crushed lime 7.5 Salt 1.2 NaHCO3 1.4 L-lysine HCl 1.5 Natuphos 1000G 0.05 Butyrate with fat coating 0 Wax coated butyrate 0 CAB 0 TOTAL 1000

Premix provided per kg diet: vitamin A, 12,000 IU (International Unit); vitamin D3, 2,500 IU; vitamin E, 50 mg; vitamin B2, 7.5 mg; vitamin B6, 3.5 mg; vitamin B1, 2.0 mg; vitamin K3, 1.5 mg; vitamin B12, 20 mcg; choline chloride, 460 mg; antioxidant (oxytrap PXN), 125 mg; niacin, 35 mg; pantothenic acid, 12 mg; biotin 0.2 mg; folic acid, 1 mg; Mn, 85 mg; Fe, 80 mg; Zn, 60 mg; Cu, 12 mg; I, 0.8 mg; Se, 0.15 mg.

Starter diets were fed from days 0 to 21, grower diets were fed from days 22 to 35, and final diets were given from days 36 until slaughter on day 42. The diets were formulated to meet or exceed the requirements of broiler chickens and were produced by Research Diet Services (Wibby-Doorstek, The Netherlands).

Birds and experimental techniques

The experiment was carried out at the Carus research farm of Wageningen University. A total of 357 male 1-day-old broilers (initial body weight 47 g; Ross 308, Aviagen Group, Newbridge, UK) were obtained from a commercial hatchery (Morren Breeders BV, Lunteren, The Netherlands). Upon arrival, each bird was weighed and categorized by body weight (small - less than μ-0.68×σ; medium - more than μ-0.68×σ and less than μ+0.68×σ; large - more than μ+0 .68×σ). Birds in each category were randomly assigned to one of 42 chicken pens in two climate-controlled rooms. Each pen contained 8 to 9 birds and measured 1.85 × 1 m (L × W) and contained a pole. Wood shavings were used as bedding material. The ambient temperature was maintained at 32°C until day 3 and then gradually decreased to 22°C on day 23. Photoperiod 23L:1D was applied until day 3 and then changed to 16L:8D. The birds were given free access to food and water. The weight of birds in the pen, as well as feed consumption per pen, were recorded on days 21, 35 and 42. Mortality was monitored daily and the body weight of dead birds was recorded.

results

The tables below show feed conversion ratio (FCR), average daily weight gain (ADG) and average daily feed intake (ADFI), as well as mortality rate. FCR was calculated at the paddock level using ADG and ADFI. Results were also recalculated as percent change relative to their respective controls.

The tables below show the results for mortality, average daily weight gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) obtained from this experiment. Data demonstrate that feeding a butyryl ester polysaccharide such as CAB provides superior growth performance compared to other butyrate-based delayed-release formulations or products. The butyryl ester polysaccharide of the present invention provides a highly sought after combination of improved ADG and ADFI coupled with reduced FCR and mortality during the supplementation period without exhibiting adverse events during the finishing period.

The results also demonstrate that in the starter and grower phases, increasing ester and butyric acid incorporation (4-L vs. 4-H) results in increased productivity (in contrast to the effect observed for 3-L vs. 3-H wax formulations) .

The effect of dietary therapy on performance indicators in the starting period, the rearing period and the final finishing period.

Product Diet therapy 1 CTR 2 3 4 L H L H L H 0-21 days Mortality, % 5.9 2.0 0.0 0.0 0.0 0.0 0.0 ADG, g/bird 39.9 39.7 42.0 41.8 40.4 40.6 41.4 ADFI, g/bird 54.5 53.8 56.7 56.6 54.2 55.3 56.9 FCR, g:g 1.37 1.36 1.35 1.35 1.34 1.36 1.38 22-35 day Mortality, % 0.0 0.0 3.9 3.9 2.0 0.0 0.0 ADG, g/bird 89.8 90.8 90.5 89.4 87.6 93.8 96.4 ADFI, g/bird 140.3 139.9 154.0 147.0 139.0 141.0 146.3 FCR, g:g 1.58 1.55 1.73 1.65 1.59 1.51 1.52 35-42 day Mortality, % 4.2 0.0 0.0 2.0 4.0 0.0 2.0 ADG, g/bird 131.9 107.3 125.0 120.3 116.4 115.7 116.9 ADFI, g/bird 224.1 197.6 211.5 198.6 214.7 213.0 211.2 FCR, g:g 1.71 1.85 1.71 1.66 1.84 1.85 1.81

