Use of extract of beta-glucan-rich saccharomyces cerevisiae cell wall for prevention of toxic effects of mycotoxin deoxynivalenol (DON)

Abstract

The present invention relates to an extract of the cell wall of Saccharomyces cerevisiae rich in beta-glucan, and to the use thereof in the prevention of mycotoxin poisoning caused by the mycotoxin deoxynivalenol, in particular in the prevention of liver and intestinal injuries and immunotoxic effects caused by the mycotoxin, and to the use thereof in the prevention of mycotoxin poisoning caused by the mycotoxin deoxynivalenol.

Description

[0001] Parent patent application

[0002] This patent application claims the priority of French patent application FR2203260 filed on April 8, 2022. The contents of the French patent application are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of health, in particular to the field of animal health. More specifically, the present invention relates to a cell wall extract of Saccharomyces cerevisiae rich in β-glucan and its use for preventing the toxic effects, in particular the immunotoxic effects, of the mycotoxin deoxynivalenol (DON) in animals, as well as preventing liver and intestinal damage induced by the DON mycotoxin. Prior art

[0004] Mycotoxins are toxic fungal secondary metabolites produced by various molds, mainly belonging to the genera Aspergillus, Penicillium or Fusarium. They are natural contaminants of cereals, but also of fruits, vegetables, oily nuts and seeds, and spices. Contamination greatly affects the production and marketing of cereals worldwide and also impairs the quality of products made from contaminated seeds. In addition, mycotoxins in food may have harmful effects on human and animal health, which can range from gastrointestinal and kidney diseases to immune deficiencies or cancer.

[0005] Deoxynivalenol (DON) is a mycotoxin of the trichothecene family (Class B), produced by various fungal contaminants of Fusarium such as F.culmorum, F.graminearum and F.pseudograminearum. It is a mycotoxin produced on field plants, particularly in cereal crops such as corn, wheat, barley and oats, and also in rice, rye and sorghum. Because trichothecenes are heat stable and sterilization-resistant, they can be present not only in derivative products such as animal feed, but also in flour, bread, pasta, etc., and even in beer. Animals ingesting feed contaminated by DON can cause damage to the immune system and intestinal mucosa, food refusal, and vomiting or diarrhea, which may lead to growth retardation. In humans, DON food poisoning causes abdominal pain, dizziness, headache, throat irritation, nausea, vomiting, diarrhea and fecal blood loss. Recently, DON has been shown to increase the risk of inflammatory bowel disease (IBD) and aggravate its symptoms (Payros et al., Archives of Toxicology (2020), DOI: 10.1007 / s00204-020-02817-z).

[0006] To prevent the risk of contamination with mycotoxins (including deoxynivalenol), food safety authorities (FDA, EFSA, CSAH, JECFA, etc.) have published regulations on maximum permissible concentrations in human and animal foods.

[0007] Since it is currently impossible to control and mitigate the contamination of grains with DON mycotoxins after harvest, the best way to prevent and control DON is field prevention. In fact, DON is produced by different species of Fusarium that grow in grains under very specific temperature and humidity conditions. Under conditions that are conducive to the growth of Fusarium fungi, the application of appropriate fungicides to prevent fungal growth helps prevent the secretion of DON and other mycotoxins. One way to reduce animal exposure to mycotoxins is to reduce their bioavailability by adding antidotes to feed. However, in the case of DON mycotoxins, there are almost no detoxification solutions, and those that exist are not very effective. Biotransformation schemes have been described, such as microorganisms or enzymes that degrade DON into less toxic metabolites, but there is currently only one microorganism on the market (Coriobacteriaceae BBSH797). Regarding detoxification by adsorption, there are very few products that can adsorb DON.

[0008] Therefore, there remains a need in the art for new methods to prevent the toxic effects caused by ingestion of DON mycotoxins.

[0009] Brief description of the invention

[0010] Surprisingly, the inventors have found that an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan significantly improves the transepithelial impedance of the epithelial barrier of porcine intestinal cells exposed to the DON mycotoxin; causes a decrease in DON-induced expression of the IL8 gene (a pro-inflammatory cytokine) in porcine intestinal cells in vitro, in porcine explants ex vivo, and in chicken intestinal tissue in vivo; prevents these cells from undergoing structural disruptions observed in untreated cells; prevents the reduction in intestinal villus size and changes in intestinal cell morphology in porcine intestinal explants; and effectively prevents DON mycotoxin-induced liver damage and reduction in villus size in chickens.

[0011] The object of the present invention is therefore an extract of the cell walls of Saccharomyces cerevisiae enriched in β-glucan for use in preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises β-glucan and mannan, wherein the β-glucan is present in a total amount greater than or equal to 50% by weight as a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount less than 5% by weight.

[0012] The present invention also relates to an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan, which is used to prevent the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract contains β-glucan and mannan, wherein the β-glucan is present in a total amount greater than or equal to 50% by weight in the form of a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount less than 5% by weight.

[0013] The present invention also relates to an extract of Saccharomyces cerevisiae cell walls rich in β-glucan for preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises β-glucan and mannan, wherein the β-glucan is present in a total amount greater than or equal to 50% by weight in the form of a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount less than 5% by weight.

[0014] In certain embodiments, the total amount of β-glucan in the β-glucan-enriched Saccharomyces cerevisiae cell wall extract is 50%-90% by weight of the extract, such as 50%-80%, or 50%-70%, or even 50%-60% by weight of the extract.

[0015] In certain embodiments, the total amount of mannan in the β-glucan-enriched Saccharomyces cerevisiae cell wall extract is 1% to 5% by weight of the extract.

[0016] In certain embodiments, the amount of mannan in the β-glucan-enriched Saccharomyces cerevisiae cell wall extract is such that the β-glucan / mannan ratio is 12-40 (wt / wt). In particular, the β-glucan / mannan ratio may be 15-30 (wt / wt), or even more particularly 18-22 (wt / wt).

[0017] In certain embodiments, the extract of Saccharomyces cerevisiae cell walls enriched in β-glucan is characterized by a dry matter content greater than or equal to 94 wt %. In particular, the dry matter content may be greater than or equal to 96 wt %.

[0018] In certain embodiments, the β-glucan-enriched Saccharomyces cerevisiae cell wall extract further comprises less than or equal to 10% by weight of protein and less than or equal to 10% by weight of glycogen.

[0019] In certain embodiments, β-glucan and mannan are the only active components of the β-glucan-enriched Saccharomyces cerevisiae cell wall extract.

