Extract of Saccharomyces Cerevisiae Yeast Cell Walls Rich in β-Glucans in the Prevention of the Toxic Effects of the Mycotoxin Deoxynivalenol (DON)

Abstract

Extract of Saccharomyces Cerevisiae Yeast Cell Walls Rich in β-Glucans for the Prevention of the Effects of the Mycotoxin Deoxynivalenol (DON) The present invention relates to extracts of Saccharomyces cerevisiae yeast cell walls rich in β-glucans, and their use in the prevention of mycotoxicoses caused by the mycotoxin deoxynivalenol, in particular in the prevention of liver and intestinal damage, and the immunotoxic effects caused by this mycotoxin. (no figure)

Description

Title of the invention: Extract of Saccharomyces Cerevisiae Yeast Cell Walls Rich in p-Glucans for the Prevention of the Toxic Effects of the Mycotoxin Deoxynivalenol (DON) Technical field

[0001] The present invention relates to the field of health, in particular animal health. The invention relates more particularly to cell wall extracts of Saccharomyces cerevisiae yeast rich in [3-glucans, and their use in the prevention of toxic effects, in particular immunotoxic effects, of the mycotoxin deoxynivalenol (DON) and in the prevention of liver and intestinal lesions induced by the mycotoxin DON in animals. Previous technique

[0002] Mycotoxins are toxic fungal secondary metabolites produced by various molds belonging primarily to the Aspergillus, Penicillium, or Fusarium species. They are natural contaminants of cereals, as well as fruits, vegetables, nuts and oilseeds, and spices. Contamination significantly affects the production and marketing of cereals worldwide and also impairs the quality of products derived from contaminated grains. Furthermore, the presence of mycotoxins in food can have significant effects on human and animal health, ranging from gastrointestinal and kidney disorders to immune deficiency or cancer.

[0003] Deoxynivalenol (DON) is a mycotoxin of the trichothecene family (class B) produced by various fungal contaminants of the genus Fusarium, such as F. culmorum, F. graminearum, and F. pseudograminearum. It is a mycotoxin that develops on the plant in the field and is found particularly in cereal crops such as maize, wheat, barley, and oats, but also on rice, rye, and sorghum. Because trichothecenes are heat-stable and resistant to sterilization, they can be found in derived products such as animal feed, but also in flour, bread, pasta, and even beer. In animals, ingestion of DON-contaminated feed is associated with alterations of the immune system and intestinal mucosa, refusal to feed, and vomiting or diarrhea, which can result in stunted growth.In humans, DON food poisoning causes abdominal pain, dizziness, headaches, throat irritation, nausea, vomiting, diarrhea, and bloody stools. DON has recently been shown to increase the risk of chronic inflammatory bowel disease. the intestine (IBD) and exacerbates its symptoms (Payros et al., Archives of Toxicology (2020), DOI: 10.1007 / s00204-020-02817-z).

[0004] To prevent the risk of contamination by mycotoxins, including deoxy-nivalenol, the authorities in charge of food safety (FDA, EFSA, CSAH, JECFA, etc...) have issued regulations on maximum tolerable concentrations in foodstuffs intended for human and animal consumption.

[0005] Since post-harvest control and mitigation of DON mycotoxin contamination in cereals are currently unfeasible, the best way to control DON is through prevention in the field. DON is produced by various Fusarium species that develop in cereals under very specific temperature and humidity conditions. Stopping fungal growth by applying appropriate fungicides during conditions favorable to Fusarium development prevents the secretion of DON and other mycotoxins. One approach to reducing animal exposure to mycotoxins is to reduce their bioavailability by including detoxifying agents in animal feed. However, in the case of DON mycotoxin, there are few detoxification solutions, and those that exist are not very effective.Biotransformation solutions have been described, such as microorganisms or enzymes that degrade DON into less toxic metabolite(s), but only one microorganism (Coriobacteriaceae BBSH797) is currently commercially available. Regarding detoxification by adsorption, very few products are capable of adsorbing DON.

[0006] There is therefore still a need in art for new strategies to prevent the toxic effects caused by the ingestion of the DON mycotoxin. Summary of the invention

[0007] The present Inventors have found, surprisingly, that Saccharomyces cerevisiae yeast cell wall extracts rich in [3-glucans] significantly improve the transepithelial resistance of the epithelial barrier of porcine intestinal cells exposed to the mycotoxin DON; cause a decrease in the expression of the IL8 gene (a pro-inflammatory cytokine) induced by DON in porcine intestinal cells in vitro, in porcine expiants ex vivo and in chicken intestinal tissue in vivo; prevent these cells from the breakdown observed in untreated cells; prevent the reduction in intestinal villus size and the alteration of enterocyte morphology in porcine intestinal expiants; and effectively prevent liver damage as well as the reduction in villus size induced by the mycotoxin DON in chicken.

[0008] Therefore, the present invention relates to a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans] for use in the prevention of mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises [3-glucans and mannans, where the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount less than 5% by weight.

[0009] The present invention also relates to a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans for use in preventing the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises [3-glucans and mannans, where the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount less than 5% by weight.

[0010] The present invention also relates to a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans for use in the prevention of liver and / or intestinal damage induced by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises [3-glucans and mannans, where the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount less than 5% by weight.

[0011] In some embodiments, the total amount of [3-glucans present in a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] is between 50% and 90% of the weight of the extract, for example between 50% and 80%, or between 50% and 70%, or between 50% and 60%, of the weight of the extract.

[0012] In some embodiments, the total amount of mannans present in a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] is between 1% and 5% of the weight of the extract.

[0013] In certain embodiments, the amount of mannans in the Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] is such that the [3-glucan / mannan] ratio is between 12 and 40 (weight / weight). In particular, the [3-glucan / mannan] ratio may be between 15 and 30 (weight / weight), or even more particularly between 18 and 22 (weight / weight).

[0014] In certain embodiments, the Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] is characterized in that it has a dry matter content greater than or equal to 94% by weight. In particular, the dry matter content may be greater than or equal to 96% by weight.

[0015] In certain embodiments, the Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] further comprises a protein content of less than or equal to 10% by weight, and a glycogen content of less than or equal to 10% by weight.

[0016] In some embodiments, [3-glucans and mannans are the only active ingredients of the [3-glucan-rich] Saccharomyces cerevisiae yeast cell wall extract.

[0017] In some embodiments, the Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] is Safglucan® which comprises [3-glucans] and mannans, where the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans] in a total amount of between 52 and 60% by weight; and the mannans are present in a total amount of between 1 and 5% by weight, and where Safglucan® also comprises a protein content of less than or equal to 10% by weight, and a glycogen content of less than or equal to 10% by weight.

[0018] The present invention also relates to a composition comprising a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans and at least one bioactive agent for use in the prevention of mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject or in the prevention of the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject or in the prevention of liver and / or intestinal damage induced by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract is as defined above.