Effect of dietary therapy on performance measures during the supplementation period and the entire experimental period.

Product Diet therapy 1 CTR 2 3 4 L H L H L H Period of administration of the supplement (0-35 days) Mortality, % 7.8 2.0 3.9 5.9 3.9 0.0 0.0 ADG, g/bird 59.51 60 61.3 60.8 59.1 61.9 63.4 ADFI, g/bird 88.26 88 95.5 92.5 87.8 89.6 92.7 FCR, g:g 1.49 1.47 1.56 1.53 1.49 1.45 1.46 The entire period of the experiment (0-42 days) Mortality, % 9.8 2.0 3.9 5.9 5.9 0.0 2.0 ADG, g/bird 70.42 67.9 71.6 70.3 68.4 70.9 72.3 ADFI, g/bird 108.9 106 114 110 108 110 112 FCR, g:g 1.55 1.57 1.60 1.56 1.58 1.56 1.56

Effect of dietary therapy on performance measures expressed as percentage change relative to their respective controls during the supplementation period (0-35 days)

2 3 4 L H L H L H ADG, % change 0.9 3.0 2.0 Minus 0.7 3.8 6.1 ADFI, % change Minus 0.3 7.6 4.6 Minus 0.6 1.5 4.7 FCR, % change Minus 1.4 4.8 2.3 Minus 0.2 Minus 2.7 Minus 1.8

Example 6 Determination of in vivo feed conversion ratio

To evaluate the ability of the butyryl polysaccharide esters of the present invention to influence feed conversion ratio under suboptimal farming practices, an in vivo experiment was conducted. The product of the invention was added to a nutritional diet of rapeseed meal (RSM), the effect on feed conversion rate was determined, statistical analysis was performed and compared with results obtained with other delayed release (hydroxy)butyrate formulations or products.

Experimental plan

The experimental design was complete block randomization. Experimental groups were allocated to 10 birds/pen x 8 replicates x 4 treatments: control and 3 butyrate-based drugs or delayed-release products (containing 1 g (hydroxy)butyrate/kg feed). The diets were based on the RSM-corn-wheat starter and grower diets detailed in the tables below. Experimental starter and grower diets were derived from the base recipe by adding feed additives instead of soybean oil.

Product Provider Characteristics 1 Control - no additive ND ND 2 Admix 30 Coated® Nutri-Ad® 30% sodium butyrate coated with palm oil 3 Polyhydroxybutyrate (PHB) Nutri-Ad® 0.10% butyrate biomass 4 Butyryl ester polysaccharide: cellulose acetobutyrate (CAB) Fisher-Scientific, Waltham, USA Butyryl content 50-54%, acetyl content 4%

Starter composition

Product 1-CTR 2 3 4 Ingredient Corn 296 296 296 296 Wheat 200 200 200 200 Rapeseed meal flour 350 350 350 350 Fish flour 75 75 75 75 Soybean oil 55.3 52.0 54.3 53.5 Premix 5 5 5 5 Crushed lime 9 9 9 9 Monocalcium phosphate 5.5 5.5 5.5 5.5 Salt 0.9 0.9 0.9 0.9 NaHCO3 2.8 2.8 2.8 2.8 L-lysine HCl 0.5 0.5 0.5 0.5 Natuphos 1000G 0.05 0.05 0.05 0.05 Admix 30 Coated® 0 3.33 0 0 PHB 0 0 1 0 CAB 0 0 0 1.8 TOTAL 1000 1000 1000 1000

