WO2023079167A1 - Micronized cell walls of saccharomyces cerevisiae yeast as a mycotoxin binding agent - Google Patents

Abstract

The present invention relates to micronized cell walls of dry Saccharomyces cerevisiae yeast useful as a binding agent for mycotoxins, in particular fumonisins, and their use for detoxifying mycotoxins in order to limit their assimilation by the organism of animals and thus prevent mycotoxicoses.

Description

Micronised Yeast Husks Saccharomyces Cerevisiae as Mycotoxin Binding Agent

Parent Patent Application

This patent application claims priority from French patent application number FR 21 11819 filed November 8, 2021. The contents of the French patent application are incorporated by reference in their entirety.

Technical area

The present invention relates to the field of animal health. The invention relates more particularly to micronized cell walls of Saccharomyces cerevisiae yeast useful as a binding agent for mycotoxins, in particular fumonisins, and their use for detoxifying mycotoxins in order to limit their assimilation by the organism of animals and thus prevent mycotoxicosis.

Technological Background

Fumonisins (FB) are among the most frequently detected mycotoxins in animal feed and feed ingredients. They are associated with various diseases in humans and animals. Contamination significantly affects the production and marketing of cereals worldwide. The ingestion of mycotoxins results in degraded animal health, reduced productivity and substantial economic losses.

Strategies are therefore needed to reduce the exposure of animals to fumonisins. Pre-harvest testing for the presence of mycotoxins and post-harvest mitigation of contamination are the main strategies for limiting mycotoxins in food and feed. However, the total elimination of molds and their toxins from agricultural products seems impractical, if not impossible. Another approach to reducing exposure to mycotoxins in animal feeds is to reduce their bioavailability by including mycotoxin detoxifying agents in animal feeds. This method is the most commonly used today (Devreese et al., Vlaams Diergeneeskundig Tijdschrif, 2013, 82).

These detoxifiers can be divided into two distinct classes, namely: mycotoxin binders and mycotoxin modifiers or transformers. The binders of mycotoxins (adsorbents or sequestrants) are compounds of high molecular weight, which are able to bind to mycotoxins in the gastrointestinal system of the animal. In this way, the binder-mycotoxin complex passes through the animal and is excreted. This prevents or minimizes animal exposure to mycotoxins. Mycotoxin binders are themselves mainly divided into two categories: inorganic silica-based compounds, on the one hand (“Statement on the establishment of guidelines for the assessment of additives from the functional group substances for reduction of the contamination of feed by myctotoxins”, EFSA journal, 2010, 8(7): 1693) and organic binders, on the other hand. The organic binders of mycotoxins are yeasts (live yeast of Saccharomyces cerevisiae or yeast cell walls) and lactic acid bacteria. For example, fumonisin B1 was successfully eliminated by a strain of Saccharomyces cerevisiae by physisorption (Pizzolitto et al., Int. J. Food Microbiol., 2012, 156: 214-221). Organisms that detoxify fumonisin are exploitable in agriculture and an optimization study for the profitable production of biomass by a probiotic strain of Saccharomyces cerevisiae has been published (Armondo et al., J. Appl. Microbiol., 2013, 114: 1338 -1346). However, the adsorption is carried out with low efficiency.

There is therefore still a need in the art for mycotoxin detoxifiers, particularly fumonisin detoxifiers.

Summary of the invention

The present inventors have surprisingly found that the micronized cell walls of Saccharomyces cerevisiae yeast have the ability to effectively absorb various mycotoxins in vitro, and to reduce the bioavailability of mycotoxins, including the bioavailability of fumonisin B1, in alive.

Consequently, the subject of the present invention is a mycotoxin-binding agent characterized in that it consists of micronized hulls of dry Saccharomyces cerevisiae yeast.

In certain embodiments, the micronized cell walls of dry Saccharomyces cerevisiae yeast have an average diameter of less than 20 μm. In certain preferred embodiments, the micronized cell walls of dry Saccharomyces cerevisiae yeast have an average diameter of about 15 µm. The present invention also relates to the use of a mycotoxin binding agent for detoxifying at least one mycotoxin from animal feed, the use being characterized in that the mycotoxin binding agent is a binding agent such as described here.

The present invention also relates to a mycotoxin binding agent for use in the prevention of mycotoxicosis in an animal, characterized in that the mycotoxin binding agent is a binding agent as described herein.

In certain embodiments, the at least one mycotoxin comprises a mycotoxin from the group consisting of type B fumonisins, zearalenone, ochratoxins, in particular ochratoxin A, and type B aflatoxins. , the at least one mycotoxin is fumonisin B1 or zearalenone.

In some embodiments, the mycotoxin binding agent is used in combination with at least one bioactive agent. In some embodiments, the bioactive agent is selected from the group consisting of mycotoxin binders, mycotoxin biotransformers, vitamins, minerals, trace elements, hepatoprotectants, immunoprotectants, and combinations thereof.

The present invention also relates to a composition comprising a mycotoxin binding agent as described here and at least one bioactive agent. The bioactive agent can be chosen from the group defined above.

The present invention also relates to an animal feed comprising a mycotoxin binding agent as described here or a composition as described here.

The present invention further relates to a method for detoxifying at least one mycotoxin from an animal feed for the purpose of preventing mycotoxicosis in an animal, the method comprising: the step of mixing the animal feed with a mycotoxin binding agent as described herein or with a composition as described herein, prior to administration of the mixture to an animal; or the step of administering a mycotoxin binding agent as described herein or a composition as described herein to an animal to which the animal feed is also administered. In some embodiments, the composition used in the method for detoxifying at least one mycotoxin from animal feed for the purpose of preventing mycotoxicosis in an animal further comprises at least one bioactive agent. In some embodiments, the bioactive agent is selected from the group consisting of mycotoxin binders, mycotoxin biotransformers, vitamins, minerals, trace elements, hepatoprotectors, immunoprotectors, and combinations thereof.

A more detailed description of certain preferred embodiments of the invention is given below.

Brief Description of Figures

Figure 1 shows adsorption isotherms of YCW product at pH3 and pH7.

Figure 2 shows the effect of pH on the adsorption of fumonisin B1 by the YCW product.

Figure 3 shows the effect of adsorbent dose on the adsorption efficiency of multi-mycotoxins by YCW.

Figure 4 shows the effect of mycotoxin detoxifier on reducing urinary excretion of FBI in rats exposed to 3 mg FBI and 125 mg YCW. The control animals received the toxin in the absence of the detoxifier. The results are the mean ± standard deviation (n=6).

Detailed description

As mentioned above, the present invention relates to micronized cell walls of Saccharomyces cerevisiae yeast for use as a binding agent for mycotoxins, including fumonisins, such as fumonisin B1 (FBI).

I - Micronized Yeast Barks Saccharomyces cerevisiae

Yeast cell walls constitute the insoluble fraction of yeasts, i.e. the wall of yeasts and the plasma membrane of yeasts. It is therefore neither whole yeast nor the cellular content of yeast such as yeast extract.

In the context of the present invention, yeast hulls are isolated hulls. The term "isolated", as used herein to characterize yeast cell walls, refers to yeast cell walls that are separated from at least one component to which they are usually associated in their natural form, and preferably separated from the rest of the yeast cell. Conversely, non-isolated yeast hulls are hulls as found in their natural state. Generally, the yeast cells according to the present invention are purified. The term "purified", as used herein to characterize yeast hulls, refers to yeast hulls which have been substantially freed of any contaminants and any other agents or cellular components which are not yeast hulls.