[0019] The present invention also relates to a pharmaceutical composition comprising an extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans and at least one physiologically acceptable excipient for use in the prevention of mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject or in the prevention of the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject or in the prevention of liver and / or intestinal lesions induced by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract is as defined above.

[0020] In certain particular embodiments, the subject to which the invention applies is an animal, in particular an animal chosen from among farm animals and companion animals.

[0021] The present invention further relates to an animal feed comprising a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucan] for use in the prevention of mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in an animal or in the prevention of the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in an animal or in the prevention of liver and / or intestinal lesions induced by the mycotoxin deoxynivalenol (DON) in an animal, characterized in that the extract is as defined above or in that the extract is part of a composition as defined above.

[0022] In some embodiments, the bioactive agent is selected from the group consisting of mycotoxin binders, mycotoxin biotransformers, vitamins, minerals, trace elements, hepatoprotectors, immunoprotectors, and their combinations.

[0023] A more detailed description of some preferred embodiments of the invention is given below. Brief description of the drawings Fig. 1

[0024] [Fig. 1] Monitoring of transepithelial resistance (TEER) over time (T) in hours in porcine intestinal cells (IPEC-J2 cell line) continuously pretreated with Safglucan®, with the addition of the mycotoxin DON. TEER variations are compared to those of cells treated with the mycotoxin DON alone, Safglucan® in the presence of the mycotoxin DON, or two control conditions (epithelial cells alone or cells with Safglucan® alone). Fig. 2

[0025] [Fig.2] Expression level of z78 mRNA, determined by quantitative PCR, by porcine intestinal cells (IPEC-J2 line) pretreated or not with Safglucan ® continuously, in the absence or presence of the DON mycotoxin. Fig. 3

[0026] [Fig.3] Microscopy of untreated porcine intestinal cells (IPEC-J2 line) (left image), treated with DON mycotoxin alone (middle image), and treated with Safglucan® and DON mycotoxin (right image). Fig. 4

[0027] [Fig.4] Expression level of 1' z78 mRNA by intestinal cells pigs (IPEC-J2 line) pretreated with different "[3-glucan] products", an algal [3-glucan (ALG), Safmannan® and Safglucan® in the absence and presence of the mycotoxin DON (P value *<0.05). Fig. 5

[0028] [Fig. 5] Histological analyses of pig jejunum explants (n=7). (A) Images Microscopic images of 5 sq m sections taken from paraffin blocks containing untreated jejunal excipients, or jejunal excipients treated with DON mycotoxin alone, Safglucan® alone, or Safglucan® and DON mycotoxin. (B) Graphs showing the histological score, enterocyte morphology, and intestinal villus height according to the treatments of pig jejunal excipients. Fig. 6

[0029] [Fig.6] Expression level of inflammation-related genes determined from mRNA isolated from jejunum explants of untreated pigs, or treated with my- DON mycotoxin alone, with Safglucan® alone, or with Safglucan® and DON mycotoxin (n=5 - 10). (P value *<0.05). Fig. 7

[0030] [Fig.7] Images of chicken liver treated with DON mycotoxin (right) and untreated (left). Fig. 8

[0031] [Fig.8] Graphs showing the percentage of liver lesions observed in chickens on days 13 and 28 (B) as a function of the presence or absence of DON mycotoxin and / or Safglucan® in the diet. Fig. 9

[0032] [Fig.9] Graph showing the height of intestinal villi in jejunum from untreated chickens, or treated with DON mycotoxin alone, or with Safglucan® and DON mycotoxin after 13 days post-treatment. Fig. 10

[0033] [Fig. 10] Expression levels of the genes il8 (A) and ifiiy (B) related to inflammation determined from jejunum mRNA of untreated chickens, or chickens treated with DON mycotoxin alone, or with Safglucan® and DON mycotoxin. Description of the implementation methods

[0034] As mentioned above, the present invention relates to Saccharomyces cerevisiae yeast cell wall extracts rich in [3-glucans] for use in the prevention of toxic effects, in particular immunotoxic effects, of the DON mycotoxin in a subject, and in the prevention of liver and / or intestinal damage induced by the DON mycotoxin in a subject.

[0035] I - Extracts of cell walls of Saccharomyces cerevisiae yeast rich in p-glucans

[0036] The 3-glucans of an extract according to the invention are obtained from the yeast Saccharomyces cerevisiae, and more particularly from a strain of Saccharomyces cerevisiae yeast. Many strains of Saccharomyces cerevisiae are known in the art. They are widely used in the food industry for their role in the manufacture of several foods, particularly breads and fermented beverages. As used here, the term “yeast strain” refers to a relatively homogeneous population of yeast cells obtained by culturing (or multiplying) the starting strain. A yeast strain is obtained from a clone, a clone being a population of yeast cells obtained from a single yeast cell. The culture of a Saccharomyces cerevisiae yeast strain can be carried out by any suitable method.Yeast cultivation 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 nature. Thus, a Saccharomyces cerevisiae yeast can be obtained by multiplying a strain in an appropriate culture medium, for example, as described in the reference book "Yeast Technology", 2nd edition, 1991, G. Reed and TW Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8.

[0037] The terms “yeast cell walls” and “yeast hulls” are used interchangeably herein and refer to the insoluble fraction of yeast cells, i.e., the cell wall and the plasma membrane of the yeast. Conventionally, yeast cell walls are obtained by a process comprising an autolysis or enzymatic hydrolysis step, primarily by proteases, followed by a separation step of the soluble and insoluble fractions, the isolated insoluble fraction corresponding to the yeast cell walls. The insoluble fraction can then be dried. The process for obtaining yeast cell walls is such that it preserves the structural polysaccharides of the cell wall, i.e., 3-glucans and mannans, the latter being in the form of mannoproteins.The methods for obtaining yeast cell walls are known in the art (see, for example, the reference work “Yeast Technology”, 2nd edition, 1991, G. Reed and TW Nogodawithana, published by Van Nostrand Reinhold, New York, ISBN 0-442-31892-8).

[0038] A Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] according to the invention refers to a fraction that has been extracted (or isolated) from the cell walls and that contains predominantly [3-glucans]. The [3-glucans from the yeast cell wall are called “cell wall [3-glucans]. They are essentially glucose polymers in which the glucose units of the main chain are linked by [3-1,3] bonds and the branches are linked by [3-1,6] bonds. Yeast [3-glucans] are insoluble and have low viscosity. Those skilled in the art know how to extract [3-glucans] from the yeast cell wall.A common method includes successive hot extractions with a base and with an acid (such as acetic acid), followed by aqueous washes to remove any soluble compound from the cell walls, and recovery of the insoluble material consisting of cell wall [3-glucans].