[0020] In certain embodiments, the β-glucan-rich Saccharomyces cerevisiae cell wall is β-glucan It comprises β-glucan and mannan, wherein the β-glucan is present in a total amount of 52 wt% to 60 wt% in the form of a mixture of β-1,3-glucan and β-1,6-glucan; the mannan is present in a total amount of 1 wt% to 5 wt%, and wherein the It also contains protein in an amount of less than or equal to 10% by weight and glycogen in an amount of less than or equal to 10% by weight.

[0021] The present invention also relates to a composition comprising an extract of Saccharomyces cerevisiae cell walls rich in β-glucan and at least one bioactive agent for preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, or preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, or preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract is as defined above.

[0022] The present invention also relates to a pharmaceutical composition comprising an extract of Saccharomyces cerevisiae cell walls rich in β-glucan and at least one physiologically acceptable excipient for preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, or for preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, or for preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract is as defined above.

[0023] In certain specific embodiments, the subject of the present invention is an animal, in particular an animal selected from farm animals and pets.

[0024] The present invention also relates to an animal feed comprising an extract of Saccharomyces cerevisiae cell walls rich in β-glucan for use in preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in animals, or for preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in animals, or for preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in animals, characterized in that the extract is as defined above, or the extract is part of a composition as defined above.

[0025] In certain embodiments, the bioactive agent is selected from the group consisting of mycotoxin binders, mycotoxin biotransformers, vitamins, minerals, trace elements, liver protectants, immune protectants, and combinations thereof.

[0026] A more detailed description of certain preferred embodiments of the invention is given below. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :Monitored in porcine intestinal cells (IPEC-J2 cell line), using Changes in transepithelial impedance (TEER) over time (T) (in hours) after continuous pretreatment and addition of DON mycotoxin. TEER changes were compared with those of cells treated with DON mycotoxin alone, and in the presence of DON mycotoxin. treated cells or under two control conditions (epithelial cells only or The TEER changes of cells were compared.

[0028] Figure 2 : In the presence or absence of DON mycotoxins, Porcine intestinal cells (IPEC-J2 cell line) were continuously pretreated or not pretreated, and the expression level of il8 mRNA was determined by quantitative PCR.

[0029] Figure 3: Micrographs of porcine intestinal cells (IPEC-J2 cell line), untreated (left), treated with DON mycotoxin alone (center), and treated with and DON mycotoxin treatment (right).

[0030] Figure 4 : Various “β-glucan products”, algal β-glucan (ALG), cyfluthrin, and il8 mRNA expression level in pretreated porcine intestinal cells (IPEC-J2 cell line) (P value*<0.05).

[0031] Figure 5 : Histological analysis of porcine jejunal explants (n=7). (A) Images of 5 μm sections taken by microscope from jejunal explants containing untreated jejunal explants, or treated with DON mycotoxin alone, Or use (B) The figure shows the histological scores, intestinal cell morphology and intestinal villus height of porcine jejunal explants according to the treatment.

[0032] Figure 6 : From untreated porcine jejunal explants, or treated with DON mycotoxin alone, Or use Expression levels of genes associated with inflammation determined from mRNA isolated from porcine jejunal explants treated with DON and DON mycotoxins (n=5-10). (p value*<0.05).

[0033] Figure 7 : Images of chicken livers treated with (right) and untreated (left) the DON mycotoxin.

[0034] Figure 8 : Graph showing the percentage of liver lesions observed in chickens at day 13 (A) and day 28 (B), depending on the presence or absence of DON mycotoxins and / or

[0035] Fig. 9 : Shows untreated chicken or chicken treated with DON mycotoxin alone, Figure 3. Graph of intestinal villus height in the jejunum of chickens 13 days after DON mycotoxin treatment.

[0036] Fig.10 : Untreated chicken or treated with DON mycotoxin alone or with Expression levels of inflammation-related i18 (A) and ifnγ (B) genes measured by jejunal mRNA in chickens treated with DON and DON mycotoxins. DETAILED DESCRIPTION OF THE INVENTION

[0038] As described above, the present invention relates to an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan for use in preventing the toxic effects, in particular the immunotoxic effects, of DON mycotoxins in a subject, and in preventing liver and / or intestinal damage caused by DON mycotoxins in a subject.

[0039] The β-glucan of the extract according to the present invention is obtained from Saccharomyces cerevisiae, more particularly from a strain of Saccharomyces cerevisiae. Many strains of Saccharomyces cerevisiae are known in the art. Due to its role in the manufacture of several foods (including bread and fermented beverages), it is widely used in the food industry. As used herein, the term "yeast strain" refers to a relatively homogeneous group of yeast cells obtained by culturing (or proliferating) a starting strain. Yeast strains are obtained from clones, and clones are groups of yeast cells obtained from single yeast cells. The cultivation of Saccharomyces cerevisiae strains can be carried out by any suitable method. Yeast culture methods are known in the prior art, and those skilled in the art know how to optimize the culture conditions for each strain according to its properties. Therefore, Saccharomyces cerevisiae can be obtained by proliferating strains in a suitable culture medium, for example, as described in the reference book "Yeast Technology", 2 nd edition, 1991, G. Reed and TW Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8.

[0040] The terms "yeast cell wall" and "yeast shell" are used interchangeably herein and refer to the insoluble portion of the yeast cell, i.e., the yeast cell wall and plasma membrane. Typically, the yeast cell wall is obtained by a process comprising an autolysis or enzymatic hydrolysis step, mainly by proteases, followed by a step of separating a soluble fraction and an insoluble fraction, wherein the separated insoluble fraction corresponds to the yeast cell wall. The insoluble fraction may then be dried. The method for obtaining the yeast cell wall is such that it retains the structural polysaccharides of the cell wall, i.e., β-glucans and mannans, which are in the form of mannoproteins. Methods for obtaining yeast cell walls are known in the art (see, e.g., reference "Yeast Technology", 2nd edition, 1991, G. Reed and TW Nagodawithana, published by Van Nostrand Reinhold, New York, ISBN 0-442-31892-8).

[0041] The extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan according to the present invention refers to a fraction that has been extracted (or separated) from the cell wall and mainly contains β-glucan. β-glucans derived from yeast cell walls are called "cell wall β-glucans". They are mainly glucose polymers whose main chain glucose units are connected by β-1,3 bonds and whose side chains are connected by β-1,6 bonds. Yeast β-glucans are insoluble and have low viscosity. Those skilled in the art know how to extract β-glucans from yeast cell walls. A commonly used method includes continuous hot extraction with an alkali and an acid (such as acetic acid), followed by washing with water to remove any soluble compounds from the cell wall, and recovering the insoluble material consisting of cell wall β-glucans.

