Glycoside inhibitors in yeast

Abstract

The present application relates to a composition of yeast polysaccharides, preferably also containing β(1,6)-glucan, preferably mannan and α-glucan, characterized in that said yeast polysaccharide is an extract of yeast wall fragments. Typically, the β-glucan is β-(1,6)-glucan. The present invention also relates to a method for obtaining such a composition, comprising at least one step of fractionating the yeast wall composition and extracting an insoluble fraction, and at least one step of extracting a soluble fraction from the insoluble fraction obtained in step a). The application finally relates to a composition having a human or veterinary therapeutic purpose for the treatment of gastrointestinal pathologies associated with pathogenic microorganisms, as well as to its non-therapeutic use for improving intestinal comfort.

Description

[Technical field]

[0001] The present disclosure relates to methods for obtaining compositions of yeast cell wall polysaccharides and their use for the preparation of food supplements aimed at treating gastrointestinal pathology or improving intestinal comfort. [Background technology]

[0002] An imbalance between pro- and anti-inflammatory bacterial species in the microbiota, as well as the predominance of certain bacterial families (Enterobacteriaceae, Fusobacterium) or the rarity of others (Clostridia, Faecalibacterium) have been described in people with chronic inflammatory bowel disease (CIBD). Specialized strains of Escherichia coli (E. coli) also cause diarrhea, food poisoning, urinary tract infections, sepsis and meningitis (Kaper JB, Nataro JP, Mobley HL - Pathogenic Escherichia coli, Nature Review Microbiology, 2004, 2, 123-140). Moreover, many studies now support the concept that the gut microbiota is a major environmental factor that may modulate the risk of colorectal cancer. Thus, commensal bacteria of the microbiota may be directly carcinogenic. This is the case, for example, of certain strains of E. coli that produce in vivo in the intestinal lumen a genotoxic substance called colibactin, which induces DNA damage in intestinal cells similar to that induced by irradiation with ionizing gamma rays (Cuevas-Ramos G et al., "Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells". PNAS USA, 2010, 107, 11537-11542).

[0003] Many microorganisms have already been described in the literature for their beneficial application to the human digestive tract and their nutritional value, for example as described in WO 2006 / 021965. These microorganisms are then generally referred to by the term probiotics, which corresponds to live microorganisms that can provide a health benefit to the host when administered in sufficient amounts (Joint FAO / WHO Expert Consultation, Probiotics in food, FAO Food and nutrition paper No. 85, ISBN 92-5-105513-0).

[0004] More specifically, the use of the yeast Saccharomyces cerevisiae (S. cerevisiae) for treating pathologies associated with Enterobacteriaceae is known. Thus, the earlier application FR 2928652 teaches that the yeast strain S. cerevisiae (CNCM I-3856, ScProl) is of therapeutic and prophylactic interest for the treatment of gastrointestinal pathologies associated with pathogenic microorganisms, in particular by limiting the colonization and / or invasion of the intestine by these microorganisms. In particular, the administration of this yeast leads to a reduction of Enterobacteriaceae in the colon. The results show that mannoprotein-rich fractions extracted from yeast strains (ScProl and / or SCB1) are able to inhibit in vitro the adhesion and invasion of human epithelial cells by Escherichia coli strains (AIEC (adherent invasive E. coli) strains) associated with Crohn's disease, one of the main CIBDs. A study by Sivignon et al. (Inflamm Bowel Dis. 2015; 21(2):276-286) also teaches that cell wall compounds from the CNCM I-3856 strain reduce intestinal colonization by AIEC bacteria and associated colitis symptoms in a mouse model mimicking Crohn's disease. Inhibiting AIEC adhesion to the intestinal wall significantly reduces AIEC invasion into intestinal tissue and therefore its infectivity.

[0005] In recent years, more and more attention has been paid to the glucan isolated from yeast cell wall.Therefore, yeast cell wall glucan has been used to enhance or stimulate immune response in humans and animals with normal or reduced immunological function (G. Hetland. Curr. Med. Chem. - Anti-infective Agents 2003, 2:135;PJ Rice, BE Lockhart, LA Barker, EL Adams, HE Ensley, DL Williams. Int. Immunopharmacol. 2004, 33:829).Patent application WO 2009 / 103884 more specifically teaches that yeast cell wall β-glucan (especially CNCM I-3856 strain) suppresses enteritis. Jawahara et al. (PLoS One 2012; 7(7):e40648) further disclose that the β-glucan fraction of S. cerevisiae yeast (specifically strain LYSC 318.2) can inhibit the pathogenic action of Candida albicans. Finally, Pengkumsri et al. (Food Sci Technol, Campinas 2017, 31(1):124-130) disclose that the β-glucan fraction has immunomodulatory properties that may be useful in the treatment of human colitis.