Yeast hulls mainly comprise carbohydrates (between 40% and 60% of carbohydrates by mass of dry matter) consisting mainly of P-glucans and mannans. They also contain from 5% to 70%, in particular from about 10 to 50%, for example from 15 to 30%, of protein materials by mass of dry matter.

Yeast hulls can be in liquid form, in dry form or in viscous form. They are considered to be in dry form when their dry matter content is at least 85%, preferably at least 90%, and even more preferably at least 95% by mass. On the contrary, if their dry matter content is less than 20% by mass, they are considered to be in liquid form. From 20% and below 85% by mass of dry matter, it is considered that the yeast hulls are in viscous form. In the context of the present invention, the yeast cell walls are in dry form.

The micronized cell walls of dry Saccharomyces cerevisiae yeast according to the present invention can be obtained by any suitable method known in the art. Conventionally, the yeast hulls are obtained by a process comprising a yeast autolysis step followed by a step of separating the soluble fraction from the insoluble fraction, the isolated insoluble fraction corresponding to the yeast hulls then being dried. This process is followed by a micronization step to obtain dry micronized yeast cell walls. Alternatively, drying and micronization can be combined in one step.

In certain particular embodiments, the yeast cell walls are obtained according to a process comprising the following steps: production of yeast in a fermenter to obtain a yeast cream, autolysis of the yeast cream at a fixed or variable temperature between 45°C and 70°C, optionally in the presence of proteolytic enzymes, separation of the soluble fraction from the insoluble fraction of the autolyzed cream of yeast in order to recover the insoluble fraction which corresponds to the yeast hulls, acidification of the insoluble fraction to a pH between 1 and 5, heating of the acidified insoluble fraction, drying by pulverization, and sieving to obtain yeast cell walls.

The proteolytic enzymes which can be implemented in this process are EC3 hydrolases, more particularly hydrolases acting on ester bonds (EC3.1), glycosidases (EC3.2), peptidases and proteases (EC3.4) and enzymes acting on carbon-nitrogen bonds (EC3.5). The proteolytic enzymes can be chosen from the group comprising exopeptidases (such as, for example, aminopeptidase, dipeptidase, dipeptidylpeptidase, tripeptidylpeptidase, peptidyl-dipeptidase, serine-type carboxypeptidase, cysteine-type carboxypeptidase, metallocarboxypeptidase, and omega peptidase), endopeptisases or proteinases (particularly cysteine endopeptidase, aspartic endopeptidase, and metalloendopeptidase) and mixtures thereof.

The term "spray drying" is used conventionally and refers broadly to processes involving the breaking down of liquid mixtures into small droplets (atomization) and the rapid removal of solvent from the mixture in a vessel (drying chamber) where there is a strong driving force for the evaporation of the solvent from the droplets. The strong driving force for solvent evaporation is usually provided by maintaining the solvent partial pressure in the spray drying apparatus well below the vapor pressure of the solvent at the droplet drying temperature. This is accomplished by (1) mixing the liquid droplets with a hot drying gas, (2) maintaining the pressure in the spray-drying apparatus at partial vacuum (e.g., 0.01 atm to 0.50 atm ), or (3) both. Generally, the temperature and flow rate of the drying gas are chosen so that the droplets of spray solution are sufficiently dry by the time they reach the walls of the apparatus that they are essentially solid, form a fine powder, and do not not stick to the walls of the drying unit. The time required to reach this level of drying depends on the size of the droplets and the conditions under which the process is operated. There Droplet size can vary from 1 µm to 500 µm in diameter, the size depending on the desired particle size of the spray-dried powder.

The term "sieving" refers to the separation of a mixture of particles of different sizes into two or more portions, by passing through a sieve of mesh, netting or other filtration systems. Sieving can also be done using sieving machines well known in the art. In general, a sieve separates the desired elements from the undesirable ones. Sieving is done to obtain desired medium sized yeast cell walls. In certain embodiments, the desired average size of the yeast cell walls is between about 50 μm and about 70 μm, preferably between about 55 μm and about 65 μm (for example 55 μm, 56 μm, 57 μm, 58 μm, 59 μm). μm, 60 μm, 61 μm, 62 μm, 63 μm, 64 μm or 65 μm), more preferably still the desired average size of the yeast cell walls is about 60 μm. Since the yeast cells obtained by the process described above are generally substantially spherical in shape, the desired average size corresponds to the average diameter of the spherical particle.

The process for obtaining micronized yeast cell walls comprises a step of micronizing the yeast cell walls. The term “micronization” refers to the process of reducing the average particle diameter of a solid material. Traditional micronization techniques are based on the use of friction to reduce particle size. These methods include milling and grinding. Particle size reduction can also take place as a result of collision and impact, for example by crushing or cutting – these methods produce coarser particles than the previous two techniques. Modern methods of micronization include atomization (a method of dehydrating a liquid into powder form by passing a stream of hot air), and methods based on the use of supercritical fluids to induce a state of saturation, which leads to the precipitation of individual particles (e.g. the RESS method - Rapid Expansion of Supercritical Solutions, the SAS method (Supercritical Anti-Solvent) and the PGSS method (Particles from Gas Saturated Solutions). The micronization method does not is not a limiting factor. Those skilled in the art know how to select the micronization method best suited to the preparation situation. The micronization step can be followed by a sieving step. The micronized yeast cell walls are substantially spherical in shape . In the context of the present invention, micronization (optionally followed by sieving) is carried out to obtain micronized yeast cell walls with an average diameter of less than 20 μm. More specifically, micronization provides a population of yeast cell walls having an average diameter of less than 20 μm, where more than 50% of the micronized yeast cells have an average diameter of less than 20 μm. For example, the micronization is carried out to obtain a population of micronized cell walls of yeast having an average diameter of about 20 μm, or about 19 μm, or about 18 μm, or about 17 μm, or d approximately 16 μm, or even approximately 15 μm, or approximately 14 μm, or approximately 13 μm, or alternatively approximately 12 μm. Preferably, the micronization is carried out to obtain a population of yeast cell walls having an average diameter of approximately 15 μm, that is to say a population where more than 50% of the micronized cell walls of yeast have an average diameter of about 15 μm (for example between 16 μm and 17 μm, or even between 14.5 μm and 15.5 μm).

After manufacture, the yeast cell walls can be packaged and/or stored under suitable conditions before use.

II - Compositions Comprising Yeast Shells Saccharomyces cerevisiae

In some embodiments, the micronized dry Saccharomyces cerevisiae yeast cell walls described herein are used alone. In other embodiments, the micronized dry Saccharomyces cerevisiae yeast cell walls described herein are used in combination with another bioactive agent. Consequently, the present invention relates to a composition comprising micronized cell walls of Saccharomyces cerevisiae yeast described here in admixture with at least one bioactive agent. In some embodiments, the bioactive agent is a mycotoxin detoxifying agent.

The mycotoxin detoxifying agent present in a composition according to the invention can be selected from the group consisting of mycotoxin binders, mycotoxin biotransformers, and combinations thereof.