[0042] The extract of the cell wall of Saccharomyces cerevisiae enriched in β-glucan according to the present invention comprises a total amount of β-glucan greater than or equal to 50% by weight, in the form of a mixture of β-1,3-glucan and β-1,6-glucan. As used herein, the term "a total amount of β-glucan greater than or equal to 50% by weight" means that β-glucan accounts for at least half of the weight of the extract according to the present invention. Therefore, the total amount of β-glucan in the extract according to the present invention may account for 50%-90% by weight of the extract, for example, 50%-80% by weight of the extract, or 50%-70% by weight, or even 50%-60% by weight. In certain specific embodiments, the total amount of β-glucan in the extract according to the present invention accounts for 50%-60% by weight of the extract, for example, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58% or about 59%.

[0043] The extract of the cell wall of Saccharomyces cerevisiae enriched in β-glucan according to the invention also contains a total amount of less than 5% by weight of mannan. Mannans from yeast cell walls are called "cell wall mannans". These are yeast polysaccharides consisting mainly of mannose, more precisely of copolymers of neutral or acidic sugars (having 5 or 6 carbon atoms) linked by glycosidic bonds and linked to proteins. In the cell wall mannans of Saccharomyces cerevisiae, mannose is present in the form of a main chain of mannose residues (50 or more), α-(1,6)-linked, branched by short chains of α-(1,2) and α-(1,3)-linked mannose. Those skilled in the art know how to extract mannans from yeast cell walls. Common methods include the use of preparations of β-glucanase (e.g., the industrial preparation GLUCANEX TM ) Enzymatic hydrolysis of yeast cell walls followed by separation of the hydrolysate by centrifugation and purification by ultrafiltration. Another method is based on thermochemical extraction.

[0044] As used herein, the term "less than 5% by weight of total mannan" means that the mannan accounts for at most 5% by weight of the extract according to the present invention. Therefore, the total amount of mannan in the extract according to the present invention may account for 0.5%-5% by weight of the extract. In certain specific embodiments, the total amount of mannan in the extract according to the present invention accounts for 1%-5% by weight of the extract, such as about 1%, about 2%, about 3%, about 4% or about 5%.

[0045] Preferably, the β-glucan-rich extract of the cell wall of Saccharomyces cerevisiae according to the present invention comprises β-glucan and mannan, wherein the β-glucan is present in a total amount greater than or equal to 50% by weight in the form of a mixture of β-1,3-glucan and β-1,6-glucan; and the total amount of mannan is such that the β-glucan / mannan ratio is 12-40 (wt / wt). In particular, the β-glucan / mannan ratio in the extract according to the present invention may be 15-30 (wt / wt), and more particularly 18-22 (wt / wt).

[0046] In certain embodiments, the β-glucan-enriched Saccharomyces cerevisiae cell wall extract according to the present invention further comprises a protein content of less than or equal to 10 wt % and a glycogen content of less than or equal to 10 wt %.

[0047] In certain embodiments, the β-glucan-rich extract of Saccharomyces cerevisiae cell walls according to the present invention comprises β-glucan and mannan as the only active ingredients, wherein the β-glucan is present in a total amount greater than or equal to 50% by weight in the form of a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount less than 5% by weight, such that the β-glucan / mannan ratio is 12-40 (wt / wt), particularly 15-30 (wt / wt), and more particularly 18-22 (wt / wt).

[0048] In certain specific embodiments, the extract of the cell wall of Saccharomyces cerevisiae according to the invention has a dry matter content greater than or equal to 94% by weight, preferably greater than or equal to 96% by weight. A dry matter content greater than or equal to 94% by weight, preferably 96% by weight, allows for better preservation of the yeast shell, in particular better bacteriological stability, and better stability against undesirable reactions (whether or not enzyme source).

[0049] In certain specific embodiments, the extract of Saccharomyces cerevisiae cell wall according to the present invention is in the form of powder.

[0050] In certain specific embodiments, the β-glucan-rich Saccharomyces cerevisiae cell wall extract according to the present invention is produced by the applicant Lesaffre Said The invention comprises β-glucan and mannan as the sole active ingredients, wherein the β-glucan is present in a total amount of 52-60 wt % in the form of a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount of 1-5 wt %. It also contains protein in an amount of less than or equal to 10% by weight and glycogen in an amount of less than or equal to 10% by weight.

[0051] After preparation, the extract of the cell wall of Saccharomyces cerevisiae according to the invention can be packaged and / or stored under suitable conditions before use, usually in a dry and cool place. The shelf life of the cell wall extract according to the invention in suitable packaging is 2 years from the date of production.

[0052] II- Compositions comprising a cell wall extract of Saccharomyces cerevisiae rich in β-glucan

[0053] In certain embodiments, the extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan described herein is used as such. In other embodiments, the extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan described herein is used in combination with at least one other bioactive agent. Therefore, the present invention relates to a composition comprising an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan as described herein and at least one bioactive agent.

[0054] The bioactive agent may be selected, for example, from agents that act on mycotoxins other than DON, since animal feed may be contaminated with more than one mycotoxin. Alternatively, the bioactive agent is known to have a beneficial effect on animal health. Thus, the bioactive agent may be a mycotoxin detoxifier, an antioxidant, a liver protectant, an immunoprotectant, or a combination of these agents.

[0055] As used herein, the term "mycotoxins other than DON" refers to toxins produced by various microscopic fungi, such as molds (Aspergillus sp., Fusarium sp., Stachybotrys sp., Penicillium sp., etc.), wherein the toxin is not deoxynivalenol. In practice, contaminated foods often contain a mixture of mycotoxins, since (1) fungi can produce several different mycotoxins simultaneously (e.g., Fusarium fungi that secrete DON are known to co-produce other mycotoxins, in particular zearalenone), (2) foods can be contaminated by several fungi (cereals are often affected by Aspergillus sp.), and (3) each raw material used in food may contain at least one fungus that produces at least one mycotoxin. Mycotoxins can be produced on many substrates: feed, cereals, oilseeds, protein crops, silage, secondary molasses, and mineral vitamin feeds. The main mycotoxins are aflatoxins, ochratoxin A, patulin, citrinin, Fusarium toxins (such as fumonisins, zearalenone, trichothecenes including T2 and HT2 toxins), Alternaria toxins, ergot alkaloids, aflatoxin, paxillin, enfusin and beauveria toxins.

[0056] The mycotoxin detoxifier in the composition according to the present invention may be selected from the group consisting of mycotoxin binders, mycotoxin bioconverters and combinations thereof.