[0006] To the best of the inventor's knowledge, currently there is still no satisfactory treatment for CIBD. Sometimes surgery is considered to remove the damaged part of the small intestine, but this is a disabling operation that frequently recurs. Therefore, generally, only symptoms such as inflammation (with steroidal or nonsteroidal anti-inflammatory drugs and antibodies targeting inflammatory cytokines) and chronic pain (typically with analgesics such as cannabinoids) are treated. In patients with a progressive disease, doctors promptly start immunomodulatory therapy to stop attacks, prevent the appearance of new lesions and prevent the risk of tumor development associated with the chronic inflammatory state. However, these treatments also have significant side effects in the medium and long term. Therefore, corticosteroids are used less and less. It is therefore essential to develop and improve existing preventive treatments, on the one hand, making it possible to improve the intestinal comfort of healthy subjects, and on the other hand, to prevent the onset of pathologies in at-risk subjects, and to develop effective and well-tolerated treatments for patients suffering from gastrointestinal pathologies such as CIBD. Summary of the Invention

[0007] Therefore, it remains important to continue innovative approaches to identify new solutions that increase efficacy in improving the quality of life for patients suffering from gastrointestinal conditions, especially those involving infectious agents. In particular, it would be of great importance to identify new active ingredients or compositions with greater efficacy in terms of anti-adhesion and / or anti-invasive effects.

[0008] A composition of yeast polysaccharides comprising β-glucan and / or α-glucan and / or mannan is proposed, characterized in that the yeast polysaccharides are extracted from yeast cell walls. Typically, the β-glucan is β(1,6)-glucan. In some embodiments, the composition further comprises mannan and α-glucan. In particular, the present patent application proposes a composition comprising 5-25%, in particular 10-20% α-glucan, 30-50%, in particular 35-45% β-glucan (in particular β6-glucan), and 30-55%, in particular 40-50% mannan. Typically, the yeast is selected from among yeasts of the genus Saccharomyces. They may therefore be selected from the group comprising the strains deposited at the Collection Nationale de Cultures de Microorganismes (National Collection of Microorganism Cultures) under the numbers CNCM I-3799, CNCM I-3856, CNCM I-4407, CNCM I-4563, CNCM I-4812, CNCM I-4978, CNCM I-5128, CNCM I-5129, CNCM I-5268 and CNCM I-5269, as well as the strain DBVPG 6763 deposited at the Collezione dei Lieviti Industriali (Industrial Yeasts Collection).

[0009] According to another aspect, there is provided a method for obtaining a yeast composition, comprising the steps of: a) at least one step of fractionating the yeast cell wall composition (typically by high temperature incubation) and collecting (extracting) the insoluble fraction (referred to as "insoluble fraction a"); b) at least one step of extracting a soluble fraction (called "soluble fraction b") from the insoluble fraction a), said fraction containing β-glucans (in particular β6-glucans), preferably also mannans and generally α-glucans, which typically comprises at least one step of incubating the soluble fraction a) in a weak acid solution and collecting (extracting) the soluble fraction ("soluble fraction b"); A method is provided, comprising:

[0010] Typically, the method includes a preliminary step of obtaining an insoluble yeast cell wall fraction.

[0011] The method preferably comprises a step a1) of incubating the insoluble fraction a) in a strong base solution and then collecting the insoluble fraction (called insoluble fraction a1). Once this intermediate step has been performed, the fraction (or composition) then incubated in the weak acid solution of step b) above becomes "insoluble fraction a1".

[0012] The present disclosure also relates to yeast polysaccharide compositions, including β-glucans, mannans, and α-glucans, as described in this patent application, in particular yeast polysaccharide compositions obtainable according to the methods described herein, for use in the treatment and / or prevention of gastrointestinal conditions.

[0013] The present invention finally relates to the non-therapeutic use of the compositions defined in this disclosure, in particular the compositions obtained according to the methods described herein, for the preparation of food compositions aimed at improving gastrointestinal comfort and / or improving and maintaining the homeostasis of the intestinal microflora.

[0014] The features described in the following paragraphs can be implemented as options. They can be implemented independently of each other or in combination with each other. [Brief description of the drawings]

[0015] Other features, details and advantages will become apparent from reading the following detailed description and examining the accompanying drawings.