The terms "mycotoxin binding agent" and "mycotoxin binder" are used interchangeably herein. They refer to an agent which adsorbs and/or deactivates mycotoxins, for example mycotoxins present in an animal feed or an animal feed ingredient, thus reversing the harmful effects of mycotoxins. Mycotoxin binders reduce exposure to mycotoxins by decreasing their bioavailability, which results in reduced absorption of mycotoxins, but also decreased distribution to blood and target organs. The term "bioavailability", as used herein in reference to a mycotoxin, refers to the fraction of mycotoxin that is absorbed/absorbable or assimilated/assimilable by an animal. The adsorption of mycotoxins by a binding agent decreases the bioavailability of the mycotoxins because the binder-mycotoxin complex crosses the digestive tract of the animal and is excreted by the animal without there having been assimilation.

Examples of mycotoxin binders include, without limitation, mycotoxin adsorbents selected from the group consisting of aluminosilicates (e.g., kaolinite, hydrated calcium/potassium/sodium aluminosilicate, hydrated sodium calcium aluminosilicate). (HSCAS)), bentonites (for example sodium bentonite, calcium bentonite), montmorillonites (for example sodium and calcium montmorillonite), zeolites (for example zeolite clinoptilolite), activated carbons, micronized fibers and polymers (eg, cholestyramine, polyvinylpyrrolidone), activated diatomaceous earth, plant fibers (eg, wheat bran and alfalfa fibers), polysaccharides (eg, glucomannan or its esterified forms) and their combinations.

The term “mycotoxin biotransformer” refers to an agent (usually an enzyme, bacterium, fungus), which deactivates or inactivates mycotoxins, for example mycotoxins present in animal feed or animal feed ingredients, thus reversing the effects harmful mycotoxins.

Examples of mycotoxin biotransformers include enzymes which degrade mycotoxins, for example chosen from the group consisting of esterases, lipases, proteases, oxidases, cellulases, epoxidases, dehydrogenases, hemicellulases (or xylases), catalases, peroxidases, laccases, xylanases, carboxylesterases, amino-/acetyltransferases, pancreatin, lactonases, lactonohydrolase and combinations thereof.

Other examples of mycotoxin biotransformers include microorganisms that are known to be deleterious to mycotoxins, e.g. selected from: Eubacterium sp. BBSH 797 (a coriobacteriaceae), Nocardia asteroids, Mycobacterium fluoranthenivorans sp., Rhodococcus erythropolis, bacilli of the genus Alcaligenes, Bacillus, Lactobacillus, Achromobacter thermophilus C5 and NG40Z, Lactobacillus paraplantarum, Stenotrophomonas maltophila, Saccharomyces cerevisiae, Exophiala spinifera, Cupriavidus basilensis OR16 Aspergillus niger, Eurotium herbariorum, Rhizopus, Trichosporon mycotoxinivorans, Phaffia rhodozymyllces , Phaffia rhodozymy lices, Phaffia rhodozymhousces, Nocardia corynebacterioides NRRL 24037, Mycobacterium fluoranthenivorans or Myxococcus fulvus ANSM068, Rhodococcus erythropolis, Brevibacterium sp., Slack ia sp. DG6, Deviosa mutans, Deviosa insulae Al 6, and combinations thereof.

In some embodiments, the bioactive agent is an antioxidant known to reduce the toxicity of mycotoxins in animals. Examples of such bioactive agents include, without limitation, rutin, quercetin, lutein, lecithin, melatonin, mannitol, curcumin, curcumoids, lycopene, allyl sulphides, fructose, chlorophyll and its derivatives, sodium thiosulfate, glutathione, methionine, aspartame, trace elements (selenium, zinc, magnesium), catechin (epigallocatechin gallate, epicatechin gallate), morine, kaempferol, fisetin, naringin, vitamins (vitamins E, C, A and B), coenzyme Q10, provitamins (carotene and carotenoids), eugenol, vanillin, caffeic acid, cholinergic acid, and their combinations.

In some embodiments, the bioactive agent is a hepatoprotectant or immunoprotectant known to be active in animals. Terms

“hepatoprotector” and “liver protector” are used interchangeably herein. They refer to an agent which improves the integrity and regeneration of hepatocytes, by optimizing the detoxification capacity of the liver and/or which promotes hepatic synthesis by stimulating the activity of digestive enzymes which ensure optimal use of nutrients by increasing their intestinal absorption and, therefore, their bioavailability. Examples of hepatoprotectors include, without limitation, hepatoprotectors of natural origin composed of a combination of a variable number of plants having different hepatoprotective properties such as Phyllantus niruri, Azadirachta indica, Andrographis paniculata, Achyrantes aspera, etc., and hepatic protectors donors of methyl groups based on the ability of methyl groups to unite with toxins, thus promoting their elimination from the organism. Among the compounds capable of donating methyl groups, certain amino acids and their derivatives (for example, methionine, carnitine, betaine, etc.), vitamin derivatives (for example, choline) are distinguished. The term “immunoprotectant” refers to an agent which, regardless of its mechanism of action, protects against the effects of an antigen. Examples of immunoprotective agents include, without limitation, envelope proteins derived from animal viruses; oligonucleotides such as for example CpG oligonucleotides. Immunoprotectants can also be chemical immunoprotectants which include but are not limited to cytokines, chemokines and lymphokines including but not limited to interferon alpha, interferon gamma and interleukin 12 , or immunoprotectors of plant origin, such as for example the plants: Eclipta alba, Aloe vera, Ocimum sanctum, Viscum album, Urtica dioica and Zingiber officinale, Solanum trilobatum, Astragalus radix and Scutellaria radix, and Achyranthes aspera.

In a composition according to the invention, the bioactive agent(s) are generally present in an amount which is sufficient to achieve the desired goal (for example the reduction of the bioavailability of mycotoxins and/or the inactivation of mycotoxins). A person skilled in the art knows how to determine such a quantity. For example, the enzyme(s) which degrade mycotoxins may be present in an amount less than or equal to 5% by weight of enzyme relative to the total weight of the composition, preferably less than 1%, more preferably between 0.01% and 0.5%, more preferably still between 0.15% and 0.25% by weight of enzyme relative to the total weight of the composition. One or more bioactive mycotoxin-binding agents may, for example, be present in an amount up to about 90-95% by weight based on the total weight of the composition, for example about 80%, by total weight per relative to the total weight of the composition, for example approximately 70%, approximately 60%, approximately 50%, approximately 45%, approximately 40%, approximately 35%, approximately 30%, approximately 25%, approximately 20%, approximately 15%, about 10%, or less than 10% by weight relative to the total weight of the composition.

In certain embodiments, a composition according to the invention can also comprise components which are generally found in supplements, supplements or food additives for animals, such as for example vitamins, minerals, trace elements, etc. A person skilled in the art knows select the appropriate components according to the animal for which the composition is intended, and knows how to determine the adequate quantities to include in such a composition.

The compositions according to the invention can be in any form, for example in the form of powder, granules, a gel or a liquid. Preferably, the compositions according to the invention are in solid form, preferably in powder form.