Growth composition

Product 1-CTR 2 3 4 Ingredient Corn 215.4 215.4 215.4 215.4 Wheat 300.0 300.0 300.0 300.0 Rapeseed meal 350.0 350.0 350.0 350.0 Fish flour 55.0 55.0 55.0 55.0 Soybean oil 63.0 59.7 62.0 61.2 Premix 5 5 5 5 Crushed lime 8 8 8 8 Monocalcium phosphate - - - - Salt 1.2 1.2 1.2 1.2 NaHCO3 1.4 1.4 1.4 1.4 L-lysine HCl 1 1 1 1 Natuphos 1000G 0.05 0.05 0.05 0.05 Admix 30 Coated® 0 3.33 0.00 0 PHB 0 0 1 0 CAB 0 0 0 1.8 TOTAL 1000 1000 1000 1000

The premix is given per kg of diet: vitamin A, 12,000 IU; vitamin D3, 2,500 IU; vitamin E, 50 mg; vitamin B2, 7.5 mg; vitamin B6, 3.5 mg; vitamin B1, 2.0 mg; vitamin K3, 1.5 mg; vitamin B12, 20 mcg; choline chloride, 460 mg; antioxidant (oxytrap PXN), 125 mg; niacin, 35 mg; pantothenic acid, 12 mg; biotin 0.2 mg; folic acid, 1 mg; Mn, 85 mg; Fe, 80 mg; Zn, 60 mg; Cu, 12 mg; I, 0.8 mg; Se, 0.15 mg.

Starter diets were fed from days 0 to 21, and grower diets from days 22 to 35. The diets were formulated to meet or exceed the requirements of broiler chickens and were produced by Research Diet Services (Wibby-Doorstek, The Netherlands).

Birds and experimental techniques

The experiment was carried out in experimental poultry house no. 12 ILVO-DIER (Burg, Merelbeke, Belgium). A total of 320 male one-day-old broilers (Ross 308, Aviagen Group, Newbridge, UK) were obtained from a commercial hatchery (Belgabroed NV, Merksplas, Belgium). Upon arrival, the birds were randomly assigned to one of 32 pens to house chicks in a single climate-controlled room. Each pen contained 10 birds and measured 2.1 x 1 m. Ambient temperature was maintained at 29–30°C until day 7 and then decreased by 2°C per week. A photoperiod of 23L:1D was applied until day 7 and then changed to 16L:8D. The birds were given free access to food and water. Weights of each bird and pen weights, as well as feed consumption per pen, were recorded weekly.

Statistical analysis

Productivity parameters were analyzed using PROC GLM from SAS (version 9.3, SAS Institute Inc., Cary, NC) using the following model

Y _ijk = μ+D _i +W _j +B _k +AW _ij +ε _ijk

where Y _ijk is the observed response of the k-th parallel assay (k = 1-8) fed the i-th diet (i = CTR, FCB, PHB, CAB) during the j-th week (j = 1-5), D _i is the i-th fixed effect of diet, B _k is the k-th fixed effect of block, W _j is the j-th random effect of measurement period, and AD _ij is the interaction effect between diet and measurement period, and ε _ijk is the residual error term k -th parallel analysis, receiving the i-th region in the j-th measurement period. When a significant effect of diet was identified, means were separated using Tukey's post hoc test. When a significant interaction between diet and measurement period was identified, means were separated using Tukey's post hoc test, and interaction term was separated by week using a simple effect test.

results

The effect of butyrate supplementation on feed conversion ratio (FCR) during the supplementation period of days 0-35 is shown in the table below.

Data demonstrate that the addition of a polysaccharide butyric acid ester, such as CAB, provides superior feed conversion rates compared to other butyrate-based delayed-release formulations or products. The improvement in feed conversion ratio for CAB was statistically significant.