[0016] [Figure 1] Figures illustrating the various protocols carried out. The upper diagram (A) illustrates the extraction of different yeast polysaccharide fractions from whole yeast or from the yeast cell wall. The lower diagram (B) corresponds to the method of the invention, where different yeast polysaccharide fractions are extracted from the yeast cell wall. [Diagram 2] Diagram of the pre-incubation protocol for T84 and Caco-2 intestinal epithelial cells. Fractions extracted from yeast are first incubated with AIEC bacteria and then added to cultures of intestinal epithelial cells organized into epithelia. Residual adhesion levels of the bacterial strain AIEC LF82 are then estimated with decreasing concentrations of the yeast fraction (1; 0.5; 0.25; 0.1 mg / mL). [Diagram 3] Residual adhesion levels (percentage) of strain AIEC LF82 to T84 cells in the presence of "soluble fraction a" (also called Fehling mannan, due to the phosphopeptide mannan extraction protocol) (fraction 1, also called "soluble fraction a"), or "soluble fraction b" (fraction 3) obtained from whole yeast strain CNCM I-3856 (pre-incubation protocol) (mean ± SEM; **: p<0.01, ***: p<0.001, t-test). [Figure 4] Residual adhesion levels (percentage) of strain AIEC LF82 to T84 cells in the presence of β3-glucan-phosphate (fraction 6) obtained from whole yeast strain CNCM I-3856 (preincubation protocol) (mean ± SEM; **: p) < 0.01, t test). [Diagram 5] Residual adhesion levels (percentage) of strain AIEC LF82 to T84 cells in the presence of whole yeast strain CNCM I-3856 or "soluble fraction b" (fraction 3) obtained from CNCM I-3856 yeast cell walls (preincubation protocol) (mean ± SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; t test). [Figure 6] Residual adhesion levels (percentage) of strain AIEC LF82 to T84 cells in the presence of "soluble fraction b" (fraction 3) obtained from CNCM I-3856 or CNCM I-5268 yeast cell walls (preincubation protocol) (mean ± SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; t test). [Figure 7]Residual adhesion levels (percentage) of AIEC LF82 cells to T84 cells in the presence of “soluble fraction b” (fraction 3), α-glucan (fraction 5), or β6-glucan (fraction 4) derived from the CNCM I-5268 yeast cell line (preincubation protocol) (mean ± SEM; *: p < 0.05; ***: p < 0.001; t test). [Figure 8] Residual adhesion levels (percentage) of strain AIEC LF82 to TC7 / Caco-2 in the presence of “soluble fraction a” (fraction 1) obtained from whole yeast strain CNCM I-3856 or yeast strain LV04, or “soluble fraction b” (fraction 3) obtained from whole yeast strain CNCM I-3856 or CNCM I-5268 yeast cell walls (pre-incubation protocol) (mean ± SEM; **: p < 0.01, ***: p < 0.001, t test). [Figure 9] Residual invasion levels (percentage) of TC7 / Caco-2 cells by bacterial strain AIEC LF82 in the presence of "soluble fraction b" (fraction 3) derived from CNCM I-5268 yeast cell walls (pre-incubation protocol). [Figure 10] Graph showing the in vivo administration protocol for yeast fractions (YF: yeast fraction) in mice (mouse model of AIEC colonization). Yeast fractions are administered orally at a dose of 5 mg / mouse. Fractions tested: "soluble a" (fraction 1) obtained from whole yeast strain CNCM I-3856, or "soluble b" (fraction 3) obtained from whole yeast strain CNCM I-3856 or from CNCM I-5268 yeast cell wall. [Figure 11] Estimation of the number of AIEC LF82 bacteria in the feces of mice 2 or 3 days after infection of the animals as a function of the yeast fraction administered. Results are shown as the number of AIEC bacteria per gram of feces (box plot, from minimum to maximum) (WF: cell wall fraction). [Figure 12] Quantification of AIEC LF82 bacteria associated with the intestinal mucosa of treated or untreated mice by yeast fraction 4 days after infection. Results are shown as number of AIEC bacteria (box plot, min to max) per gram of tissue (WF: cell wall fraction). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Most of the drawings and the following description contain elements that are certain in nature, and therefore may serve not only to provide a better understanding of the present disclosure, but also to contribute to its definition, if desired.

[0018] "About" in the present application is understood to mean within a variance of 20%, in particular 15%, 10% or 5% relative to the indicated numerical value. As an example, the expression "a temperature of about 100°C" is to be understood as a temperature between 80°C and 120°C, in particular between 85°C and 115°C, more in particular between 90°C and 110°C, preferably between 95°C and 105°C.

[0019] The inventors of the present application have developed a new method by which compositions of yeast cell wall fragments can be obtained, the effectiveness of which in particular in inhibiting bacterial adhesion and invasion, in particular adherent and invasive Escherichia coli (AIEC), is significantly increased compared to whole yeast or derivatives described in the prior art.

[0020] More specifically, the inventors have demonstrated, in a totally surprising way, that yeast polysaccharide compositions extracted from yeast cell wall fragments (also called yeast walls or cell walls) exhibit adhesion and bacterial invasion inhibitory activity that is significantly superior to yeast polysaccharide compositions in which the polysaccharides are obtained from whole yeast. Also surprisingly, the yeast polysaccharide compositions according to the invention, which contain in particular β1,6-glucan, and optionally mannan and α-glucan, also exhibit higher activity than the mannan fractions previously described. This result is unexpected, since it was thought that mannosidic structures exposed by glycoproteins expressed on the surface of enterocytes were only involved in attachment to bacterial cells via fimbrial structures (FimH) exposed on the surface of type 1 pili (Barnich et al. JCI, 2007).

[0021] Thus, the present disclosure relates to a composition of yeast cell wall polysaccharides comprising β-glucan. Typically, the β-glucan comprises β(1,6)-glucan (hereinafter, also referred to as β6-glucan in particular in the examples). Typically, the composition further comprises mannan and / or α-glucan.

[0022] A yeast cell is generally composed of an envelope, also called the shell or wall, and an interior portion. Thus, in this application, the terms "wall", "shell" or "wall" are used interchangeably to modify the polysaccharides or yeast fragments described herein. Thus, the term "cell wall polysaccharides" refers to the polysaccharides that make up the wall (or shell) of yeast.