[0039] A Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] according to the present invention comprises a total amount greater than or equal to 50% by weight of [3-glucans] in the form of a mixture of [3-1,3-glucans and [3-1,6-glucans]. As used herein, the term “total amount greater than or equal to 50% by weight of [3-glucans]” means that the [3-glucans represent at least half the weight of the extract according to the invention. Thus, the total amount of [3-glucans] present in an extract according to the invention may represent between 50% and 90% of the weight of the extract, for example between 50% and 80%, or between 50% and 70%, or even between 50% and 60%, of the weight of the extract. In certain particular embodiments, the total quantity of [3-glucans] present in an extract according to the invention represents between 50% and 60%, for example, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, or about 59% of the weight of the extract.

[0040] A cell wall extract of Saccharomyces cerevisiae yeast rich in 3-glucans according to the present invention also contains mannans in a total amount of less than 5% by weight. Mannans from yeast cell walls are called “cell wall mannans.” These are yeast polysaccharides consisting mainly of mannose, and more precisely, copolymers of neutral or acidic sugars (with 5 or 6 carbon atoms), linked together by glycosidic bonds and bound to proteins. In Saccharomyces cerevisiae cell wall mannans, mannose is present as a skeleton of mannose residues (50 or more) linked by α-(1,6), branched by short chains of mannose linked by α-(1,2) and α-(1,3). Those skilled in the art know how to extract mannans from yeast cell walls.A common method involves enzymatic digestion of the yeast cell walls with a 3-glucanase preparation (e.g., the industrial preparation GLUCANEX™), followed by separation of the hydrolysate by centrifugation and purification by ultrafiltration. Another method is based on a hot chemical extraction.

[0041] As used herein, the term “total quantity less than 5% by weight of mannans” means that the mannans represent at most 5% of the weight of the extract according to the invention. Thus, the total quantity of mannans present in an extract according to the invention may represent between 0.5% and 5% of the weight of the extract. In certain particular embodiments, the total quantity of mannans present in an extract according to the invention represents between 1% and 5%, for example, approximately 1%, approximately 2%, approximately 3%, approximately 4%, or approximately 5% of the weight of the extract.

[0042] Preferably, a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans] according to the present invention comprises [3-glucans] and mannans, wherein the [3-glucans] are present as a mixture of [3-1,3-glucans and [3-1,6-glucans] in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount such that the [3-glucan / mannan] ratio is between 12 and 40 (weight / weight). In particular, the [3-glucan / mannan] ratio in an extract according to the invention may be between 15 and 30 (weight / weight), and more particularly between 18 and 22 (weight / weight).

[0043] In certain embodiments, a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] according to the present invention further comprises a protein content of 10% by weight or less, and a content of glycogen less than or equal to 10% by weight.

[0044] In certain embodiments, a Sac-charomyces cerevisiae yeast cell wall extract rich in [3-glucans according to the present invention comprises, as the only active ingredients, [3-glucans and mannans, wherein the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount less than 5% by weight such that the [3-glucans / mannans ratio is between 12 and 40 (weight / weight), in particular between 15 and 30 (weight / weight), and more particularly between 18 and 22 (weight / weight).

[0045] In certain particular embodiments, a cell wall extract of Saccharomyces cerevisiae yeast 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%, preferably 96% by weight, allows for better preservation of the yeast hulls, in particular better bacteriological stability and better stability with respect to undesirable reactions of enzymatic or non-enzymatic origin.

[0046] In certain particular embodiments, an extract of cell walls of Saccharomyces cerevisiae yeast according to the invention is in powder form.

[0047] In certain particular embodiments, the Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] according to the invention is Safglucan®, which is produced by the Applicant, Lesaffre. Safglucan® comprises [3-glucans] and mannans as the sole active ingredients, where the [3-glucans] are present as a mixture of [3-1,3-glucans] and [3-1,6-glucans] in a total amount of between 52 and 60% by weight; and the mannans are present in a total amount of between 1 and 5% by weight. Safglucan® also comprises a protein content of less than or equal to 10% by weight, and a glycogen content of less than or equal to 10% by weight.

[0048] After manufacturing, a cell wall extract of Saccharomyces cerevisiae yeast according to the invention can be packaged and / or stored under suitable conditions, generally a cool, dry place, before use. The shelf life of a cell wall extract according to the invention, in suitable packaging, is 2 years from the date of production.

[0049] II - Compositions Comprising an Extract of Cell Walls of Saccharomyces cerevisiae Yeast Rich in p-Glucans

[0050] In some embodiments, the [3-glucan-rich] Saccharomyces cerevisiae yeast cell wall extracts described herein are used as such. In other embodiments, the [3-glucan-rich] Saccharomyces cerevisiae yeast cell wall extracts described herein are used in combination with at least one other bioactive agent. Accordingly, the present invention relates to a composition comprising a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans, as described herein, combined with at least one bioactive agent.

[0051] The bioactive agent may, for example, be chosen from among agents having an effect on mycotoxins other than DON—animal feed being able to be contaminated by more than one mycotoxin. Alternatively, the bioactive agent may be known to have a beneficial effect on animal health. Thus, the bioactive agent may be a mycotoxin detoxifier, an antioxidant, a hepatoprotective agent, an immunoprotective agent, or a combination of these agents.

[0052] As used herein, the term “mycotoxins other than DON” refers to toxins produced by various species of microscopic fungi, such as molds (Aspergillus sp., Fusarium sp., Stachybotrys sp., Penicillium sp., etc.), the toxins not being deoxynivalenol. Indeed, contaminated foods often contain a mixture of mycotoxins because (1) a fungus can produce several different mycotoxins at the same time (Fusarium fungi, which secrete DON, are, for example, known to co-produce other mycotoxins, notably zearalenone), (2) a food can be contaminated by several fungi (cereals are often affected by Aspergillus spp.), and (3) each of the raw materials used in food can harbor at least one fungus producing at least one mycotoxin.Mycotoxins can develop on many substrates: forages, cereals, oilseeds, protein crops, silage, and to a lesser extent molasses and vitamin-mineral feed. 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, sterigmatocystin, paxillin, enniatin and beauvericin.

[0053] The mycotoxin detoxifying agent present in a composition according to the invention can be selected from mycotoxin binders, mycotoxin biotransformers, and combinations thereof.

[0054] The terms “mycotoxin binding agent” and “mycotoxin binder” are used interchangeably herein. They refer to an agent that adsorbs and / or deactivates mycotoxins other than DON, for example, mycotoxins present in animal feed or an animal feed ingredient, thereby reversing the adverse effects of the mycotoxins. Mycotoxin binders reduce exposure to mycotoxins by decreasing their bioavailability, which leads to reduced absorption of the mycotoxins by the animal, and thus a decrease in distribution to the blood and target organs. The term “bioavailability,” as used here in reference to a mycotoxin, refers to the fraction of mycotoxin that is absorbed / absorbed sorbable or assimilated / assimilable by an animal. Adsorption of mycotoxins by a binding agent decreases the bioavailability of mycotoxins because the binding-mycotoxin complex passes through the animal's digestive system and is excreted by the animal without assimilation.