III - Uses of Micronised Peels of Dry Saccharomyces cerevisiae Yeast as a Binding Agent for Mycotoxins

The micronized cell walls of dry Saccharomyces cerevisiae yeast described herein, which bind effectively to mycotoxins, in particular fumonisins, such as fumonisin B1, can be used to decrease the bioavailability of mycotoxins in animal feed, thereby improving the performance and health of animals and reducing the incidence of syndromes or diseases associated with mycotoxins in these animals. The invention therefore relates to the use of the micronized cell walls of dry Saccharomyces cerevisiae yeast described here, alone or combined with at least one bioactive agent, for detoxifying mycotoxins, for example in animal feed, and/or for preventing mycotoxicosis in an animal.

As used herein, the term “mycotoxins” refers to toxins produced by various species of microscopic fungi, such as molds (Aspergillus sp., Fusarium sp. Stachybotrys sp., Penicillium sp., etc.). They can develop on many substrates: fodder, cereals, oilseeds, protein crops, silage, incidentally molasses and vitaminized mineral food. The main mycotoxins are aflatoxins, ochratoxin A, patulin, citrinin, Fusarium toxins (such as fumonisins, zearalenone, trichothecenes), Altemaria toxins, ergot alkaloids, sterigmatocystin, and paxillin. In certain embodiments of the invention, the mycotoxins are preferably chosen from fumonisins (in particular type B fumonisins, such as fumonisins B1, B2 and B3, preferably fumonisin B1), zearalenone, ochratoxins, in particular ochratoxin A.

The term "mycotoxicosis" means a condition arising from the ingestion of a mycotoxin, which comes from food contaminated with said mycotoxin. produced by a microscopic fungus. Zearalenone is a hormonal decoy, endocrine disruptor, which "mimics" estrogen and can cause reproductive problems in animals fed with fusarium-ridden food (going as far as infertility and spontaneous abortion, especially in pigs) . In cattle, zearalenone can cause reduced growth, decreased milk production and reduced feed efficiency. In poultry, increased mortality is observed. Ochratoxin A produces immunosuppression in farm animals. Fumonisins produce negative effects via reduced blood flow and cardiac output. In this way, they reduce growth and cause pulmonary edema in pigs and poultry. This reduction in blood flow affects all major organs, including the liver, and can exacerbate and enhance the effects of other toxins that may also be present.

As used herein, the term "animal" refers to a living being of the kingdom Animalia. In the context of the present invention, the term refers more specifically to livestock (such as cattle (cow, buffalo, zebu, bison, aurochs, yak), sheep (sheep), goats (goat), pigs (pig ), equids (horse, donkey, mule), camelids (camel, dromedary, llama, alpaca), and deer (reindeer, deer)) and other livestock (hen, turkey, duck, goose, pigeon, quail, pheasant, partridge, ostrich, emu, rhea, ratite, guinea fowl, rabbit, guinea pig); aquatic animals (marine, pond or freshwater fish farming, shellfish farming, crustacean farming and mollusc farming); pets (cats, dogs, rabbits, horses, etc.); and laboratory animals. The term animal, as used herein, does not denote a particular age. In certain specific embodiments, the animal for which the micronized yeast Saccharomyces cerevisiae cell walls described here are intended is chosen from among the avian, bovine, porcine, equine, ovine, caprine, canine and feline species. For example, the animal can be chosen in particular from pigs, dairy or beef cattle, sheep, goats, hens, and turkeys. In other particular embodiments, the animal for which the micronized yeast cell shells Saccharomyces cerevisiae described here are intended is chosen from fish, in particular those used in aquaculture, and crustaceans (for example shrimps, etc.).

As used herein, the terms “pet food”, “pet food ingredient” and “pet food product” mean any natural or processed organic material that is susceptible to biodegradation and can be consumed. by an animal. Examples of such organic materials range from freshly harvested grain to pelleted feed, and include, for example, any compound, grain, nut, fodder, silage, preparation, composition or mixture suitable or intended for ingestion by an animal. The term “forage” refers to plant material (mainly leaves and stems of plants) consumed by grazing animals. The term “silage” refers to fermented, high-moisture fodder that can be fed to ruminants. Formulas, compositions and mixtures, e.g. commercial formulas, compositions and mixtures, may be in any suitable form, e.g. as concentrates, premixes, supplements, total mixed ration (TMR), etc. . The animal feed may contain cereals (eg corn, wheat, barley, rye, rice, sorghum, millet or a combination thereof), roughage, silage, legumes (eg soybeans), spent grains, animal products, etc. Dregs mainly come from industrial processes such as brewing and distilling, intended for the production of drinking alcohol (whisky, vodka or gin) or biofuels. Brewers grains are produced from malt, itself derived from barley. Cereals other than barley may occasionally be used in brewing, such as sorghum, especially in Africa. The spent grains from the production of bioethanol are corn or wheat. Wet spent grains are dried to produce dry spent grains, which are mainly used as animal feed.

As used herein, the term "detoxify mycotoxins" means to decrease the bioavailability of mycotoxins, for example mycotoxins present in animal feed. A mycotoxin binder detoxifies by annihilating mycotoxin toxicity. More specifically, a mycotoxin binder detoxifies by binding to mycotoxins such that the binder-mycotoxin complex, which is non-toxic, passes through the digestive tract of animals and is excreted.

The expression “to prevent mycotoxicosis in an animal” means to reduce or inhibit, partially or completely, the probability that an animal develops one or more symptoms related to contact (ingestion) with a mycotoxin (reduction of growth, immunosuppression, reduced feed efficiency, infertility, spontaneous abortion, decreased milk production, pulmonary edema, or death, depending on the mycotoxin and the animal). In certain embodiments of the present invention, the use of the micronized cell walls of dry Saccharomyces cerevisiae yeast described herein, alone or combined with at least one bioactive agent, to detoxify mycotoxins in an animal feed and/or to prevent mycotoxicosis in an animal, includes mixing such yeast cell walls with animal feed. Mixing can be done by any suitable method known in the art.

In some embodiments, the animal feed to which micronized yeast Saccharomyces cerevisiae hulls described herein are added has been determined to be contaminated with molds capable of producing mycotoxins or to contain mycotoxins. In other embodiments, the animal feed has been determined to be likely to be contaminated with molds capable of producing mycotoxins or to be likely to contain mycotoxins. In still other embodiments, nothing is known about the potential contamination of the animal feed by molds capable of producing mycotoxins or by mycotoxins.

The micronized yeast cells Saccharomyces cerevisiae described herein, alone or in combination with at least one bioactive agent, can be added to animal feed in amounts of from about 0.01% to about 3% by total feed weight ( which corresponds to quantities of approximately 0.1 kg to approximately 30 kg per ton of food) subject to compliance with the legislation in force. For example, in some embodiments, the micronized cell walls of Saccharomyces cerevisiae yeast described herein, alone or in combination with at least one bioactive agent, are added to an animal feed in amounts from about 0.01% to about 0.01% .5% by total weight of the feed (corresponding to amounts of about 0.1 kg to about 5 kg per ton of feed), for example in amounts of about 0.03% to about 0 .3% by weight of feed (which corresponds to about 0.3 kg to about 3 kg per ton of feed).

Alternatively or additionally, the micronized yeast cells Saccharomyces cerevisiae described herein, alone or in combination with at least one bioactive agent, can be administered directly to animals in supplement form in amounts ranging from about 0.01 to about 200 grams per animal per day, depending on the animal species, the animal's size and feed intake, and the type of feed with which the supplement is administered. For example, the micronized cell walls of Saccharomyces cerevisiae yeast described here, alone or in combination with at least one bioactive agent, are administered to animals in amounts ranging from about 0.1 to about 30 grams per animal per day, depending on the animal species, size and food intake of the animal, and the type of food with which the supplement is administered.