[0023] Three main groups of polysaccharides form the yeast cell wall, especially in the yeast Saccharomyces cerevisiae: polymers of mannose (or mannan), which make up about 40% of the yeast cell wall's dry mass, polymers of glucose (β-glucan and α-glucan), which make up about 60% of the cell wall's dry mass, and polymers of N-acetylglucosamine (chitin), which make up about 2% of the cell wall's dry mass.

[0024] Glucans are polysaccharides composed of glucose monomers that may be branched or unbranched. They can be classified into different subtypes depending on the glucose linkage mode. α-glucans are polymers of glucose monomers that are linked mainly by α-linkages (1-4).

[0025] Yeast β-glucan is a polysaccharide composed of glucose monomers and can be classified into two subtypes according to the glucose linkage: long chains of about 1500 β-1,3-glucose units, which account for about 85% of yeast β-glucan, and short chains of about 150 β-1,6-glucose units, which account for about 15% of yeast β-glucan (Klis, F., Mol, P., Hellingwerf, K. and Brul, S. (2002) "Dynamics of cell wall structure in Saccharomyces cerevisiae". FEMS Microbiology Reviews 26, 239-256).

[0026] Short chains of β-1,6-glucan are involved in covalent bonds with β-1,3-glucan, mannoproteins, and chitin. These crosslinks may also contribute to the modular structure of the cell wall (Kollar, R. et al. (1995) "Architecture of the yeast cell wall. β-(1,6)-glucan interconnects mannoprotein, β-(1,3)-glucan, and chitin". Journal of Biological Chemistry 270,17762-17775).

[0027] Mannans are polymers or oligomers of mannose linked through an N-glycan core. In particular, Saccharomyces cerevisiae yeast mannan is a polymer of α1,6 mannose branched at α1,2 with terminal α1,3 (see, in particular, Sendid et al., Med Sci (Paris). 2009; 25(5): 473-482). The mannan-rich fraction according to the present patent application comprises at least 30% mannan, in particular at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% mannan.

[0028] A β6-glucan fraction is understood to mean a fraction rich in β-1,6-glucan polysaccharides. Typically, such a fraction contains at least 30% β-1,6-glucan, in particular at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% β-1,6-glucan polymers.

[0029] β3-glucan fraction is understood to mean a fraction rich in β1,3-glucan. Typically, such fraction contains at least 30% β-1,3-glucan polymers, in particular at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% β-1,3-glucan polymers.

[0030] An α-glucan (or glycogen) fraction is understood to mean a fraction rich in α1,4 α1,6 glucan. Typically, such a fraction contains at least 60% α1,4 α1,6 glucan polymers, in particular at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% α1,4-α1,6 glucan polymers.

[0031] Yeast polysaccharide compositions described in the prior art and typically obtained from whole yeast typically contain 60-80% α-glucan, usually 65-75% α-glucan, 5-25% β-glucan, usually 10-20% β-glucan, and 5-25% mannan, usually 10-20% mannan.

[0032] The inventors have shown that the polysaccharide composition obtained from the yeast cell wall fraction is rich in β-glucan, typically β6 glucan, and preferably also rich in mannan. Without wishing to be bound by any theory, the inventors believe that the composition obtained from the yeast cell wall fraction comprises a strong, non-dissociable, typically covalently linked heterogeneous polymer composed of α-glucan, β-glucan (particularly β-6 glucan), and mannan, as described above.

[0033] Thus, the yeast polysaccharide composition of the present invention typically contains at least 30% β-glucan, particularly 30-50% β-glucan, preferably 35-45% β-glucan, particularly β(1,6)-glucan.

[0034] Preferably, the yeast polysaccharide composition of the present disclosure further comprises mannan. Typically, the composition of the present patent application is rich in mannan and may comprise 30-55% mannan, particularly 40-50% mannan.

[0035] Preferably, the yeast polysaccharide composition according to the present disclosure comprises α-glucan, in particular 5-25%, in particular 10-20% α-glucan.

[0036] In certain embodiments, the composition comprises the following proportions of polysaccharides: 5 to 25% α-glucan, preferably 10 to 20% α-glucan; 30 to 50% β-glucan, preferably 35 to 45% β-glucan, particularly β(1,6)-glucan; It contains 30 to 55% mannan, preferably 40 to 50% mannan.

[0037] The walls (or shells) of yeasts used in this application may be derived from one or more types of yeast. Yeasts are unicellular eukaryotic microorganisms belonging to the fungi kingdom. Yeasts particularly suitable for carrying out the present disclosure are yeasts of the genus Saccharomyces. These yeasts constitute a taxonomic genus of non-mycelium-forming ascomycetes and include several species used as fermentation agents in the food industry. Traditionally, yeasts belonging to the genus Saccharomyces may be selected among food yeasts such as S. cerevisiae, S. uvarum, S. bayanus or S. pasteurianus. The yeast is preferably a strain of the species Saccharomyces cerevisiae (brewer's yeast or brewer's yeast or baker's yeast, S. cerevisiae var. boulardii). For example, Saccharomyces cerevisiae yeast strains deposited at the Collection Nationale de Cultures de Microorganismes under numbers CNCM I-3799, CNCM I-3856, CNCM I-4407, CNCM I-4563, CNCM I-4812, CNCM I-4978, CNCM I-5128, CNCM I-5129, CNCM I-5268, and CNCM I-5269 are particularly well suited for the present disclosure.