[0057] The terms "mycotoxin binding agent" and "mycotoxin binder" are used interchangeably herein. They refer to agents that adsorb and / or inactivate mycotoxins other than DON, such as mycotoxins in animal feed or animal feed ingredients, thereby reversing the harmful effects of the mycotoxins. Mycotoxin binders reduce exposure to mycotoxins by reducing their bioavailability, resulting in reduced absorption of the mycotoxin by the animal and, therefore, reduced distribution to the blood and target organs. As used herein, the term "bioavailability" with respect to mycotoxins refers to the portion of the mycotoxin that is absorbed / absorbable or assimilable / assimilatable by an animal. Adsorption of the mycotoxin by a binder reduces the bioavailability of the mycotoxin, since the binder-mycotoxin complex passes through the digestive system of the animal and is excreted by the animal without assimilation.

[0058] Examples of mycotoxin binders include, but are not limited to, mycotoxin adsorbents selected from the group consisting of aluminosilicates (e.g., kaolinite, hydrated calcium / potassium / sodium aluminosilicates, hydrated sodium calcium aluminosilicates (HSCAS)), bentonites (e.g., sodium bentonite, calcium bentonite), montmorillonites (e.g., sodium montmorillonite and calcium montmorillonite), zeolites (e.g., clinoptilolite), activated carbon, micronized fibers and polymers (e.g., cholestyramine, polyvinylpyrrolidone), activated diatomaceous earth, plant fibers (e.g., wheat and alfalfa bran fibers), polysaccharides (e.g., glucomannan or its esterified forms), certain algae, certain bacteria (lactic acid bacteria), and combinations thereof.

[0059] The term "mycotoxin bioconverter" refers to an agent (usually an enzyme, bacteria, fungus) that inactivates or kills mycotoxins other than DON, such as mycotoxins in animal feed or animal feed ingredients, thereby reversing the harmful effects of the mycotoxins.

[0060] Examples of mycotoxin bioconverters include enzymes that degrade mycotoxins other than DON, for example selected from the group consisting of esterases, lipases, proteases, oxidases, cellulases, cyclooxygenases, dehydrogenases, hemicellulases (or xylanases), catalases, peroxidases, laccases, xylanases, carboxylesterases, amino / acetyltransferases, pancreatins, lactonases, lactone hydrolases, and combinations thereof.

[0061] Other examples of mycotoxin bioconverters include microorganisms known to be harmful to mycotoxins, such as selected from: Eubacterium sp. BBSH797 (Coriobacteriaceae), Nocardia asteroides, Mycobacterium Fluoranthenivorans sp., Rhodococcus erythropolis, Bacillus, Lactobacillus, Achromobacter thermophilus C5 and NG40Z, Lactobacillus paraplantarum, Stenotrophomonas maltophila, Saccharomyces cerevisiae, Exophiala spinifera, Cupriavidus baselense. basilensis) OR16 Aspergillus niger, Eurotiumher bariorum, Rhizopus, Trichosporon mycotoxinivorans, Phaffiarhodozymyllces, Phaffia rhodozymyllces, Phaffia rhodozymousces, Nocardia corynebacterioides NRRL 24037, Mycobacterium fluoranthenivorans or Myxococcus fulvus ANSM068, Rhodococcus erythropolis, Brevibacterium sp., Slackia sp. DG6, Deviosa mutans, Deviosa insulae A16, Solanum tuberosum), Aspergillus oryzae, Eggerthella sp. DII-9, Pseudomonas sp. Y1, Lysobacter sp.)S1, Sphingomonas, Nocardioides, Citrobacter, Marmaricolasp. MIM116 and combinations thereof.

[0062] In certain embodiments, the bioactive agent in the composition according to the present invention is a known antioxidant that can reduce the toxicity of mycotoxins other than DON in animals. Examples of such bioactive agents include, but are not limited to, rutin, quercetin, lutein, lecithin, melatonin, mannitol, curcumin, turmeric, lycopene, allyl sulfide, fructose, chlorophyll and its derivatives, sodium thiosulfate, glutathione, methionine, aspartame, trace elements (selenium, zinc, magnesium), catechins (epigallocatechin gallate, epicatechin gallate), morin, kaempferol, fisetin, naringin, vitamins (vitamin E, C, A and B), coenzyme Q10, provitamins (carotenes and carotenoids), eugenol, vanillin, caffeic acid, cholinergic acid and combinations thereof.

[0063] In certain embodiments, the bioactive agent is a known liver protectant or immunoprotectant active in animals. The terms "liver protectant" and "liver protectant" are used interchangeably herein. They refer to agents that improve the integrity and regeneration of liver cells, optimize the detoxification ability of the liver and / or promote liver synthesis by stimulating the activity of digestive enzymes, which increase their intestinal absorption and thus increase their bioavailability, thereby ensuring the best use of nutrients. Examples of liver protectants include, but are not limited to, liver protectants of natural origin composed of a combination of various plants with different liver protection properties, such as Phyllanthus niruri, Azadirachtaindica, Andrographis paniculata, Achyrantes aspera, etc., and liver protectants that provide methyl groups based on the ability of methyl groups to bind to toxins, thereby promoting their removal from the body. Among the compounds that can provide methyl groups, we distinguish certain amino acids and their derivatives (e.g., methionine, carnitine, betaine, etc.), vitamin derivatives (e.g., choline). The term "immunoprotectant" refers to an agent that prevents the action of an antigen regardless of its mechanism of action. Examples of immunoprotectants include, but are not limited to, envelope proteins derived from animal viruses; oligonucleotides, such as CpG oligonucleotides. Immunoprotectants can also be chemical immunoprotectants, including, but not limited to, cytokines, chemokines and lymphokines, including, but not limited to, interferon α, interferon γ and interleukin 12, or plant-derived immunoprotectants, such as plants: Eclipta alba, Aloe vera, Ocimum sanctum, Viscumalbum, Urtica dioica and Zingiber officinale, Solanum trilobatum, Astragalus radix and Scutellaria radix, and Achyranthes aspera.

[0064] In the composition according to the present invention, the bioactive agent is usually present in an amount that can achieve the desired goal (e.g., reduce the bioavailability of mycotoxins and / or inactivate mycotoxins). Those skilled in the art know how to determine such an amount. For example, the enzyme that degrades mycotoxins outside DON can be less than or equal to 5% by weight relative to the total weight of the composition, preferably less than 1% by weight relative to the total weight of the composition, more preferably 0.01% by weight-0.5% by weight, more preferably 0.15% by weight-0.25% by weight. One or more bioactive agents in conjunction with mycotoxins other than DON can be present, for example, in an amount of up to about 90-95% by weight relative to the total weight of the composition, for example, about 80% by weight relative to the total weight of the composition, for example, about 70% by weight, about 60% by weight, about 50% by weight, about 45% by weight, about 40% by weight, about 35% by weight, about 30% by weight, about 25% by weight, about 20% by weight, about 15% by weight, about 10% by weight, or less than 10% by weight relative to the total weight of the composition.