A person skilled in the art will recognize that the micronized cell walls of Saccharomyces cerevisiae yeast described here, alone or in combination with at least one bioactive agent, can be added to animal litter comprising organic matter (for example straw which is traditionally used in livestock farms and stud farms, or other vegetation such as hemp bedding, flax bedding, corn bedding, cotton bedding, peat, cellulose, dead leaves, hay, etc.). Indeed, some animals consume their litter, and when it is contaminated, they can be victims of mycotoxicosis. When incorporated into animal litter, the micronized yeast cell walls Saccharomyces cerevisiae described herein, alone or in combination with at least one bioactive agent, may be added in amounts ranging from about 0.0125% to about 10% per litter weight.

Those skilled in the art will also recognize that the micronized cell walls of Saccharomyces cerevisiae yeast described here, alone or in combination with at least one bioactive agent, can be added to water intended for animal consumption or to water used in fish farming. When they are added to water, the micronized cell walls of Saccharomyces cerevisiae Saccharomyces cerevisiae yeast described here, alone or in combination with at least one bioactive agent, can for example be added in quantities ranging from approximately 0.0125% 30% by weight of water.

Unless otherwise defined, all technical and scientific terms used in the Description have the same meaning as commonly understood by one ordinarily skilled in the field to which this invention belongs. Likewise, all publications, patent applications, patents and other references mentioned herein are incorporated by reference.

Examples

The following examples describe some embodiments of the present invention. However, it is understood that the examples and figures are presented for illustrative purposes only and in no way limit the scope of the invention. In each of the following examples, the abbreviation “YCW” stands for micronized yeast cell walls.

Example 1: In vitro evaluation of Multi-Mycotoxin Binding Agents.

The experiments reported in this example were carried out with the team of the Institute of Science of Food Production (CNR-ISPA) National Research in Italy.

Determination of physico-chemical adsorption parameters (maximum adsorption capacity and affinity) related to the adsorption of FBI and ZEA by the YCW product. These values were calculated using the isothermal method.

Experimental procedure.

The adsorption isotherms were carried out at constant temperature (37°C), at pH 3 and at pH 7, for a fixed content of YCW product in buffered solutions containing increasing concentrations of FBI; the FBI concentrations (10 concentrations tested) ranging from 0.25 to 20 pg/mL. The product content was set at 0.5% (w/v) and the contact time was 90 minutes. After centrifugation, the supernatants were collected, derivatized with o-phthaldialdehyde (OPA) reagent, and then analyzed by HPLC-FLD. All experiments were performed in triplicate. From the experimental adsorption data, the rate of toxin remaining in solution at equilibrium (Cs) as well as the rate of toxin adsorbed per unit weight of adsorbent (Ca) were calculated, and integrated into SigmaPlot (Systat .corn, version 12), then modeled according to the linear equation. Adsorption isotherms were used to calculate parameters related to adsorption: maximum capacity and affinity of the adsorption process.

The effect of pH on the adsorption of mycotoxins by the YCW product was evaluated as follows: 10 mg of YCW was mixed with 2 mL of FBI solutions prepared in different buffer solutions in the pH range 3 to 7. The product was introduced at 0.5% (weight/volume). The FBI concentration was set at 1 µg/mL. The samples were incubated at 37°C for 90 minutes at 250 rpm, then centrifuged, the supernatants collected and the analysis of the residual FBI carried out.

The effect of the YCW dosage on the adsorption of FBI and ZEA was evaluated at pH 3 and pH 7 as follows: different quantities of YCW were weighed and then mixed with the mycotoxin solutions containing 1 pg/mL of each toxin, in order to obtain the following adsorbent contents: 0.001; 0.0025; 0.005; 0.01; 0.05; 0.1; 0.5; 1.0 and 2.0% (weight/volume). Samples were incubated at 37°C for 90 minutes under agitation, then centrifuged, the supernatants were then analyzed for their residual mycotoxin content. The FBI is analyzed as indicated above, the ZEA was analyzed by HPLC with fluorimetric detection (excitation wavelength 274 nm, emission wavelength 440 nm).

Results

The results of the in vitro adsorption studies are presented in Figures 1 to 3.

Data shown are the mean value of three replicates per sample. The standard deviation is shown in the figures. As shown in Figure 1 and Table 1 below, the yeast wall (sample YCW) shows its ability to adsorb FBI with greater FBI adsorption capacity at pH 3 compared to its capacity at pH of 7, the optimum pH for adsorption being located at 4.

Table 1. YCW product adsorption isotherms at pH3 and pH7.

Figure 3 shows the variation in adsorption for fumonisin B1 and zearalenone (ZEA) as a function of yeast wall content; the results demonstrate that the detoxifier YCW has a greater adsorption capacity for ZEA than for FB 1.

Example 2: In vitro efficacy of Multi-Mycotoxin Binding Agents.

The experiments reported in this example were carried out with teams from the Institute of Sciences of Food Production (CNR-ISPA) National Research in Italy and the Department of Pharmacology, Toxicology and Biochemistry of the Faculty of Veterinary Medicine in Ghent University in Belgium.

Materials and Methods

Approval of in vivo studies by Ethics Committees and Protection Organizations. All permissions and approvals have been obtained to perform the in vivo studies. Animal studies took place in European countries (Italy and Belgium) where they are regulated by national and international legal and ethical rules. The animal studies were designed in accordance with the guidelines of current European legislation (EU Directive 2010/63) and national laws on the care and use of experimental animals (Ministero Italiano della Ricerca Scientifica, D.Lgs. March 4, 2014 n° 26, and Belgian Royal Decree of May 27, 2013). All pharmacokinetic experiments involving piglets and rats were presented to Ugent University (Belgium) and Bari University (Italy), respectively, for approval by internal ethics committees and animal welfare organizations (Organismi Preposti al Benessere degli Animali) of each university.

My cotoxins detoxifiers. YCW was supplied in powder form and stored in the testing facilities in a dry, clean space at room temperature. A dose of 0.25 g/kg body weight and 0.50 g/kg body weight of YCW was administered by intragastric bolus to pigs and rats, respectively. Considering a feed intake of 50 g feed/kg body weight in 4-week-old pigs and 100 g feed/kg body weight in rats, this corresponds in all cases to an inclusion rate food of 0.5% (w/w). This feed additive inclusion rate was chosen based on previous in vitro studies showing that the YCW product has a high mycotoxin adsorption capacity.

Evaluation of the Efficacy of Selected Multi-mycotoxin Binding Agents in Reducing the Bioavailability of Mycotoxins in Rats.

Mycotoxins. The FBI (purity >99%) used for this study was purchased from Sigma-Aldrich (Milan, Italy). The mycotoxin was administered daily at the following dose: 12 mg FB1/kg body weight. Considering a food intake of 100 g of food/kg of body weight in rats, this dose corresponds to a daily intake of contaminated food containing 120 pg/g of FBI.