[0055] Examples of mycotoxin binders include, without limitation, selected mycotoxin adsorbents from the group consisting of aluminosilicates (e.g., kaolinite, hydrated calcium / potassium / sodium aluminosilicate, hydrated sodium and calcium aluminosilicate (HSCAS)), bentonites (e.g., sodium bentonite, calcium bentonite), montmorillonites (e.g., sodium and calcium montmorillonite), zeolites (e.g., clinoptilolite zeolite), activated carbons, micronized fibers and polymers (e.g., cholestyramine, polyvinylpyrrolidone), activated diatomaceous earth, plant fibers (e.g., wheat bran and alfalfa fibers), polysaccharides (e.g., glucomannan or its esterified forms), certain algae, certain bacteria (lactic acid bacteria) and their combinations.

[0056] The term “mycotoxin biotransformer” refers to an agent (usually an enzyme, a bacterium, a fungus), which deactivates or inactivates mycotoxins other than DON, for example mycotoxins present in animal feed or feed ingredients, thereby reversing the harmful effects of mycotoxins.

[0057] Examples of mycotoxin biotransformers include enzymes that degrade mycotoxins other than DON, for example selected 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 their combinations.

[0058] Other examples of mycotoxin biotransformers include microorganisms that are known to be deleterious to mycotoxins, for example selected from: Eubacterium sp. BBSH 797 (a coriobacteriaceaef Nocardia asteroids, Mycobacterium fluoranthenivorans sp., Rhodococcus erythropolis, bacilli of the genus Alcaligenes, Bacillus, Lactobacillus, Achromobacter thermophilus C5 and NG40Z, Lactobacillus paraplantarum, Stenotrophomonas maltophila, Sac-charomyces cerevisiae, Exophiala spinifera, Cupriavidus basilensis OR16 Aspergillus niger, Eurotium herbariorum, Rhizopus, Trichosporon mycotoxinivorans, Phaffia rho-dozymyllces, Phaffia rhodozymy lices, Phaffia rhodozymhousces, Nocardia corynebac-terioides NRRL 24037, Mycobacterium fluoranthenivorans or Myxococcus fulvus ANSM068, Rhodococcus erythropolis, Brevibacterium sp., Slackia sp. DG6, Deviosa mutons, Deviosa insulae A16, Solarium tuberosum, Aspergillus oryzae, Eggerthella sp DII-9, Pseudomonas sp Yl, Lysobacter sp SI, Sphingomonas, Nocardioides, Citrobacter, Marmaricola sp MIM116 and combinations thereof.

[0059] In some embodiments, the bioactive agent present in a composition according to the present invention is an antioxidant known to reduce the toxicity of mycotoxins other than DON in animals. Examples of such bioactive agents include, without limitation, rutin, quercetin, lutein, lecithin, melatonin, mannitol, curcumin, curcumoids, lycopene, allyl sulfides, fructose, chlorophyll and its derivatives, sodium thiosulfate, glutathione, methionine, aspartame, trace elements (selenium, zinc, magnesium), catechin (epigallocatechin gallate, epicatechin gallate), morin, kaempferol, fisetin, naringin, vitamins (vitamins E, C, A and B), coenzyme Q10, provitamins (carotene and carotenoids), eugenol, vanillin, caffeic acid, cholinergic acid, and combinations thereof.

[0060] In some embodiments, the bioactive agent is a hepatoprotective or immunoprotective agent known to be active in animals. The terms “hepatoprotective” and “liver protectant” are used interchangeably herein. They refer to an agent that improves the integrity and regeneration of hepatocytes, optimizing the liver's detoxification capacity and / or promoting hepatic synthesis by stimulating the activity of digestive enzymes that ensure optimal nutrient utilization by increasing their intestinal absorption and, consequently, their bioavailability.Examples of hepatoprotective agents include, but are not limited to, naturally derived liver protectants composed of a combination of various plants with different hepatoprotective properties, such as Phyllanthus niruri, Azadirachta indica, Andrographis paniculata, Achyrantes aspera, etc., and methyl group-donating liver protectants based on the ability of methyl groups to bind to toxins, thus promoting their elimination from the body. Among the compounds capable of donating methyl groups are certain amino acids and their derivatives (e.g., methionine, carnitine, betaine, etc.), and vitamin derivatives (e.g., choline). The term “immunoprotective agent” refers to an agent that, regardless of its mechanism of action, protects against the effects of an antigen.Examples of immunoprotective agents include, but are not limited to, envelope proteins derived from animal viruses; oligonucleotides such as CpG oligonucleotides. Immunoprotectors may also be chemical immunoprotectors 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 plants: Eclipta alba, Aloe vera, Ocimum sanctum, Viscum. album, Urtica dioica and Zingiber officinale, Solanum trilobatum, Astragalus radix and Scutellaria radix, and Achyranthes aspera.

[0061] In a composition according to the invention, the bioactive agent(s) are generally present in an amount sufficient to achieve the desired purpose (e.g., reducing the bioavailability of mycotoxins and / or inactivating mycotoxins). Those skilled in the art can determine such an amount. For example, the enzyme(s) that degrade mycotoxins other than DON 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%, and more preferably still between 0.15% and 0.25% by weight of enzyme relative to the total weight of the composition.One or more mycotoxin-binding bioactive agents other than DON may, for example, be present in an amount of up to about 90-95% by weight relative to the total weight of the composition, e.g. about 80%, about 70%, about 60%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or less than 10% by weight relative to the total weight of the composition.

[0062] In certain embodiments, a composition according to the invention may also include components commonly found in animal feed supplements, additives, or feed supplements, such as vitamins, minerals, trace elements, etc. Those skilled in the art know how to select the appropriate components according to the animal for which the composition is intended, and how to determine the appropriate quantities to include in such a composition.

[0063] The compositions according to the invention can be in any form, for example as a powder, granules, gel, or liquid. Preferably, the compositions according to the invention are in solid form, preferably as a powder.

[0064] The invention also relates to a pharmaceutical composition comprising a cell wall extract of Saccharomyces cerevisiae yeast rich in 3-glucans, as described herein, and at least one physiologically acceptable excipient in veterinary medicine. In the pharmaceutical composition, the extract may be present as the sole active ingredient. Alternatively, the pharmaceutical composition may further comprise at least one other bioactive agent, such as those listed above.

[0065] A pharmaceutical composition according to the invention can be classified as a veterinary pharmaceutical preparation available by prescription or over the counter.

[0066] In the context of the present invention, “physiologically acceptable excipient in veterinary medicine” means any medium or additive that does not interfere not with the efficacy of the biological activity of the active ingredient (here the cell wall extract rich in [3-glucans), and which is not excessively toxic to the animal, at the concentrations at which it is administered.

[0067] The veterinary pharmaceutical compositions according to the present invention can be administered using any combination of dosage and route of administration effective in achieving the desired prophylactic effect. Those skilled in the art will recognize that the exact amount to be administered may vary from one animal species to another, and from the effects of the DON mycotoxin on the animal species being treated.