[0065] In certain embodiments, the composition according to the present invention may also contain components commonly used in supplements, tonics or feed additives for animals, such as vitamins, minerals, trace elements, etc. Those skilled in the art know how to select appropriate components according to the animals for which the composition is used, and know how to determine the appropriate amounts to be included in such a composition.

[0066] The composition according to the invention may be in any form, such as in the form of a powder, granules, gel or liquid. Preferably, the composition according to the invention is in solid form, preferably in powder form.

[0067] The present invention also relates to a pharmaceutical composition comprising an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan as described herein and at least one physiologically acceptable excipient in veterinary medicine. In the pharmaceutical composition, the extract may be present as the only active ingredient. Alternatively, the pharmaceutical composition may further comprise at least one other bioactive agent, such as the bioactive agents listed above.

[0068] The pharmaceutical composition according to the invention can be classified as a veterinary pharmaceutical preparation, available by prescription or over the counter.

[0069] In the context of the present invention, the term "physiologically acceptable excipient in veterinary medicine" refers to any medium or additive that does not interfere with the effectiveness of the biological activity of the active ingredient (here, yeast cell wall extract enriched in β-glucan) and is not overly toxic to animals at the administered concentration.

[0070] The veterinary composition according to the invention can be administered using any combination of dosages and routes of administration effective to achieve the desired prophylactic effect. The skilled person will know that the exact amount to be administered may vary depending on the animal species and will know the effects of the DON mycotoxin on the animal species to be treated. III - Use of the extract of the cell wall of Saccharomyces cerevisiae enriched in β-glucan

[0071] As mentioned above, DON has negative effects on the intestinal tract and immune responses. This mycotoxin induces intestinal histological damage, including necrosis of the intestinal epithelium. DON also disrupts the intestinal barrier function, which may lead to increased transport of pathogens and greater susceptibility to enteric infectious diseases. DON also modulates the immune reactivity of the intestinal mucosa, can interact in the association of epithelial cells and intestinal immune cells, and represents a predisposing factor for inflammatory diseases.

[0072] It has been shown that the β-glucan-rich Saccharomyces cerevisiae cell wall extract described herein does not adsorb DON mycotoxin, is able to significantly improve the transepithelial impedance of the epithelial barrier of porcine intestinal cells exposed to DON mycotoxin; causes a decrease in the expression of the DON-induced il8 gene (a pro-inflammatory cytokine) in porcine intestinal cells in vitro, in porcine explants ex vivo, and in chicken intestinal tissue in vivo; prevents these cells from undergoing the structural disruption observed in untreated cells; prevents the reduction in intestinal villus size and intestinal cell morphological damage in porcine intestinal explants; and effectively prevents liver damage and DON mycotoxin-induced villus size reduction in chickens.

[0073] Therefore, the present invention also relates to an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan as described herein, alone or in combination with at least one biologically active agent, for use in preventing mycotoxicosis caused by the DON mycotoxin in a subject. The present invention also relates to an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan as described herein, alone or in combination with at least one biologically active agent, for use in preventing the toxic effects, in particular the immunotoxic effects, of the DON mycotoxin in a subject. The present invention also relates to an extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan as described herein, alone or in combination with at least one biologically active agent, for use in preventing liver and / or intestinal damage induced by the DON mycotoxin in a subject.

[0074] The term "mycotoxicosis" refers to a condition that occurs as a result of ingesting mycotoxins from food contaminated with said mycotoxins produced by microscopic fungi. Thus, mycotoxicosis caused by DON mycotoxins is a condition that occurs as a result of ingesting DON mycotoxins. As used herein, the term "preventing mycotoxicosis caused by DON mycotoxins in a subject" means partially or completely reducing or inhibiting the likelihood that a subject will develop one or more symptoms associated with ingesting DON (depending on the subject's growth reduction, immunosuppression, vomiting, diarrhea, intestinal mucosal damage, food refusal, liver damage, abdominal pain, etc.).

[0075] As used herein, the term "immunotoxic effects of DON mycotoxins" refers to the deleterious effects induced on the immune system by the ingestion of DON. There may be different types of immunotoxic effects, including immunosuppression, immunostimulation, hypersensitivity, and autoimmunity, which can promote infections and tumors. When animals are exposed to low doses of DON, certain components of the immune system are stimulated. In contrast, when animals are exposed to higher doses of DON, immunosuppressive reactions are generally observed. The term "preventing the immunotoxic effects of DON mycotoxins" refers to partially or completely reducing or inhibiting the likelihood of a subject developing an immunotoxic effect after ingesting DON.

[0076] As used herein, the term "liver and / or intestinal damage" refers to any pathological changes in the tissues or cells of the liver and / or intestines, which are then in an abnormal state. Such damage can lead to liver and / or intestinal dysfunction.

[0077] As used herein, the term "subject" refers to a human or an animal. As used herein, the term "animal" refers to an organism of the animal kingdom. In the context of the present invention, the term more specifically refers to livestock (such as cattle (cows, buffaloes, shoulder cattle, bison, European bison, yaks), Ovis (sheep), Ovis subfamily (goats), swine (pigs), Equine (horses, donkeys, mules), Camelidae (camels, dromedaries, llamas, alpacas) and Cervidae (reindeer, deer)) and other livestock (chickens, turkeys, ducks, geese, pigeons, quail, pheasants, partridges, ostriches, emus, rheas, ratites, guinea fowls, rabbits, guinea pigs); aquatic animals (marine, pond or freshwater fish farming, farmed shellfish, farmed crustaceans and farmed mollusks); pets (cats, dogs, rabbits, horses, etc.); and experimental animals. As used herein, the term animal is not a specific age.

[0078] In certain specific embodiments, the animals used for the Saccharomyces cerevisiae cell wall extract described herein are selected from pets and farm animals, in particular livestock (pigs, cattle, sheep, goats) and poultry (chickens, turkeys, roosters, guinea fowls, quails, ducks, geese).

[0079] Animals do not have the same sensitivity to DON mycotoxins. The difference in sensitivity is notably due to the differences in the absorption mechanism, metabolism and distribution of DON and elimination. Pigs are known to be very sensitive to DON because their diet is rich in grains, and also because mycotoxins are quickly and effectively absorbed before being distributed to organs, and only weakly metabolized to detoxify. Poultry are less sensitive to DON than pigs. The first zootechnical effect was observed from a concentration of 5 mg DON per kilogram of feed. However, acute poisoning leads to animal death 3.5 hours-13.5 hours after ingesting contaminated feed. For ruminants, the contamination of DON may involve grain concentrates or corn silage containing DON. Other feeds (hay, forage) may also be contaminated, but the growth of Fusarium and the production of DON after harvest are considered negligible. Ruminants seem to be almost insensitive to DON up to 12 mg / kg in feed.