Study design. The study was carried out using 20 healthy rats of the same age (6-9 months), of both sexes, and of approximately the same body weight at the start of the experiment. An acclimatization period of 2 weeks was respected. The rats were housed two per cage. Rats were randomly assigned to different groups and treated with mycotoxin (provided as a single dose) or mycotoxin in combination with mycotoxin detoxifier (as detailed below). The rats were housed individually in cages to collect urine 4, 8, 10, 24, 32, 48, 56, and 72 hours. Urine samples were weighed and stored at -20°C before analysis.

Animals and Housing Conditions. 20 (male and female) healthy Wistar rats (supplied by Envigo RMS S.R.L) were used. Supplier surveillance records indicated that the rats were free of known bacterial, viral and parasitic pathogens. The following criteria for abandonment and exclusion of animals were treated: change in body weight > 10% with diarrhea and/or anorexia, dehydration, convulsions, piloerection, changes in behavior and change in rectal temperature of ± 4° C that persist for more than 48 hours have been considered human endpoints predictive of toxicity and leading to animal exclusion and sacrifice (Morton; NCR3s).

The rats were randomly divided into 2 groups of 6 animals (3 males and 3 females). Groups of animals were used for each mycotoxin/detoxifier combination, labeled C (mycotoxin control), T (treatment, mycotoxin + Lesaffre detoxifier).

The housing conditions of the rats were in accordance with EU guidelines. Upon receipt, rats were randomized into study groups and housed in polycarbonate cages (2 rats per cage, 1500 cm ² x 18 cm), on a 12-hr/12-hr reverse light/dark cycle. The temperature was controlled and maintained at 22°C ± 1°C with a relative humidity of 50% ± 5%.

Throughout the acclimatization period, rats were maintained on a standard laboratory diet (Dicta Tekland 2018, Envigo RMS S.R.L.) and water ad libitum. During feeding trials with mycotoxins (± detoxifying) and urine collection, food was removed twelve hours before performing the experiment, but water mixed with apple juice (1/1 , v/v) was still available. Animals in these groups were fed again 72 hours after administration.

Bolus delivery. Each rat was first sedated by inhalation using 3% isoflurane mixed with 1.5% LC 1/minute. Then the head and body were held vertically as required for oral gavage and held for a count of 2 seconds (based on the maximum time required to gavage any rat) before releasing the rat into a metabolic cage. where rats were housed for 72 hours for urine collection. A flexible oral gavage tube and disposable polypropylene (Instech) was used for gavage. Personnel performing oral gavage were experienced in the procedure.

FBI stallions were used for the study. The FBI standard was dissolved in water to prepare a 3 mg/ml stock solution, and 1 ml of this solution was used as such for animal testing. Animals treated with toxin plus binder received 125 mg of additive dissolved in 1 ml of water toxin solution, in order to have an additive intake of 0.5 g additive/kg body weight. These solutions were administered to the animals immediately after their preparation. To further protect the animals during the experiment, each morning animals in the treated groups received an additional daily dose of the additive dissolved in a drinkable mixture of water and apple juice and supplied ad libitum. The use of apple juice improved the palatability of the aqueous suspension of the additives.

Zootechnical Parameters. Rats were weighed individually upon arrival, then twice weekly during the acclimation period, before bolus administration and at the end of the caged metabolic period. The following parameters were monitored daily: general condition, breathing, excreta status, appetite, hydration, mobility, piloerection and behavioral changes. Daily information on animal behavior was collected in a specific technical sheet.

FBI Analysis in Rat Urine. Analysis for the presence of FBI in rat urine was performed by UPLC-FLD/PDA method, which has been successfully validated. In addition to mycotoxin analysis, urine samples were analyzed for creatinine content using the creatinine (urinary) colorimetric assay kit from Kayman (Cabru, sas, Milan, Italy). The FBI content in urine was normalized for the respective creatinine concentration (mol/L). Normalized urinary excretion data were presented as mean ± SD. The area under the curve (AUCo->t) over 72 hours and the maximum urine concentration (C _max ) were calculated and used to compare the control and the treatment groups. The AUCo->t and Cmax comparisons in the positive control rats (fed with the mycotoxin without the additive) and in the treated rats (fed with the mycotoxin in the presence of the additive) were made by unidirectional analysis of the variance. Statistical analysis was performed using SigmaPlot® (version 12.0). A post-hoc analysis was performed by TukeyTest to find means significantly different from each other. In addition, normality test (Shapiro-Wilk) and variance equality test were used to test, respectively, the normally distributed population and the constant variance hypothesis. The significance level threshold for the tests of normality and equality of variance was set at P <0.05. One-way Kruskal-Wallis analysis of rank variance was used when tests for normality and/or equality of variance failed.

Evaluation of the Efficacy of Selected Multi-mycotoxin Binding Agents in Reducing the Bioavailability of Mycotoxins in Piglets.

Mycotoxins. The administered dose of each mycotoxin was based on previous studies (Devreese et al., J. Chrom., 2012, 1257: 75-80; Catteuw et al., J. Agric. Food Chem., 2019, D01:10.1021/ acs.jafc.8b05838). ZEA was obtained from Fermentek Ltd (Jerusalem, Israel). The FBI was obtained after aqueous extraction of a corn fungal culture prepared at CNR-ISPA and containing 0.571 mg/mL of FBI and 0.226 mg/mL of FB2. The following doses were administered to each animal: 0.331 mg ZEA/kg body weight; and 2.00 mg FB1/kg body weight. Given a feed intake of 50 g feed/kg body weight in 4-week-old pigs, these doses correspond to a daily intake of feed contaminated with multiple mycotoxins containing 6.62 pg/g of ZEA and 40 pg/g of FB 1.

Power Control. Animals were fed a commercial swine diet that was assessed for possible mycotoxin contamination prior to the start of the study using a validated multi-mycotoxin liquid chromatography-tandem mass spectrometry (LC-MS) screening method. /MS). Only DON and ZEA could be detected, at a level well below the maximum European reference level (2006/576/EC). This diet was given ad libitum, except during the abstention period.

Study design. Each study was conducted with 32 healthy pigs, equally divided between sexes, of the same age (4 weeks) and approximately the same body weight at the start of the experiment. An acclimatization period of one week was respected (days 1 to 7). The piglets were randomly allocated to 2 groups of 16 animals. The first group, the control group (C), received the mycotoxin mixture (day 8 and 9). The second group, the treatment group (T), received the mycotoxin mixture in combination with the mycotoxin detoxifiers (days 8 and 9). In order to To study the effect of food withdrawal on the absorption of mycotoxins, the two groups were divided into 2 subgroups of 8 animals. For the first subgroup (C1 and T1), feeding was suspended for 12 hours (h) before administration until 4 hours after administration. The second subgroup (C2 and T2) received food ad libitum. Two days of bolus administration were organized (days 8 and 9). Each day, half of each subgroup received the boluses. Blood samples from the piglets were taken from the external jugular vein. Blood collection time points were 0 (before administration) and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48 and 72 hours after administration.

Animals and Housing Conditions. 32 (male and female) healthy hybrid Seghers piglets (Landrace x Pietrain) (provided by RA-SE Genetics, Belgium) body weight: 9.31 ± 0.82 kg at the start of the experiment at the start of the experience, were used. The animal experiments have been approved by the ethics committee of the Faculty of Veterinary Medicine Medicine and Faculty of Bioscience Engineering of the University of Ghent (application number EC 2017/12).