[0038] The compositions of the present disclosure may be derived from one or more yeasts of different strains or different species.

[0039] The compositions of the present disclosure are typically obtained from yeast wall preparations or yeast wall fragments separated from the cell contents (cytoplasm), in particular by extraction of cell wall polysaccharides from yeast wall fragments. Methods for obtaining yeast walls or yeast wall fragments are well known in the art of the present disclosure. In this regard, reference can be made to the literature "Yeast Technology", 2nd edition, 1991, published by G. Reed and TW Nagodawawithana, Van Nostrand Reinhold, New York, ISBN 0-444-31892-8.

[0040] Briefly, yeast cell wall fragments or yeast shell fragments can be obtained by chemical (solvents, acids, bases), physical (sonication, high pressure), enzymatic (typically using proteases and nucleases) or autolytic (endogenous enzymes) lysis of yeast cells followed by separation of the soluble and insoluble parts by physical means, e.g., centrifugation and collection of the insoluble fraction (or part). Typically, centrifugation of the lysed yeast cell biomass results in a supernatant and a centrifugation pellet. The supernatant is mainly composed of free amino acids and peptides resulting from the degradation of proteins, and nucleotides resulting from the degradation of nucleic acids (RNA, DNA). The centrifugation pellet contains intact or partially degraded yeast walls in the form of emptied yeast cells.

[0041] Yeast autolysis is the hydrolysis of the yeast cell contents by the yeast's own enzymes. It is typically obtained by placing a suspension of yeast cells under specific physical medium conditions and / or contacting them with activators that cause apoptosis of the yeast cells and the release of their enzymes into the cell body. Hydrolysis of the cell contents produces soluble compounds. The insoluble fraction collected after the separation step constitutes a product called the yeast cell wall, which contains the yeast cytoskeleton as well as membranes and components that were not solubilized by autolysis or heterolysis. This insoluble fraction is often recovered in the form of an aqueous yeast cell wall suspension (or composition).

[0042] The yeast cell walls can be in liquid form (15-20% dry matter), dry form (>85% dry matter), or paste form (25-85% dry matter). The yeast cell walls are preferably present in dry form. In some embodiments, wall fragments of CNCM I-5268 yeast are used as an example of the compositions and / or methods described herein.

[0043] Yeast cell walls or shells, or any composition comprising same, from the same type of yeast or from different types (strains) or species of yeast can be used to practice the invention described herein.

[0044] The present disclosure also relates to a method for obtaining the above-mentioned composition of yeast cell wall polysaccharides, which preferably comprises, inter alia: (a) at least one step of fractionating the yeast cell wall composition and collecting the insoluble fraction, in particular at least one step of thermal extraction of the insoluble fraction (also referred to in the examples of the present patent application as "insoluble fraction a") from the yeast cell wall composition, and (b) at least one step of extraction in a weak acid solution, typically by incubating "insoluble fraction a" in a weak acid solution and then collecting the soluble fraction (called "soluble fraction b").

[0045] Typically, the method includes a preliminary step consisting of collecting a yeast cell wall fraction. In practice, this step is typically carried out by obtaining an insoluble fraction from a yeast hydrolysate (in particular a yeast autolysate), as described above. This step is particularly aimed at removing all or part (at least 60%, in particular at least 75%, more in particular at least 80%, at least 85%, at least 90%, even more in particular at least 95%) of compounds that are not bound to the wall or that are only weakly bound to the wall (see in this respect the method for obtaining the yeast cell wall fraction described above).

[0046] Step a) of thermal extraction (or thermohydrolysis) can be carried out by high-temperature incubation of the yeast cell wall fragments in a solution buffered to neutral pH, typically at a temperature above 75 ° C, in particular at 80-180 ° C, more particularly at 90-150 ° C, more particularly at 95-145 ° C. Typically at a temperature of about 120 ° C. Neutral pH is understood to mean a pH of about 7, in particular a pH of 6-8. The high-temperature incubation is typically maintained for at least 30 minutes, in particular at least 1 hour, typically from 1 hour to 4 hours, or from 1 hour to 3 hours. Preferably, the incubation is maintained for about 1.5 hours. The buffer is typically a solution based on a citrate buffer (about 20 mM), or any other equivalent buffer in situ. After incubation, the soluble and insoluble fractions are typically separated by centrifugation and the insoluble fraction (insoluble a) is collected. Typically, centrifugation may be carried out at a speed of 4000-6000 rpm, preferably about 5000 rpm, for at least 20 minutes, in particular 20-40 minutes, preferably about 30 minutes. This step may be repeated 2-4 times, preferably twice. The soluble fraction collected at the end of this thermohydrolysis step typically contains phosphopeptide mannans that are not tightly bound (typically non-covalently bound) to cell wall polysaccharides.