[0080] In certain embodiments of the present invention, the use of a cell wall extract of Saccharomyces cerevisiae rich in β-glucan alone or in combination with at least one bioactive agent for preventing mycotoxicosis caused by DON mycotoxins in a subject and / or preventing the immunotoxic effects of DON mycotoxins in a subject and / or preventing liver and / or intestinal damage induced by DON mycotoxins in a subject comprises mixing the cell wall extract of Saccharomyces cerevisiae rich in β-glucan with animal feed. The mixing can be performed by any suitable method known in the art.

[0081] As used herein, the terms "animal feed," "animal feed ingredients," and "animal feed products" refer to any natural or processed organic material that is susceptible to biodegradation and can be consumed by animals. Examples of such organic materials range from newly harvested grains to pelleted feeds, and include, for example, any compound, grain, nut, feed, silage, preparation, composition, or mixture suitable for or intended for ingestion by animals. The term "feed" refers to plant material (primarily leaves and stems of plants) for ruminants. The term "silage" refers to a fermented feed with a high moisture content that can be used as a ruminant feed. Preparations, compositions, and mixtures, such as commercial preparations, compositions, and mixtures, can be in any suitable form, such as concentrates, premixes, supplements, total mixed rations (TMRs), and the like. Animal feed can contain grains (e.g., corn, wheat, barley, rye, rice, sorghum, millet, or any combination thereof), feed, silage, legumes (e.g., soybeans), spent grains, animal products, and the like. Spent grains come primarily from industrial processes such as brewing and distilling for the production of drinkable alcohol (whisky, vodka or gin) or biofuels. Brewers' grains come from malt, which itself comes from barley. Cereals other than barley, such as sorghum, are sometimes used for brewing, especially in Africa. Spent grains from bioethanol production are made from corn or wheat. Wet spent grains are dried to produce dry grains that are primarily used as animal feed.

[0082] In certain embodiments, it has been determined that the animal feed supplemented with the β-glucan-enriched Saccharomyces cerevisiae cell wall extract as described herein is capable of producing DON mycotoxin or is contaminated with molds containing DON mycotoxin. In other embodiments, it has been determined that the animal feed is susceptible to contamination with molds capable of producing DON mycotoxin or is susceptible to contamination with molds containing DON mycotoxin. In other embodiments, the potential contamination of animal feed with molds capable of producing DON mycotoxin or DON mycotoxin is still unknown.

[0083] The cell wall extract of Saccharomyces cerevisiae rich in β-glucan as described herein, alone or in combination with at least one biologically active agent, can be added to animal feed in an amount of about 0.003% to about 0.5% of the total weight of the feed (which corresponds to an amount of about 0.03 kg to about 5 kg per ton of feed), subject to current regulations. For example, in some embodiments, the cell wall extract of Saccharomyces cerevisiae rich in β-glucan as described herein, alone or in combination with at least one biologically active agent, is added to animal feed in an amount of about 0.003% to 0.5% of the total weight of the feed (which corresponds to an amount of about 0.03 kg to about 5 kg per ton of feed), for example, in an amount of about 0.005% to about 0.05% by weight of the feed (which corresponds to an amount of about 0.05 kg to about 0.5 kg per ton of feed).

[0084] In the above, the focus has been on the use of beta-glucan-rich Saccharomyces cerevisiae cell wall extracts in the field of animal health. However, it is also planned to expand the use of these extracts to human health, since deoxynivalenol is the cause of severe mycotoxicosis in humans.

[0085] Unless otherwise defined, all technical and scientific terms used in the specification have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Likewise, all publications, patent applications, patents and other references mentioned herein are incorporated herein by reference. DETAILED DESCRIPTION

[0086] The following examples describe certain embodiments of the present invention. However, it should be understood that the examples and drawings are given for illustrative purposes only and are not intended to limit the scope of the present invention.

[0087] In all the following examples, the term "CTL" refers to "control".

[0088] Embodiment 1: In vitro evaluation

[0089] In vitro studies were performed on the porcine intestinal cell line IPEC-J2 to investigate the effects on cell membrane permeability and expression of different genes. Due to their diet rich in grains, pigs are exposed to and sensitive to DON mycotoxins to a certain extent. In addition, pigs are a good model for humans, especially in terms of digestion and immunity.

[0090] Materials and methods

[0091] Various "β-glucan products" were used in this study: It is manufactured by the applicant Lesaffre; seaweed β-glucan manufactured by Lesaffre and The characteristics of these "β-glucan products" are shown in Table 1 below.

[0092] Table 1. Characteristics of the investigated “β-glucan products”.

[0093]

[0094] IPEC-J2 cells were thawed in the culture medium DMEM / F12+5% SVF+16mM Hepes+1X ITS+5ng / ml EGF and maintained in culture for 2 passages (1 week). At passage 3, cells were cultured in the presence of porcine serum (Gibco) and maintained for 2 passages (1 week).

[0095] The "β-glucan product" was added to IPEC-J2 cells at a final concentration of 50 μg / mL and maintained at this concentration at each passage. After 15 days of culture in the presence of the "β-glucan product", various tests were performed: TEER (transepithelial / endothelial electrical impedance) test and mRNA extraction for quantification of IL8 by quantitative PCR.

[0096] DON mycotoxin is prepared as follows by freezing and paraffin film stock solution (30mM DON in acetonitrile solution) every 2-3 weeks. DON solution was removed from the freezer 2 minutes before sampling and vortexing. Confirm that there is no crystal formation. Take out 30 μL solution and add to 270 μL sterile water to obtain a storage solution of 3mM water / acetonitrile mixture (10 / 90, v / v). The storage solution is diluted one-third in water / acetonitrile mixture (10 / 90, v / v) to obtain 1mM solution. Then these two solutions are diluted 1 / 100 in the holes of the 96 microplates for the TEER test and the quantitative PCR test to a final concentration of 10 and 30 μM.

[0097] TEER Assay Protocol. For this assay, a Falcon 24-well insert was used. The wells were pre-wetted by placing 800 μL of culture medium on the basolateral side, wetting the membrane with 100 μL of culture medium, and incubating at 37 °C for 15 min. Prepare a 0.5 × 10 5 10 cells / mL cell suspension and 300 μL of each of these suspensions was placed in each pre-wetted insert. After three days, the medium was changed in each insert. The inserts were placed The next day, add DON mycotoxin and read the TEER for at least 24 hours.