The following exclusion criteria were addressed: 1. Apparent illness or any clinical condition at arrival that could interfere with study results. 2. Animals that become ill or die during the test and/or that require medication should be excluded, if any, because the influence of disease on the results is suspected.

The piglets were randomly divided into 4 groups of 8 animals: Control: Subgroup Cl: piglet 1-8 and Subgroup C2: piglet 9-16 Treatment: Subgroup Tl: piglet 17-24 Subgroup T2 : piglet 25-32.

The housing conditions for the piglets were in accordance with EU directives (European Council, 2007. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Off. J. EU, 2010/63/EU). Pen size (4 pigs/pen) was 4 m ² . The lighting schedule was a natural day and night schedule. In addition, normal lights could be switched on to be able to carry out the requested manipulations with the food and the animals (eg health observations, feeding, etc.). The temperature was controlled and maintained between 22 and 26°C (according to EU guidelines for animals with a live weight of 8 to 30 kg). Throughout the acclimatization period, the piglets had ad libitum access to feed and water. During the abstention period (group C1 and T1) was withdrawn 12 hours before the experiment was carried out, but the water was still available. Animals in these groups were fed again 4 hours after administration.

Bolus delivery. The mycotoxin detoxifier was suspended in 5 ml of tap water and administered through an intragastric tube. ZEA was dissolved in dimethyl sulfoxide (DMSO) to give a stock solution of 10 mg/ml, stored at -20°C. A liquid extract of the corn fungal culture in water was used as an FBI bolus (0.571 pg of FBl extract/mL). The appropriate amount of the stock solutions (based on the body weight of the piglets) was administered as such (FBI), diluted with ethanol (ZEN) to a volume of 1 ml and administered immediately after administration of a mycotoxin detoxifier. Then the tube was rinsed with 50ml of tap water to ensure that no mycotoxins or detoxifiers remained in the tube.

Zootechnical Parameters. Piglets were weighed individually upon arrival and before bolus administration. The following parameters were monitored daily by observation of the veterinarians responsible for animal experimentation: general condition, respiration, onset of the disease, state of excrement, appetite and mobility. During the trial, one pig met the exclusion criteria due to severe cardiac arrhythmia and was euthanized (pig #3). However, post-weaning diarrhea was noted the first days after arrival, with no decrease in general condition, mobility or appetite. Therefore, these pigs were not excluded.

Blood and serum sampling. Blood samples (± 1 ml) were taken from the external jugular vein with an 18G needle at the times described above and collected in EDTA tubes. Samples were identified by study number, animal number, time and date and centrifuged within 2 hours of collection. Plasma was stored at -20°C until analysis. For the collection of serum samples, blood was drawn in the same way and collected in serum tubes. Samples were identified by study number, animal number, time and date and centrifuged within 2 hours of collection. Serum was stored (2-6°C) until analysis. Analysis of Mycotoxins in Plasma. Quantitative plasma analysis of FBI and ZEA (use of ZEA-glucuronide as a biomarker for ZEA exposure) was performed with a validated LC-MS/MS method. The limit of quantification (LOQ) in porcine plasma of ZEA-glucuronide (ZEA-GlcA) has been set at 0.5 ng/mL, and for FBI at 1 ng/mL (Lauwers et al., Toxins, 2019, 11:171). ZEA-glucuronide for analytical experiments was provided by Dr. Huybrechts (Sciensano, Ixelles, Belgium).

Toxicokinetic analysis. Toxicokinetic modeling of plasma concentration-time profiles was performed by non-compartmental analysis (Phoenix 6.4, Pharsight Corporation, USA). The following parameters were calculated: area under the plasma concentration-time curve from time zero to the last sampling point with values above the LOQ (AUCo->t), area under the plasma concentration-time curve from time zero to infinity (AUCo-> _½ ), peak plasma concentration (C _max ), time to peak plasma concentration (T _max ), elimination half-life (Ti/2ei) and elimination rate constant ( kei).

Effect of Mycotoxin Detoxifier on P Oral Adsorption of ZEA and FBI. Relative oral availability was assessed as a marker of mycotoxin detoxifying efficacy.

The effect of mycotoxin detoxifier on the oral absorption of ZEA and FBI was evaluated by comparing toxicokinetic parameters between the control group and the treatment group (for subgroups 1 and 2), with particular emphasis on AUGHI, AUG, C _max and Tmax using two-way ANOVA with Tukey HSD post-hoc test (SPSS 25.0; IBM, USA) to exclude differences based on different days of bolus administration (Factor B: bolus, day 8 or 9). In case Levene's test indicated that no homogeneity of variances was present, a non-parametric Kruskal-Wallis test was performed.

Effect of Food Withdrawal on Oral Absorption of ZEA and FBI. The effect of food withdrawal on the oral absorption of ZEA and FBI was evaluated by comparing toxicokinetic parameters between the two control groups, with particular emphasis on AUGHI, AUGH/., C _max and T _max using an ANOVA bidirectional with post-hoc Tukey HSD test (SPSS 25.0; IBM, USA) to exclude differences based on different days of bolus administration (factor B: day of bolus administration 8 or 9). In the event that Levene's test indicated that no homogeneity of variances was present, a t-Student test was performed.

Results

A. Evaluation of the Efficacy of YCW in Reducing the Bioavailability of Mycotoxins in Rats

Biomonitoring of exposure to mycotoxins is an important tool for monitoring public health and safety. Using this tool, exposures are typically measured by collecting biological samples such as blood and urine samples. Urine sampling is a more convenient and less invasive alternative to blood sampling. This is reasonable in the case of in vivo experiments involving the rat model.

Similarly, the rat model confirmed the effectiveness of YCW to adsorb FBI efficiently (Figure 4). The total excretion of FBI in the urine of YCW-treated rats was more than 50% lower than in the urine of control rats. The toxicokinetic parameters (area under the curve (AUC) from 0 to 72 hours and maximum concentration at 24 hours calculated for the control group were significantly lower than in the FBI + YCW treated group (see Table 2 below) (P< 0.05) The yeast wall reduced AUC values by about 40%, demonstrating that urinary excretion of FBI is reduced by the YCQ MOL wall control and treated was 1.18 ± 0.52 and 0.64 ± 0.60%, respectively (P<0.05).

Table 2. Toxicokinetic parameters of urinary expression in cats.

B. Evaluation of the Efficacy of YCW in Reducing the Bioavailability of

ZEA and FBI at the Piglet 1) Results for ZEA

Since plasma concentrations of ZEA after oral bolus administration of ZEA were below LOQ (likely due to low oral bioavailability and/or first-pass biotransformation), ZEA-GlcA was used as a plasma biomarker for the exhibition at the ZEA. Table 3 shows the results of the most important toxicokinetic parameters of ZEA-GlcA after oral bolus administration of ZEA, with or without YCW and/or feed withdrawal, at 7 or 8 pigs per subgroup (Cl: n = 7; T1, C2, T2: n = 8).

Table 3. Main toxicokinetic characteristics of ZEA-GlcA after single oral bolus administration of ZEA alone (Cl and C2) or in combination with the mycotoxin detoxifier (Tl and T2) to 7 or 8 pigs per subgroup (Cl : n=7; T1, C2, T2: n=8). In subgroups C1 and T1, food was interrupted from 12 hours before until 4 hours after bolus administration. Values represent mean ± SD.