[0068] III - Uses of Extracts of Dried Cell Walls of Saccharomyces cerevisiae Yeast Rich in p-Glucans

[0069] As mentioned above, DON has a negative impact on the intestine and immune responses. This mycotoxin induces intestinal histological alterations, including necrosis of the intestinal epithelium. DON also disrupts the function of the intestinal barrier, which can lead to increased translocation of pathogens and greater susceptibility to enteric infectious diseases. DON also modulates the immune reactivity of the intestinal mucosa and can interact in the dialogue between epithelial cells and intestinal immune cells, representing a predisposing factor for inflammatory diseases.

[0070] Extracts of Saccharomyces ce-revisiae yeast cell walls rich in [3-glucans, described herein, which do not adsorb the mycotoxin DON, have been shown to significantly improve the transepithelial resistance of the epithelial barrier of porcine intestinal cells exposed to the mycotoxin DON; to cause a decrease in the expression of the il8 gene (a pro-inflammatory cytokine) induced by DON in porcine intestinal cells in vitro, in porcine expiants ex vivo and in chicken intestinal tissue in vivo; to prevent these cells from the breakdown observed in untreated cells; to prevent the reduction in intestinal villus size and the alteration of enterocyte morphology in porcine intestinal expiants; and to effectively prevent liver damage as well as the reduction in villus size induced by the mycotoxin DON in chicken.

[0071] The invention therefore also relates to a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans], as described herein, alone or in combination with at least one bioactive agent, for use in the prevention of mycotoxicosis caused by the DON mycotoxin in a subject. The invention also relates to a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans], as described herein, alone or in combination with at least one bioactive agent, for use in the prevention of the toxic effects, in particular the immunotoxic effects, of the DON mycotoxin in a subject. The invention also relates to a cell wall extract Saccharomyces cerevisiae yeast lulares rich in [3-glucans, as described herein, alone or combined with at least one bioactive agent, for use in the prevention of DON mycotoxin-induced liver and / or intestinal damage in a subject.

[0072] The term “mycotoxicosis” refers to a condition arising from the ingestion of a mycotoxin, which originates from food contaminated by said mycotoxin produced by a microscopic fungus. Mycotoxicosis caused by the mycotoxin DON is therefore a condition arising from the ingestion of the mycotoxin DON. As used here, the term “preventing mycotoxicosis caused by the mycotoxin DON in a subject” means reducing or inhibiting, partially or totally, the likelihood that a subject will develop one or more symptoms related to the ingestion of DON (reduced growth, immunosuppression, vomiting, diarrhea, intestinal mucosal damage, refusal to eat, liver damage, abdominal pain, etc., depending on the subject).

[0073] The term “immunotoxic effects of mycotoxin DON,” as used herein, refers to the adverse effects induced by the ingestion of DON on the immune system. Various types of immunotoxic effects are possible, including immunosuppression, which can promote infections and tumors, immunostimulation, hypersensitivity, and autoimmunity. When animals are exposed to low doses of DON, certain components of the immune system are stimulated. Conversely, when animals are exposed to higher doses of DON, an immunosuppressive response is generally observed. The term “preventing the immunotoxic effects of mycotoxin DON” means reducing or inhibiting, partially or completely, the likelihood that an individual will experience an immunotoxic effect after ingesting DON.

[0074] The term “liver and / or intestinal lesions,” as used herein, refers to any pathological alteration of a tissue or cell of the liver and / or intestines, which are then in an abnormal state. These alterations can lead to liver and / or intestinal dysfunction.

[0075] As used herein, the term “subject” refers to a human or an animal. As used herein, the term “animal” refers to a living being of the kingdom Animalia. In the context of the present invention, the term more specifically refers to livestock (such as bovines (cow, buffalo, zebu, bison, aurochs, yak), ovine (sheep), caprines (goat), porcine (pig), equine (horse, donkey, mule), camelids (camel, dromedary, llama, alpaca), and cervids (reindeer, deer)) and other farm animals (chicken, turkey, duck, goose, pigeon, quail, pheasant, partridge, ostrich, emu, rhea, ratite, guinea fowl, rabbit, guinea pig); to aquatic animals (marine, pond or freshwater fish farming, shellfish farming, crustacean and mollusc farming); to companion animals (cats, dogs, rabbits, equines, etc.); and to laboratory animals. The term "animal," as used here, does not refer to a specific age group. particular.

[0076] In certain particular embodiments, the animal for which the Saccharomyces cerevisiae yeast cell wall extracts described herein are intended is chosen from among companion animals and livestock, in particular cattle (pigs, cattle, sheep, goats) and poultry (chicken, turkey, rooster, guinea fowl, quail, duck, goose).

[0077] Animals do not all have the same sensitivity to the mycotoxin DON. Differences in sensitivity are primarily due to differences in the absorption mechanism, metabolism, distribution, and elimination of DON. Pigs are known to be highly sensitive to DON due to their cereal-rich diet and also because the mycotoxin is rapidly and efficiently absorbed before being distributed to the organs, where it is only weakly metabolized and therefore detoxified. Poultry are less sensitive to DON than pigs. The first zootechnical effects are observed at a concentration of 5 mg of DON per kilogram of feed. However, acute poisoning results in the animal's death between 3.5 and 13.5 hours after ingestion of the contaminated feed. For ruminants, DON contamination is possible through cereal concentrates or corn silage containing DON.Other forages (hay, silage) can also be contaminated, but the development of Fusarium and the production of DON after harvest are considered negligible. Ruminants appear to be relatively unaffected by DON up to 12 mg / kg of feed.

[0078] In certain embodiments of the present invention, the use of a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans], alone or in combination with at least one bioactive agent, in the prevention of mycotoxicosis caused by the mycotoxin DON in a subject and / or in the prevention of the immunotoxic effects of the mycotoxin DON in a subject and / or in the prevention of liver and / or intestinal damage induced by the mycotoxin DON in a subject, includes mixing a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] with animal feed. The mixing can be carried out by any suitable method known in Art.

[0079] As used herein, the terms “animal feed”, “animal feed ingredient” and “animal feed product” refer to any natural or processed organic matter that is susceptible to biodegradation and that can be consumed by an animal. Examples of such organic matter range from freshly harvested grains to pelleted feeds, and include, for example, any compound, grain, nut, fodder, silage, preparation, composition or mixture suitable or intended to be ingested by an animal. The term “fodder” refers to plant material (mainly plant leaves and stems) consumed by grazing animals. The term “silage” refers to fermented, high-moisture forage that may be used for ruminant feeding. Preparations, compositions, and mixtures, such as commercial preparations, compositions, and mixtures, may be in any suitable form, for example, as concentrates, premixes, supplements, total mixed rations (TMR), etc. Animal feed may contain grains (e.g., corn, wheat, barley, rye, rice, sorghum, millet, or combinations thereof), forage, silage, pulses (e.g., soybeans), distillers' grains, animal products, etc. Distillers' grains are primarily a byproduct of industrial processes such as brewing and distilling, intended for the production of potable alcohol (whisky, vodka, or gin) or biofuels.Brewer's spent grains are produced from malt, which is itself derived from barley. Cereals other than barley may occasionally be used in brewing, such as sorghum, particularly in Africa. Spent grains from bioethanol production are made from corn or wheat. Wet spent grains are dried to produce dry spent grains, which are primarily used as animal feed.