[0047] The method preferably comprises a step a1) of extracting the insoluble fraction in a strong base solution, typically comprising incubating the insoluble fraction obtained at the end of step a) (called "insoluble fraction a") with a strong base solution at the end of which the insoluble fraction (called "insoluble fraction a1") is collected. Once this intermediate step has been performed, the fraction (or composition) incubated in the weak acid solution of step b) above then becomes the "insoluble fraction a1".

[0048] Step a1) of extraction in a strong base solution typically comprises incubating the insoluble fraction a) in a strong base solution. The strong base solution is typically a solution of sodium hydroxide (NaOH) or any other equivalent, preferably concentrated to about 1N. The incubation in the strong base solution is typically carried out at room temperature, preferably at 16-24°C, in particular at about 20°C. It is ideally carried out with stirring for at least 16 hours, typically 16-32 hours, preferably 24 hours. The soluble and insoluble fractions are then separated, typically by centrifugation, and the insoluble fraction a1) is recovered. Typically, centrifugation can be carried out at a speed of 5000-8000 rpm, preferably about 7000 rpm, for at least 20 minutes, in particular 20-40 minutes, preferably about 30 minutes. Centrifugation is followed by a high temperature incubation step. The inventors believe that this step allows the removal of residual phosphopeptide mannans.

[0049] Step b) of extraction in a weak acid solution typically comprises a step of incubating the "insoluble fraction a" or "a1" (if an intermediate step of extraction in a strong base has been carried out) in a weak acid solution. The weak acid solution is typically a solution of acetic acid or any other equivalent, preferably concentrated to about 0.5 N. The incubation in the weak acid solution is typically carried out at a temperature of at least 70°C, in particular 75°C to 130°C, more particularly 75°C to 115°C, more particularly about 90°C. It is ideally carried out for at least 1 hour, typically 2 to 4 hours, preferably 3 hours, preferably with stirring. The soluble and insoluble fractions are then separated, preferably by centrifugation, and the soluble fraction (called soluble fraction b) is collected. Typically, centrifugation can be carried out at a speed of 4000 to 6000 rpm, preferably about 5000 rpm, for at least 20 minutes, in particular 20 to 40 minutes, preferably about 30 minutes. This step is repeated at least two times, in particular at least three, four, five, six, seven or eight times, in particular 3-8 times.

[0050] Typically, the centrifugation step of the method of the present patent application is carried out at room temperature, preferably between 16 and 24°C, in particular at about 20°C.

[0051] Typically, the "soluble fraction b" obtained according to the protocol described herein is a fraction rich in β-glucan, in particular β-6 glucan, and preferably also mannan (as defined above). Such fractions also typically contain α-glucan. Thus, the method of the present invention makes it possible to obtain a composition, in particular as described above, with the advantageous effects as claimed herein.

[0052] It should be noted that a β3-glucan fraction (i.e., typically enriched in β-3 glucan as described above) can be obtained from yeast cell wall fragments (husks) according to the present disclosure if an insoluble fraction (also called "insoluble fraction b") is collected at the end of step b). Likewise, this step b) can be repeated at least two times, in particular at least three, four, five, six, seven, eight times, in particular 3-8 times.

[0053] In a particular embodiment, a fraction rich in β6-glucan (preferably containing at least 60%, in particular at least 65, 70, 75, 80, 85, 90, 95, 99% β6-glucan) can be obtained from the soluble fraction obtained at the end of step b) ("soluble fraction b"). Thus, an isolated fraction of β6-glucan can be obtained, for example, by enzymatic treatment, typically with amyloglucosidase (e.g. from Aspergillus niger). However, preferably, the soluble fraction obtained at the end of step b) ("soluble fraction b") can be treated with an iodine solution (as shown in the examples). The incubation in the iodine solution is typically performed at room temperature (preferably 16-24 °C, in particular about 20 °C) (it is ideally performed for at least 15 min, typically 20-45 min, preferably 30 min, with stirring). After treatment with the iodine solution, the soluble and insoluble fractions are then typically separated by centrifugation and the soluble fraction (also called "soluble fraction c") is collected. Typically, centrifugation can be carried out at a speed of 4000-6000 rpm, preferably about 5000 rpm, for at least 20 minutes, in particular 20-40 minutes, preferably about 30 minutes. An ethanol solution can then be added to the supernatant and the solution can be centrifuged under the same conditions as before. A concentrated solution of strong acid (typically a hydrochloric acid solution concentrated to at least 2N, preferably about 3N) is added to the pellet to dissociate the β6-glucan and α-glucan complexes, which are then precipitated (in a conventional manner with ethanol or any equivalent). This second method makes it possible to collect the isolated β6-glucan and α-glucan-rich fraction (see also the method described in the results).

[0054] As shown above, the data obtained by the inventors showed that the "soluble fraction b" obtained from yeast cell walls very significantly inhibited the adhesion of AIEC-type pathogenic bacteria, resulting in a significant reduction in the invasion of AIEC (adhesive invasive E. coli) bacteria in intestinal tissues and therefore in their infectivity. AIEC has also been shown to adhere to intestinal cells by binding to a mannose-rich glycoprotein known as CEACAM6. Thus, transgenic mice expressing the human CEACAM6 protein become highly susceptible to AIEC infection, although this pathotype is generally less virulent for rodents. The in vitro data were validated, since treatment of mice infected with the yeast strain or the identified yeast derivatives reduced colonization of the digestive tract by AIEC bacteria.