AUCo^,: area under the curve from time zero to infinity; AUCo^t: area under the curve from time zero to the last sample time with values greater than LOQ; C _max : maximum plasma concentration; T _max : time at maximum plasma concentration; T 1/2 ei: elimination half-life; k _ei : elimination rate constant; Rel. F: relative oral bioavailability; *: significant difference between Cl and T1 or between C2 and T2.

For AUG. and AUCo->t, significant differences were found between C2 and T2 subgroups (p=0.020 and p=0.037 respectively; two-way ANOVA with HSD Tukey Post Hoc test). For Cl and Tl, no significant difference between AUG. and AUG t could not be detected at p<0.05. However, a trend could be observed towards a lower systemic exposure to ZEA in the T1 subgroup (Rel. F = 40.7%). For all other parameters, no significant difference was found at p<0.05 for the T1 subgroup compared to the Cl subgroup and for the T2 subgroup compared to the C2 subgroup. Moreover, no significant difference could not be found in the toxicokinetic parameters between the C1 and C2 subgroups, reflecting that the presence of food had no effect on the oral absorption of ZEA in pigs using the oral bolus model.

4) Results for FBI

Table 4 below shows the results of the most important toxicokinetic parameters of FBI after simple oral bolus administration of FBI (Cl and C2) whether or not associated with YCW (T1 and T2) and/or withdrawal feed, at 7 or 8 pigs per subgroup (Cl: n = 7; Tl, C2, T2: n = 8). Due to enterohepatic recirculation of FBI in pigs, Ti/2ei and k _ei could not be calculated. Therefore, relative F values were based on AUCo->t. In subgroup C1 and T1, food was withdrawn from 12 hours before until 4 hours after bolus administration. Values represent mean ± SD

Table 4. Main toxicokinetic characteristics of FBI after single oral bolus administration of FBI alone (Cl and C2) or in combination with the mycotoxin detoxifier (Tl and T2) to 7 or 8 pigs per subgroup (Cl: n=8 ; T1, C2, T2: n=7). In subgroups C1 and T1, food was interrupted from 12 hours before until 4 hours after bolus administration. Values represent mean ± SD.

AUCo^t: area under the curve from time zero to last sample time; C _max : maximum plasma concentration; T _max : time at maximum plasma concentration; Rel. F: relative oral bioavailability; *: significant difference (limit) between Cl and T1 or between C2 and T2.

For AUG t and T _ma x (limit), significant differences were observed between the Cl and Tl subgroups (p = 0.085 and p = 0.048 respectively; Kruskal-Wallis test). Moreover, for T _ma x, a borderline significant difference was observed between the C2 and T2 subgroups (p = 0.098; Kruskal-Wallis test). Between C2 and T2, no significant difference in AUCo->t could be detected at p<0.05 nor p<0.1. However, a trend could be observed towards a lower systemic exposure to FBi in the Tl subgroup (rel. F = 72.5%). For C _max , no significant difference was observed for the T1 subgroup compared to the C1 subgroup and for the T2 subgroup compared to the C2 subgroup. Moreover, no significant difference could be observed in the toxicokinetic parameters between the Cl and C2 subgroups, reflecting that the presence of food had no effect on the oral absorption of FBI in pigs using the oral bolus pattern.

Regarding the efficacy of YCW in reducing the oral bioavailability of mycotoxins in pigs, it can be concluded that YCW did not significantly alter the main toxicokinetic characteristics of AFBl and OTA in pigs. pigs at a dose of 0.25 g/kg body weight. For ZEA, YCW significantly reduced the systemic exposure (AUG and AUCo->t) of mycotoxins in pigs at a dose of 0.25 g kg body weight when food was provided ad libitum. However, when food was withdrawn from 12 hours before to 4 hours after bolus administration, a trend was observed towards lower systemic exposure (AUG and AUGHI) when the mycotoxin detoxifier was administered. This also results in reduced oral bioavailability (rel. F%), which was 40.7% when YCW was administered after food withdrawal and 43.7% when YCW was administered and food was removed. provided at will. A relative F <80% is considered non-bioequivalent to the control group, demonstrating a clear mycotoxin detoxifying effect on systemic ZEA exposure. In the case of FBI, YCW significantly reduced the systemic exposure (AUCo->t) to FBI in pigs at a dose of 0.25 g YCW/kg body weight. Again, this was reflected in reduced oral bioavailability (rel. F%), which was 27.1% when mycotoxin and YCW were given after food withdrawal, and 72.5% when the mycotoxin and YCW were administered during feeding ad libitum. A relative F <80% is considered non-bioequivalent to the control group, demonstrating a clear effect of YCW on systemic FBI exposure. However, a statistical comparison cannot be made for F because this parameter was calculated on the mean AUC values.

Regarding food withdrawal, it can be concluded that the presence of food does not significantly influence the oral absorption of ZEA and FBI. Findings

YCW significantly reduced urinary excretion and oral absorption of FBI in rats and pigs. YCW was effective in preventing the adsorption of ZEA and FBI in in vitro tests, and it significantly reduced the oral absorption of these mycotoxins in pigs.

To the knowledge of the present inventors, this is the first time that micronized cell walls of Saccharomyces cerevisiae yeast have proved effective in binding fumonisins.

Claims

Claims 1. Binding agent for mycotoxins characterized in that it consists of micronized hulls of dry Saccharomyces cerevisiae yeast. 2. Binding agent for mycotoxins according to claim 1, characterized in that the micronized cell walls of dry Saccharomyces cerevisiae yeast have an average diameter of less than 20 μm. 3. Binding agent for mycotoxins according to claim 1, characterized in that the micronized cell walls of dry Saccharomyces cerevisiae yeast have an average diameter of approximately 15 μm. 4. Mycotoxin binding agent for use in the prevention of mycotoxicosis in an animal, characterized in that the mycotoxin binding agent is a binding agent according to any one of claims 1 to 3. 5. Mycotoxin binding agent for use according to claim 4, characterized in that the at least one mycotoxin comprises a mycotoxin from the group consisting of fumonisins type B, zearalenone, ochratoxins, in particular ochratoxin A, and type B aflatoxins. 6. Mycotoxin binding agent for use according to claim 5, characterized in that the at least one mycotoxin is fumonisin B 1 or zearalenone. 7. Mycotoxin binding agent for use according to any one of claims 4 to 6, characterized in that the mycotoxin binding agent is used in combination with at least one bioactive agent. 8. A composition comprising a mycotoxin binding agent according to any one of claims 1 to 3, and at least one bioactive agent. 9. An animal feed comprising a mycotoxin binding agent according to any one of claims 1 to 3 or a composition according to claim 8. A method for detoxifying at least one mycotoxin from an animal feed, the method comprising: the step of mixing the animal feed with a mycotoxin binding agent according to any one of claims 1 to 3 or with a composition according to claim 8. Mycotoxin binding agent for use according to claim 7 or composition according to claim 8 or animal feed according to claim 9 or method according to claim 10, characterized in that the bioactive agent is selected from the group consisting of by mycotoxin binders, mycotoxin biotransformers, vitamins, minerals, trace elements, hepatoprotectors, immunoprotectors, and combinations thereof.