[0080] In some embodiments, animal feed to which a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans], as described herein, is added has been determined to be contaminated with molds capable of producing the mycotoxin DON or to contain the mycotoxin DON. In other embodiments, the animal feed has been determined to be susceptible to contamination by molds capable of producing the mycotoxin DON or to contain the mycotoxin DON. In still other embodiments, nothing is known regarding the potential contamination of the animal feed by molds capable of producing the mycotoxin DON or by the mycotoxin DON itself.

[0081] A cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans, as described herein, alone or in combination with at least one bioactive agent, may be added to animal feed in quantities of about 0.003% to about 0.5% by total feed weight (corresponding to quantities of about 0.03 kg to about 5 kg per tonne of feed) subject to compliance with applicable legislation. For example, in some embodiments, a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans], as described herein, alone or in combination with at least one bioactive agent, is added to animal feed in an amount of about 0.003% to about 0.5% by total weight of the feed (corresponding to an amount of about 0.03 kg to about 5 kg per tonne of feed), for example, in an amount of about 0.005% to about 0.05% by weight of feed (which corresponds to approximately 0.05 kg to approximately 0.5 kg per tonne of feed).

[0082] In the foregoing, the emphasis has been placed on the use of Saccharomyces cerevisiae yeast cell wall extracts rich in [3-glucans] in the field of animal health. However, it is envisaged that the use of these extracts will be extended to human health, as deoxynivalenol is responsible for serious mycotoxicoses in humans.

[0083] Unless otherwise defined, all technical and scientific terms used in the Description have the same meaning as that commonly understood by an ordinary specialist in the field to which this invention belongs. Likewise, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference. Examples

[0084] The following examples describe certain embodiments of the present invention. However, it is understood that the examples and figures are presented for illustrative purposes only and do not in any way limit the scope of the invention.

[0085] In all the examples below, the term "CTL" means "control".

[0086] Example 1: Evaluation of Salglucan / / ? vitro

[0087] In vitro studies were conducted on the porcine intestinal cell line IPEC-J2, and the effects on membrane permeability and the expression of various genes were investigated. Due to their cereal-rich diet, pigs are particularly exposed to and susceptible to the mycotoxin DON. Furthermore, pigs are a good model for humans, particularly with regard to the digestive and immune systems.

[0088] Materials and Methods

[0089] Different "[3-glucan] products" were used in this study: Safglucan®, according to the present invention, which is produced by Lesaffre, the Applicant; an algal [3-glucan]; and Safmannan® produced by Lesaffre. The characteristics of these "[3-glucan] products" are presented in Table 1 below.

[0090] Table 1. Characteristics of the “[3-glucan] products studied.

[0091] [Tables 1] Name Descriptio n Structure Origin b-glucans (in %) Mannan s (in %) Ratio [3-glucans / mannans Safglucan® [3-1,3 / 1,6-branched glucans Saccharomyce s cerevisiae 57.4 (52 - 60) 1.46 (1-5) 39 (12-40) [3-glucan from algae [3 1.3 Linear glucan Euglena gracilis 56.1 3.45 16.2 Safmannan [3-1.3 / 1.6-glucans branched Saccharomyce s cerevisiae 22.4 22.3 1

[0092] IPEC-J2 cells were thawed in DMEM / F12 + 5% FBS + 16 mM Hepes + IX ITS + 5 ng / ml EGF and maintained in culture for 2 passages (1 week). On the third passage, the cells were cultured in the presence of pig serum (Gibco) and maintained for 2 passages (1 week).

[0093] A "[3-glucan] product" was added to IPEC-J2 cells at a final concentration of 50 qg / mL, and this concentration was maintained at each passage. After 15 days of culture in the presence of the "[3-glucan] product," various assays were conducted: a TEER (transepithelial / endothelial electrical resistance) test, and mRNA extraction for quantification of IL8 by quantitative PCR.

[0094] The mycotoxin DON was prepared every 2 to 3 weeks from a frozen, parafilm-coated stock solution (30 mM DON in acetonitrile) as follows. The DON solution was removed from the freezer 2 minutes before sampling and vortexed. It was verified that no crystals had formed. 30 qL of the solution were taken and added to 270 qL of sterile water, thus giving the 3 mM stock solution in a water / acetonitrile mixture (10 / 90, v / v). The stock solution was diluted one-third in a water / acetonitrile mixture (10 / 90, v / v) to obtain a 1 mM solution. These two solutions were then used diluted to 1 / 100th in the wells of the TEER assays and the wells of the 96 micro-well plates of the quantitative PCR assays to obtain a final concentration of 10 and 30 qM.

[0095] TEER Test Protocol. In this test, Falcon 24-well inserts were The wells were pre-moistened by adding 800 qL of medium to the basolateral well, wetting the membrane with 100 qL of medium, and incubating for 15 minutes at 37°C. Cell suspensions containing 0.5 x 10⁵ cells / mL, continuously cultured with a 3-glucan product, were prepared, and 300 qL of each suspension was loaded into each pre-moistened insert. Three days later, the medium in each insert was changed. The inserts were placed in a cellZscope®, and the value The TEER was determined. The following day, the DON mycotoxin was added, and the TEER was read for at least 24 hours.

[0096] RNA for Quantitative PCR. Cell suspensions containing 0.5 x 0.5 Cultured or uncultured cells / mL with a continuous [3-glucan] product were prepared, and 200 pL of each suspension was loaded into each well of a 96-microwell plate (i.e., 10,000 cells / well). The following morning, the mycotoxin DON was added and incubated for 6 hours. The medium was then removed by aspiration, and 130 pL of RAI + 5 pL of TCEP were added to each well to lyse the cells. The plate was stored at -20°C until the day of RNA extraction with the Macherey Nagel™ Nucleospin™ 96 RNA Kit.

[0097] RNA Extraction and Reverse Transcription. RNA extraction was performed by centrifugation, following the instructions provided in the package insert for the Macherey-Nagel™ NucleoSpin™ 96 RNA Kit (reference 740709.4). Elution was carried out in 30 pL of RNase-free water. 10 pL of RNA were then used to perform reverse transcription to complementary DNA using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (reference 4368814).

[0098] A reaction, in the absence of RNase inhibitor, contains: 2 pL of 10X RT Buffer, 0.8 pL of 25X dNTP Mix (100 mM), 2 pL of 10X RT random Primers, 1 pL of Multi-TiscLibe Reverse Transcriptase and 4.2 pL of nuclease-free water.