[0055] Without wishing to be bound by any particular theory, the inventors believe that the "soluble fraction b" described herein contains alpha (1-4) and (1-6) glucosidic units as well as mannosidic units, which are important for the presentation of the binding structure or 3D conformation, thus allowing such fractions to effectively inhibit bacterial adhesion to the intestinal wall.

[0056] Thus, the present patent application relates to the pharmaceutical use of the compositions exhibiting advantageous activity described herein and / or obtained according to the method disclosed herein. In particular, the compositions described herein are particularly suitable for the treatment or prevention of gastrointestinal conditions or diseases. The compositions are typically compositions obtained preferably from yeast cell walls of the genus Saccharomyces, and are also rich in β-glucan (particularly β6 glucan), advantageously mannan. Preferably, the compositions contain at least 30% β-glucan, advantageously at least 30% mannan.

[0057] As used herein, the term "treatment" or "treating" is defined as the administration of a composition described herein to a patient in need thereof for the purpose of curing, alleviating, ameliorating, improving, and / or affecting a disease and / or any symptoms of the disease, particularly gastrointestinal disease, intestinal disorder, or functional bowel disorder. In particular, the term "treat" or "treatment" refers to the reduction or alleviation of at least one undesirable clinical symptom associated with the disease, such as pain, inflammation, diarrhea, nausea or vomiting, anorexia, or fatigue. As used herein, the term "prevent" or "prevention" is defined as the administration of a composition described herein to a patient in need thereof for the purpose of preventing the onset of a disease or at least one of its symptoms, and / or reducing the severity of at least one of its symptoms.

[0058] In certain embodiments, the compositions of the present invention can be used in clinical nutrition for the treatment or prevention of the conditions described herein.

[0059] Patient is typically understood to mean a mammal, particularly a human.In some cases, the patient may be in remission, and the administration of the composition according to the present patent application may be aimed at preventing or suppressing recurrence, particularly reducing the severity of at least one of the symptoms of disease or functional disorder in the event of recurrence.

[0060] In certain embodiments, the compositions of the invention are intended for veterinary use, typically animal health. In such embodiments, the patient is a non-human mammal, typically selected from among domestic or pet animals (such as cats or dogs) or livestock (ruminants, pigs, goats, sheep, horses, donkeys, etc.).

[0061] The gastrointestinal conditions covered by this patent application may be chronic or not and may be associated with diarrhea or constipation. They typically include functional bowel disorders, infectious bowel diseases, colorectal cancer and / or inflammatory bowel diseases.

[0062] Functional bowel disorders include, inter alia, irritable bowel syndrome, functional bloating, functional constipation, functional diarrhea, and any other unspecified functional bowel disorder (see, inter alia, P. de Saussure & D Bertolini; Rev Med Switzerland 2006; volume 2.31649, “Functional bowel disorders: contributions and limits of evidence-based medicine”). Infectious bowel diseases typically include gastroenteritis, food poisoning, and / or diarrhea caused by viruses, bacteria, or parasites. Chronic inflammatory bowel diseases (CIBD) typically include Crohn's disease and ulcerative colitis.

[0063] The compositions of the present patent application are particularly useful for preventing gastrointestinal colonization by pathogenic microorganisms, reducing adhesion to the intestinal mucosa, and further enhancing the intestinal barrier function against pathogenic microorganisms.Tests for inhibiting adhesion of pathogenic microorganisms to intestinal epithelial cultures such as T84 strains, or to intestinal cells obtained from intestinal biopsies of patients, with or without pre-incubation with the test compositions, are described in particular in the results of the present application as well as in the application WO 2009 / 103884 and the paper by Sivignon et al. (IBD, 2015, vol 21(2):276-286).

[0064] As indicated above, infectious gastrointestinal pathologies, as well as CIBD and colorectal cancer, are generally associated with an imbalance in the intestinal flora (dysbiosis) associated with the presence of pathogenic microorganisms and / or the invasiveness of certain pathogenic microorganisms (see Rahmouni O, Dubuquoy L, Desreumaux P, Neut C. “Enteric microflora in inflammatory bowel disease patients”. Med Sci (Paris). 2016 Nov; 32 (11): 968-973; Kaper JB, Nataro JP, Mobley HL - Pathogenic Escherichia coli), Cuevas-Ramos G et al., “Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells”. PNAS USA, 2010, 107, 11537-11542).

[0065] In general, the present patent application indicates that the compositions described herein are particularly useful for reducing adhesion to and penetration of the gastric and / or intestinal mucosa and associated inflammation, particularly for the conditions listed above.