Effect of butyrate as a dietary supplement on growth performance over the entire 0-35 day period

Product United SEM 1-CTR 2 3 4 Diet A week Block Diet×week B.W.G. 48.41 48.26 48.36 48.56 1.62 0.9981 Less than 0.0001 0.2511 0.0032 VFI 79.15 76.52 77.42 76.08 1.93 0.6898 Less than 0.0001 0.5882 0.5192 FCR 1.64 1.59 1.60 1.57 0.02 0.0164 Less than 0.0001 0.7997 0.0194

Claims (22)

1. Non-therapeutic method in relation to farm animals to increase the efficiency of production of livestock products, where the farm animals are selected from monogastric animals with lower intestinal fermentation that are in good health, by oral administration of a cellulose butyryl ester further containing acetyl groups of the ester, wherein the cellulose butyryl ester has a number average molar mass of 2000 to 1,000,000 g/mol. 2. Non-therapeutic method according to claim 1, wherein the cellulose butyryl ester has a number average molar mass of 5000 to 500,000 g/mol. 3. Non-therapeutic method according to claim 1, wherein the butyryl cellulose ester has a number average molar mass of 7000 to 250000 g/mol. 4. Non-therapeutic method according to claim 1, wherein the cellulose butyryl ester has a number average molar mass of 10,000 to 100,000 g/mol. 5. Non-therapeutic method according to claim 1, wherein the cellulose butyryl ester has a number average molar mass of 12,000 to 50,000 g/mol. 6. Non-therapeutic method according to claim 1, wherein the cellulose butyryl ester has a number average molar mass of 13,000 to 25,000 g/mol. 7. Non-therapeutic method according to claim 1, wherein the cellulose butyryl ester has a number average molar mass of 15,000 to 17,000 g/mol. 8. Non-therapeutic method according to any one of paragraphs. 1-7, where the average number of butyryl groups per monosaccharide unit ranges from 0.1 to 4. 9. Non-therapeutic method according to any one of paragraphs. 1-7, where the average number of butyryl groups per monosaccharide unit is from 1 to 3.5. 10. Non-therapeutic method according to any one of paragraphs. 1-7, where the average number of butyryl groups per monosaccharide unit ranges from 1.75 to 3.25. 11. Non-therapeutic method according to any one of paragraphs. 1-7, where the average number of butyryl groups per monosaccharide unit is from 2.25 to 3. 12. Non-therapeutic method according to any one of paragraphs. 1-7, where the average number of butyryl groups per monosaccharide unit ranges from 2.5 to 2.95. 13. Non-therapeutic method according to any one of paragraphs. 1-12, wherein the butyryl ester of the polysaccharide is characterized by an acetyl content of at least 1 wt.% relative to the total weight of the butyryl ester of cellulose. 14. Non-therapeutic method according to any one of paragraphs. 1-13, wherein the butyryl ester of the polysaccharide is characterized by an acetyl content of at least 2 wt.% relative to the total weight of the butyryl ester of cellulose. 15. Non-therapeutic method according to any one of paragraphs. 1-13, wherein the butyryl ester of the polysaccharide is characterized by an acetyl content of at least 3 wt.% relative to the total weight of the butyryl ester of cellulose. 16. Non-therapeutic method according to any one of paragraphs. 1-13, wherein the butyryl ester of the polysaccharide is characterized by an acetyl content of at least 4 wt.% relative to the total weight of the butyryl ester of cellulose. 17. Non-therapeutic method according to any one of paragraphs. 1-16, where the animal is a species of poultry or pig. 18. Non-therapeutic method according to any one of paragraphs. 1-17, which is designed to reduce the feed conversion ratio. 19. Non-therapeutic method according to any one of paragraphs. 1-18, which is designed to increase live weight. 20. Non-therapeutic method according to any one of paragraphs. 1-19, which is designed to increase average daily weight gain.