[0099] The thermocycling program used is as follows: 10 minutes at 25°C, 2 hours at 37°C, 5 minutes at 85°C and a maintenance at 4°C.

[0100] Quantitative PCR. The PCRs were performed on a QuantStudio 3 instrument (Applied Biosystems). The mastermixes (Applied Biosystems™ Taqman™ Fast Advanced Master Mix, ref. 4444964), primers, and Taqman™ probes (Taqman gene expression assay, HPRT pig, Ss03388274-ml-VIC-MGB-PL; Taqman gene expression assay, IL8 pig, Ss03392437_ml-FAM-MGB) were ordered from Thermo Fisher Scientific. The PCRs performed were relative quantitative PCRs that compared the expression of the IL8 gene in the presence and absence of the [3-glucan] product. The results obtained are expressed as 2ΔCt, where ΔCt is the difference in Ct between the gene of interest (U8) and the Housekeeping gene (HPRT), whose expression is stable.

[0101] Results

[0102] The results of the in vitro studies are presented in Figures 1 to 4.

[0103] The porcine intestinal cell line IPEC-J2 was continuously pretreated with Safglucan® with the addition of the mycotoxin DON. The results of the TEER assays, shown in [Fig. 1], demonstrate the transepithelial resistance of the epithelial barrier of IPEC-J2 and show that cells pretreated with Safglucan® are less affected by the effects of the mycotoxin DON than unpretreated cells.

[0104] Figure 2 shows the results of quantitative PCR on the expression of 1' il8 (a (pro-inflammatory gene that is expressed by epithelial cells in case of danger). These results show that continuous pretreatment of IPEC-J2 cells with Safglucan® leads to a decrease in DON mycotoxin-induced IL-8 production in IPEC-J2 cells.

[0105] The photos taken with a microscope (shown in [Fig.3]) show that IPEC-J2 cells treated with Safglucan® in the presence of DON mycotoxin are less destructured than cells treated with DON mycotoxin alone.

[0106] The porcine intestinal cell line IPEC-J2 was then continuously pretreated with Safglucan®, as well as with other 3-glucan products having different origins and / or 3-glucan / mannan ratios, before the addition of the mycotoxin DON. Interleukin IL-8 expression by IPEC-J2 cells was measured. The results, shown in [Fig. 4], demonstrate that cells pretreated with Safglucan® are less affected by the effects of DON than cells pretreated with the other 3-glucan products.

[0107] Example 2: In vitro adsorption of DON mycotoxin by Safglucan®

[0108] To understand the mechanism of action of Safglucan®, an in vitro test was performed to determine the ability of Safglucan® to adsorb the mycotoxin DON. Incubation was carried out at two different pH levels (3 and 7) and for two different batches of Safglucan® (batch 1 and batch 2). The results obtained are presented in Table 2 below.

[0109] Table 2. Adsorption of DON mycotoxin by Safglucan®.

[0110] [Tableaux2] % Adsorption at pH 3 % Adsorption at pH 7 Safglucan® batch 1 1% 5% Safglucan® batch 2 0.5% 2%

[0111] These results allow us to exclude a mechanism of action by adsorption of the mycotoxin DON by Safglucan®, whether under acidic pH conditions (pH 3) or under neutral pH conditions (pH 7). By comparison, it was determined that the adsorption of the mycotoxin by the Safmannan® product is <10%.

[0112] Example 3: Evaluation of the Effects of Safglucan® ex vivo

[0113] The experiments reported in this example were carried out by the team of Dr. Isabelle Oswald, whose address is: INRAE ​​- NATIONAL INSTITUTE for AGRICULTURE, FOOD and ENVIRONMENT Food Toxicology Unit UMR 1331 ToxAlim - 180 chemin de Tournefeuille - BP93173 - 31027 Toulouse cedex 03.

[0114] Ex vivo studies were conducted on porcine intestinal expiants to de The effects on intestinal tissue and the expression of different genes are studied. Explant culture is an alternative method situated between cell culture and whole-animal testing. It allows for the testing of contaminants / additives on a whole-organ sample containing the different cell types that compose it. It also allows for replicates and limits the variability of observed responses (each animal serving as its own control).

[0115] Materials and Methods

[0116] Experimental Protocol. The animals were reared under feeding and environmental conditions that closely mimic those of farm animals. Jejunal excipients were exposed to the mycotoxin DON with or without Safglucan® at 10 mg / L*. From 6 to 12 four-week-old pigs, jejunal segments were harvested and rapidly placed in washing medium (cold Williams E medium + antibiotics: penicillin, streptomycin, and gentamicin). Excipients were prepared using 8 mm diameter biopsy punches for histological analyses and 6 mm diameter punches for qPCR and Western blot analyses, taking care to identify the mucosal vs. muscularis orientation.The expiry samples were then carefully placed in 6-well plates containing complete medium (Williams E medium + glucose (25g / L), 1% ITS, 1% Ala-Glu (3mol / L), 1% penicillin / streptomycin and 0.5% gentamicin) on sponges for 4 hours (the time could be modified according to the analyses to be performed) at 39°C (the temperature could be modified according to the analyses to be performed) on a shaking tray.

[0117] Jejunal excipients were exposed or not to DON (10 µM) and co-treated or not with 10 mg / L of Safglucan® solution. The 10 µM DON dose was chosen because it corresponds to a concentration of 3 mg / L (3 ppm), which is close to concentrations found in animal feed. Indeed, the maximum recommended levels of deoxynivalenol in feed in Europe are 5 ppm for complete feeds, with the exception of feed for these pigs, which has a dose of 0.9 ppm.

[0118] At the end of the processing, the samples were collected and stored as follows:

[0119] - For histological analyses: the biological samples were transferred into Cassettes and tissues were fixed in 4% formalin before being transferred to a 70% alcohol solution and then embedded in paraffin. Crypt depth and villus length were also measured using the NIS-Elements-Ar image analysis software (Nikon System-Elements-Advanced research).

[0120] - For quantitative PCR and Western Blot analyses: biological samples were transferred into Eppendorf tubes and stored at -80°C.

[0121] The samples were generated from two sets of explants prepared from two independent experiments.

[0122] Results

[0123] The results of the ex vivo studies are presented in Figures 5 and 6.

[0124] Sections of 5 sqm were made from the paraffin blocks containing the Jejunal excipients were treated. They were then stained with hematoxylin and eosin (H&E) for histopathological analysis, which takes into account different types of lesions. The results obtained are presented in [Fig. 5]. These results show that DON increases the number of lesions on the tissue, while Safglucan® reduces villous damage in excipients co-exposed to DON. Enterocyte damage, characterized by the observation of flattening of jejunal epithelial cells in excipients co-exposed to DON, is also reduced in the presence of Safglucan®. A significant decrease of approximately 35% in villous length was observed in jejunal excipients treated with DON (10 mM). Treatment of excipients with Safglucan® tends to reduce the impact of DON on villous length.