[0098] RNA for quantitative PCR was prepared containing 0.5 × 10 5 Cell suspensions of 100 cells / mL cultured with or without continuous β-glucan products were prepared and 200 μL of each of these suspensions were deposited in each well of a 96-well microplate (i.e., 10,000 cells / well). The next morning, DON mycotoxin was added and incubated for 6 hours. The culture medium was then removed by aspiration, and 130 μL RA1+5 μL TCEP was added to each well to lyse the cells. The plates were stored at -20°C until analyzed with Macherey Nagel. TM Nucleospin TM The RNA was extracted using 96 RNA kit on the same day.

[0099] RNA extraction and reverse transcription were performed according to Macherey-Nagel TM NucleoSpin TMRNA extraction was performed by centrifugation according to the instructions provided in the 96RNA kit package insert (reference 740709.4). Elution was performed in 30 μL of RNase-free water. TM High Capacity cDNA Reverse Transcription Kit (Ref. 4368814) reverse transcribes 10 μL RNA into complementary DNA.

[0100] In the absence of RNase inhibitors, the reaction contained: 2 μL 10X RT buffer, 0.8 μL 25X dNTP mix (100 mM), 2 μL 10X RT random primers, 1 μL polyclonal reverse transcriptase, and 4.2 μL nuclease-free water.

[0101] The thermal cycling program used was as follows: 10 min at 25°C, 2 h at 37°C, 5 min at 85°C, and hold at 4°C.

[0102] Quantitative PCR. PCR was performed on a QuantStudio 3 apparatus (Applied Biosystems). Master mix (Applied Biosystems TM Taqman TM Fast Advanced Master Mix, ref.4444964), Taqman TM Primers and probes (Taqman Gene Expression Assay, HPRT pig, Ss03388274-m1-VIC-MGB-PL; Taqman Gene Expression Assay, IL8 pig, Ss03392437_m1-FAM-MGB) were ordered from ThermoFisher. The PCR performed was a relative quantitative PCR that compared the expression of the i18 gene in the presence and absence of the "β-glucan product". The results obtained are expressed as 2 -δCT ΔCt is the Ct difference between the target gene (i18) and its stable housekeeping gene (HPRT).

[0103] result

[0104] The results of in vitro studies such as Figure 1-4 shown.

[0105] Added DON mycotoxin Continuous pretreatment of the porcine intestinal cell line IPEC-J2. Figure 1 The results of the TEER assay shown indicate transepithelial resistance of the epithelial barrier of IPEC-J2 and show that the use of cytokines Pretreated cells were less affected by the DON mycotoxin than unpretreated cells.

[0106] Figure 2 The results of quantitative PCR for the expression of il8, a pro-inflammatory gene expressed by epithelial cells when under threat, are shown. Continuous pretreatment of IPEC-J2 cells resulted in a reduction in IL-8 production induced by DON mycotoxin in IPEC-J2.

[0107] Micrographs (shown in Figure 3 (middle) shows that in the presence of DON mycotoxin, The treated IPEC-J2 cells showed less structural damage than those treated with DON mycotoxin alone.

[0108] Then before adding DON mycotoxins, Other "β-glucan products" with different sources and / or β-glucan / mannan ratios were used to sequentially pre-treat porcine intestinal line IPEC-J2 cells. The expression of interleukin IL-8 by IPEC-J2 cells was determined. Figure 4 The results shown show that the The pretreated cells were less affected by DON than cells pretreated with other "β-glucan products".

[0109] Embodiment 2: In vitro adsorption of mycotoxin DON

[0110] To understand In vitro experiments were performed to determine how The ability to adsorb DON mycotoxin was measured at two different pH values ​​(3 and 7) and for two different batches of (Batch 1 and Batch 2) were incubated. The results obtained are shown in Table 2 below.

[0111] Table 2. Absorbs DON mycotoxins.

[0112]

[0113] These results make it possible to exclude the possibility that the The mechanism of action of adsorption of DON mycotoxins. The product adsorbs mycotoxins less than 10%.

[0114] Embodiment 3: In vitro assessment of effects

[0115] The experiments described in this example were performed by the team of Dr. Isabelle Oswald, whose address is as follows: INRAE-NATIONAL RESEARCH INSTITUTE for AGRICULTURE, FOOD and the ENVIRONMENT, Food Toxicology Unit UMR 1331 ToxAlim-180 chemin de Tournefeuille-BP93173-31027 Toulouse Cedex03.

[0116] In vitro studies were performed using porcine intestinal explants to determine the effects on intestinal tissue and the effects of different gene expression. Explant culture is an alternative to cell culture and animals as a whole. This allows testing of contaminants / additives on whole organ samples containing the different cell types that make it up. This also allows for repeated experiments and limits the variability of observed responses (each animal has its own control).

[0117] Materials and methods

[0118] Experimental protocol. Animals were maintained under housing and environmental conditions that best represent those of farm animals. -1 of Jejunal explants were exposed to DON mycotoxins in the absence of DON. Jejunal segments were removed from 6-12 4-week-old pigs and quickly placed in a wash medium (cold Williams E medium + antibiotics: penicillin, streptomycin and gentamicin). Explants were prepared using a biopsy punch with a diameter of 8 mm for histological analysis and a biopsy punch with a diameter of 6 mm for qPCR and Western blot analysis, taking care to identify the mucosal and muscle directions. Explants were then placed in a 6-well plate on a sponge containing complete medium (Williams E medium + glucose (25 g / L), 1% ITS, 1% Ala-Glu (3 mol / L), 1% penicillin / streptomycin and 0.5% gentamicin) on a shaking plate at 39°C (temperature can be adjusted according to the analysis to be performed) for 4 hours (time can be adjusted according to the analysis to be performed).

[0119] Jejunal explants were exposed to or not exposed to DON (10 μM) and treated with or without 10 mg / L Co-treatment. The dose of 10 μM in DON was chosen because it corresponds to a concentration of 3 mg / L (3 ppm), close to the concentration that can be present in animal feed. In fact, the maximum level of deoxynivalenol recommended in Europe for complete feeds is 5 ppm for complete feeds, but the dose of deoxynivalenol in the feed of these pigs was 0.9 ppm.

[0120] After processing is complete, collect samples and store as follows:

[0121] - For histological analysis: biological samples were transferred into cassettes and tissues were fixed in 4% formalin and then transferred to 70% alcohol solution and then embedded in paraffin. The measurement of crypt depth and villus length was also performed using the image analysis software NIS-Elements AR (Nikon System-Elements Advanced Research).

[0122] -For quantitative PCR and Western Blot analysis: transfer biological samples into Eppendorf tubes and store at -80 °C.

[0123] Samples were generated from two sets of explants prepared from two independent experiments.

[0124] result

[0125] The results of in vitro studies are as follows Figure 5 and 6 shown.