[0125] The RNA from the expiry subjects was then extracted and used to study the expression of various genes, particularly genes related to inflammation and cytokine production. The results obtained are shown in [Fig. 6]. These results show that exposure of the expiry subjects with 10 µM of DON leads to an increase in the production of transcripts of inflammation-related genes. The presence of Safglucan® significantly downmodulates the expression of transcripts for the z'Zlb and il8 genes. A downtrend was also observed for Villa.

[0126] Example 4: Evaluation of the Effects of Safglucan® in vivo

[0127] The experiments reported in this example were carried out by Regiane Santos at Schothorst Feed Research, whose address is: Meerkoetenweg, 26 8218 NA Lelystad The Netherlands.

[0128] In vivo studies were carried out on broiler chickens exposed to feed naturally contaminated with the mycotoxin DON.

[0129] Materials and Methods

[0130] The experiment lasted 28 days, with an initial start-up phase from 0 to 13 days and a growth phase from 13 to 28 days. The experiment included 3 treatments according to an experimental design in "randomized completed block design" with replicas each, each replica comprising 24 cages and each cage 13 animals.

[0131] Table 3. Food treatment.

[0132] [Tables3] Tl T2 T3 DON <0.5 3.5 ppm 3.5 ppm Safglucan® 0 0 125g / Ton

[0133] On days 13 and 28, three animals per cage and per treatment (i.e., 18 animals per treatment) were randomly selected and euthanized. The liver and jejunum were harvested. The livers were examined macroscopically, and pale color and lesions such as cysts or hemorrhagic areas were counted and expressed as a percentage relative to livers without lesions. From the jejunum, villus height and crypt depth were measured on a minimum of 5 villi per animal, per treatment, and per cage.

[0134] Results

[0135] The results of the in vivo studies are presented in Figures 7 and 8.

[0136] Figure 7 shows the lesions of a chicken liver treated with the mycotoxin DON (left) compared to an untreated liver. The liver exposed to the mycotoxin DON appears paler, slightly more yellow; a hemorrhagic lesion is clearly visible.

[0137] Figure 8 presents graphs showing the percentage of liver lesions observed in chickens on days 13 and 28 as a function of the presence or absence of the mycotoxin DON and / or Safglucan® in the diet. It is clear that the presence of DON in the feed induces a significant increase in liver lesions, and the presence of Safglucan® in the diet prevented the lesions induced by the mycotoxin DON.

[0138] Figure 9 presents a graph showing the height of intestinal villi in the jejunum of chickens on day 13 as a function of the presence or absence of the mycotoxin DON and / or Safglucan® in the diet. It appears that the presence of DON in the feed induces a decrease in the size of the intestinal villi in the jejunum, and the presence of Safglucan® in the diet prevented this decrease in villus size.

[0139] Figure 10 presents graphs showing the expression of inflammatory genes in the jejunum of chickens as a function of the presence or absence of the mycotoxin DON and / or Safglucan® in the diet. It appears that the presence of DON in the feed induces a strong increase in VUS mRNA and interferon gamma (iFIII), and the presence of Safglucan® in the diet prevented this increase in the expression of these two inflammation-related genes.

Claims

Demands

1. Extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans] for use in the prevention of mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises [3-glucans] and mannans, where the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans] in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount less than 5% by weight, and characterized in that the [3-glucans] and mannans are the only active ingredients of the extract.

2. Extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans for use in the prevention of the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises [3-glucans and mannans, where the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount less than 5% by weight, and characterized in that the [3-glucans and mannans are the only active ingredients of the extract.

3. Extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans for use in the prevention of liver and / or intestinal damage induced by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract comprises [3-glucans and mannans, where the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans in a total amount greater than or equal to 50% by weight; and the mannans are present in a total amount less than 5% by weight, and characterized in that the [3-glucans and mannans are the only active ingredients of the extract.

4. Extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans for use according to any one of claims 1 to 3, characterized in that mannans are present in such an amount that the [3-glucans / mannans ratio is between 12 and 40 (weight / weight).

5. Extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans] 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. Extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans] for use according to any one of claims 1 to 5, characterized in that the extract further comprises a content of less than or equal to 10% by weight, and a glycogen content of less than or equal to 10% by weight.

7. Extract of Saccharomyces cerevisiae yeast cell walls rich in [3-glucans] for use according to any one of claims 1 to 6, characterized in that the extract is Safglucan® which comprises [3-glucans] and mannans, wherein the [3-glucans are present as a mixture of [3-1,3-glucans and [3-1,6-glucans] in a total amount of between 52 and 60% by weight; and the mannans are present in a total amount of between 1 and 5% by weight, and wherein Safglucan® further comprises a protein content of less than or equal to 10% by weight, and a glycogen content of less than or equal to 10% by weight.

8. A composition comprising a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans and at least one bioactive agent for use in the prevention of mycotoxicosis caused by deoxynivalenol mycotoxin (DON) in a subject or in the prevention of the immunotoxic effects of deoxynivalenol mycotoxin (DON) in a subject or in the prevention of liver and / or intestinal damage induced by deoxynivalenol mycotoxin (DON) in a subject, characterized in that the extract is as defined in any one of claims 1 to 7.

9. A pharmaceutical composition comprising a cell wall extract of Saccharomyces cerevisiae yeast rich in [3-glucans] and at least one physiologically acceptable excipient for use in the prevention of mycotoxicosis caused by the mycotoxin deoxynivalenol (DON) in a subject or in the prevention of the immunotoxic effects of the mycotoxin deoxynivalenol (DON) in a subject or in the prevention of liver and / or intestinal damage induced by the mycotoxin deoxynivalenol (DON) in a subject, characterized in that the extract is as defined in any one of claims 1 to 7

10. / An extract for use according to any one of claims 1 to 7 or a composition for use according to claim 8 or a pharmaceutical composition for use according to claim 9, characterized in that the subject is an animal, in

11.

12. in particular an animal chosen from among farm animals and pets. An animal feed comprising a Saccharomyces cerevisiae yeast cell wall extract rich in [3-glucans] for use in the prevention of mycotoxicosis caused by deoxynivalenol mycotoxin (DON) in an animal or in the prevention of the immunotoxic effects of deoxynivalenol mycotoxin (DON) in an animal or in the prevention of liver and / or intestinal lesions induced by deoxynivalenol mycotoxin (DON) in an animal, characterized in that the extract is as defined in any one of claims 1 to 7 or in that the extract forms part of a composition as defined in claim 8. A composition for use according to claim 8 or claim 11, characterized in that the bioactive agent is selected from the group consisting of mycotoxin binders, mycotoxin biotransformers, vitamins, minerals, trace elements, hepatoprotectors, immunoprotectors, and combinations thereof.

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