[0126] 50 μm sections were prepared from paraffin blocks containing treated jejunal explants. They were then stained with hematoxylin-eosin (H&E) for histopathological analysis, taking into account different types of injury. The results obtained are shown in Figure 5 These results suggest that DON increases the amount of tissue damage, while The invention allows to reduce the damage to the villi of the explants co-exposed to DON. The damage to the intestinal epithelial cells is characterized by the observation of flattening of the jejunal epithelial cells in the explants co-exposed to DON. A significant decrease of about 35% in villus length was observed in jejunal explants treated with DON (10 mM). Treatment of explants tended to reduce the effect of DON on villus length.

[0127] RNA was then extracted from the explants and used to study the expression of different genes, particularly those associated with inflammation and cytokine production. The results obtained are shown in Figure 6 These results show that exposure of explants to 10 μM DON resulted in increased production of gene transcripts associated with inflammation. The presence of significantly downregulated the expression of il1b and il8 gene transcripts. A downward trend was also observed for il1α.

[0128] Embodiment 4: In vivo evaluation of the effect

[0129] The experiments described in this example were performed by Regiane Santos of Schothorst Feed Research, Meerkoetenweg, 26 8218 NA Lelystad, The Netherlands.

[0130] In vivo studies were conducted in broiler chickens exposed to feed naturally contaminated with the mycotoxin DON.

[0131] Materials and methods

[0132] The experiment lasted 28 days, with an initial start-up period of 0-13 days and a growth period of 13-28 days. The experiment included 3 treatments according to a "randomized complete block design" experimental design, each with replications, each replication including 24 cages, and 13 animals per cage.

[0133] Table 3. Dietary treatments.

[0134]

[0135]

[0136] On the 13th and 28th day, 3 animals were randomly selected per cage and per treatment group (i.e., 18 animals per treatment) and euthanized. The liver and jejunum were removed. The liver was visually observed, and pale color and lesions, such as cysts or hemorrhagic areas, were counted and expressed as a percentage relative to the liver that did not show lesions. Using the jejunum, the height of the villi and the depth of the crypts were measured for at least 5 villi per animal, per treatment, and per cage.

[0137] result

[0138] The results of in vivo studies such as Figure 7 and 8 shown.

[0139] Figure 7 Lesions in a chicken liver treated with the DON mycotoxin compared to an untreated liver are shown (left). The liver exposed to the DON mycotoxin appears whiter with a yellowish tint; hemorrhagic lesions are clearly visible.

[0140] Figure 8 The indications depend on the presence of DON mycotoxins and / or The graphs of the percentage of liver damage in chickens were observed on days 13 and 28. It can be clearly seen that the presence of DON in the feed induced a significant increase in liver damage, while The presence of makes it possible to prevent damage induced by the DON mycotoxin.

[0141] Fig. 9 Is indicated depending on the presence of DON mycotoxins and / or Figure 13 shows the height of intestinal villi in the jejunum of chickens. It can be seen that the presence of DON in the diet induced a decrease in the size of the intestinal villi in the jejunum, while the presence of DON in the diet The presence of makes it possible to prevent this villus size reduction.

[0142] Fig.10 The indication depends on the presence of DON mycotoxins in the diet and / or Figure 2 shows the expression of inflammatory genes in the jejunum of chickens. It can be seen that the presence of DON in the diet induced a significant increase in il8 and interferon gamma (ifnγ) mRNA, while The presence of α-glucose prevents the increased expression of these two inflammation-related genes.

Claims

1. An extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan for use in preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that The extract comprises β-glucan and mannan, wherein the β-glucan is present in a total amount of greater than or equal to 50% by weight as a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount of less than 5% by weight.

2. An extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan for use in preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, characterized in that The extract comprises β-glucan and mannan, wherein the β-glucan is present in a total amount of greater than or equal to 50% by weight as a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount of less than 5% by weight.

3. An extract of the cell wall of Saccharomyces cerevisiae rich in β-glucan for use in preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that The extract comprises β-glucan and mannan, wherein the β-glucan is present in a total amount of greater than or equal to 50% by weight as a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount of less than 5% by weight.

4. The extract of β-glucan-rich cell wall of Saccharomyces cerevisiae for use thereof according to any one of claims 1 to 3, characterized in that The mannan is present in an amount such that the β-glucan / mannan ratio is 12-40 (wt / wt).

5. The extract of β-glucan-rich cell wall of Saccharomyces cerevisiae for use according to any one of claims 1 to 4, characterized in that The extract has a dry matter content greater than or equal to 94% by weight.

6. The extract of β-glucan-rich cell wall of Saccharomyces cerevisiae for use according to any one of claims 1 to 5, characterized in that: The extract also contains protein in an amount less than or equal to 10% by weight and glycogen in an amount less than or equal to 10% by weight.

7. The extract of β-glucan-rich cell wall of Saccharomyces cerevisiae for use thereof according to any one of claims 1 to 6, characterized in that The β-glucan and mannan are the only active components of the extract.

8. The β-glucan-rich extract of Saccharomyces cerevisiae cell wall for use according to claim 7, characterized in that: The extract is It comprises β-glucan and mannan, wherein the β-glucan is present in a total amount of 52-60% by weight in the form of a mixture of β-1,3-glucan and β-1,6-glucan; and the mannan is present in a total amount of 1-5% by weight, and wherein the It also contains protein in an amount of less than or equal to 10% by weight and glycogen in an amount of less than or equal to 10% by weight.

9. A composition comprising an extract of Saccharomyces cerevisiae cell walls rich in β-glucan and at least one bioactive agent for preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, or for preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, or for preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that The extract is as defined in any one of claims 1-8.

10. A pharmaceutical composition comprising an extract of β-glucan-rich cell walls of Saccharomyces cerevisiae and at least one physiologically acceptable excipient for preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, or for preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, or for preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that The extract is as defined in any one of claims 1-8.

11. The extract for use according to any one of claims 1 to 8, the composition for use according to claim 9, or the pharmaceutical composition for use according to claim 10, characterized in that: The subject is an animal, in particular an animal selected from livestock and pets.

12. Animal feed comprising an extract of cell walls of Saccharomyces cerevisiae rich in β-glucan for preventing mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in animals, or for preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in animals, or for preventing liver and / or intestinal damage caused by the mycotoxin deoxynivalenol (DON) in animals, characterized in that The extract is as defined in any one of claims 1 to 8, or the extract is part of a composition as defined in claim 9.

13. The composition for use according to claim 9 or claim 12, characterized in that The bioactive agent is selected from the group consisting of mycotoxin binders, mycotoxin biotransformers, vitamins, minerals, trace elements, liver protectants, immune protectants, and combinations thereof.

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