[0066] The pathogenic microorganisms targeted by the compositions described herein are typically enteric pathogens, typically bacterial species of the family Enterobacteriaceae (such as Salmonella spp., Klebsiella spp., Serratia spp. or E. coli), bacteria of the species Clostridioides difficile or yeasts of the species Candida albicans, advantageously bacteria whose adhesion to cells involves the type 1 fimbrial adhesive FimH (adheson, adhesion). E. coli associated with the colonic mucosa (mucosa-associated E. coli) are preferably targeted, in particular the following types of E. coli: AIEC (adherent invasive E. coli), ETEC (enterotoxigenic E. coli), EIEC (enteroinvasive E. coli), EPEC (enteropathogenic E. coli), EHEC (enterohemorrhagic E. coli), EAEC (enteroaggregative E. coli), DAEC (diffusely adherent E. coli), UPEC (uropathogenic E. coli). In a particular embodiment, colibactin-producing E. coli are of particular interest, in particular in patients suffering from, suffering from or at risk of developing colorectal cancer.

[0067] The present patent application also relates to the non-therapeutic use of the above compositions in the form of a nutraceutical, dietary supplement or functional food, aimed at improving or maintaining intestinal comfort and / or improving the intestinal flora (typically by suppressing intestinal colonization by pathogenic commensal bacteria). Improving or maintaining intestinal comfort is understood to mean, in particular, suppressing or preventing intestinal bloating, suppressing or preventing air swallowing and / or regulating digestion in humans or animals.

[0068] The composition according to the present patent application may comprise, in addition to the active fraction extracted from the yeast cell walls (consisting of the polysaccharide cell wall fraction mentioned above and preferably obtained according to the method described), any excipient, carrier and / or adjuvant conventionally used in the pharmaceutical field or for the formulation of dietary supplements, nutraceuticals or functional foods and chemically compatible with said active fraction (i.e. β-glucan, mannan and α-glucan of the yeast cell walls, in particular the "soluble fraction b" given as an example). For example, the composition of the invention may comprise ingredients selected from among vitamins, trace elements, amino acids and other additives aimed at nutritional intake and / or animal or human health.

[0069] Functional foods or nutraceuticals are understood to mean foods that contain ingredients that have beneficial health effects or can improve physiological functions, particularly digestive health here. Nutraceuticals are understood to mean foods that are intended to supplement the normal diet according to EU Directive 2002 / 46 / EC. Nutraceuticals constitute concentrated sources of nutrients or other substances that have a nutritional or physiological effect when taken alone or in combination in small amounts. Foods with specific nutritional purposes are understood to mean foods with specific nutritional purposes, targeted at clearly defined population groups such as infants, young children or athletes.

[0070] Physiologically acceptable adjuvants, vehicles, and excipients are typically described in the "Handbook of Pharmaceutical Excipients", second edition, American Pharmaceutical Association, 1994. To formulate pharmaceutical compositions according to the present invention, those skilled in the art can also advantageously refer to the latest edition of the European Pharmacopoeia or the United States Pharmacopoeia (USP). Commonly used carriers, excipients, and adjuvants include, but are not limited to, saline, solvents, dispersion media, coatings, preservatives, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents.

[0071] The composition can be used as a drug or active ingredient, or in non-therapeutic use situations, typically as a dietary supplement. The formulation and dosage of the active fraction are then adapted to the selected use.

[0072] In the context of therapeutic use, the compositions may be formulated for oral or enteral administration.

[0073] In the context of a pharmaceutical composition or dietary supplement, the food composition according to the invention may be in different dosage forms, such as in liquid form or in the form of capsules, dragees, pills, powders, suppositories, or any other dosage formulation. As a functional food, the food composition according to the invention may be provided in a wide variety of food and drink forms, for example juices or milk-based preparations. EXAMPLES

[0074] 〔material and method〕 Method for Extracting "Soluble Fraction a" Phosphopeptide Mannan with Fehling's Solution 1.50 g of yeast was suspended in 300 mL of 0.02 M citrate buffer (pH 7) and sterilized in an autoclave at 121° C. for 90 minutes. 2. Centrifuge at 5000 rpm for 30 minutes at 4° C. Collect the supernatant. 3. The pellet is suspended in 300 mL of 0.02 M citrate buffer and autoclaved again for 90 minutes at 121° C. Then, it is centrifuged again to separate the supernatant and the pellet. 4. Combine the two supernatants together (X mL). 5. Add an equal volume of Fehling's solution (X mL) to the supernatant and stir overnight at 4° C. A precipitate forms. The phosphopeptidemannan-copper complex is grayish blue in color. Centrifuge at 5000 rpm for 30 min at 4° C. to collect the precipitate. 6. Add 100 mL of 3N HCl to the precipitate, then stir at 4°C until the precipitate dissolves. The copper complex decomposes and the solution turns green. 7. Precipitate the phosphopeptide mannan by adding 300 mL of ethanol and stirring overnight at 4° C. Centrifuge at 5000 rpm for 30 minutes at 4° C. Collect the precipitate (white). 8. Dissolve the precipitate of phosphopeptide mannan in 50 mL of water. Dialyze (MWCO 3500) against water overnight at 4° C. The dialyzed mannan is then dried and lyophilized.

[0075] [Preparation of Fehling's solution (mix freshly used buffers A and B)] [Buffer A] Dissolve 1.35 g of copper(II) sulfate.5H2O in 300 mL of H2O. Add 2.5 mL of 2N sulfuric acid. 3. Add water to 500mL. [Buffer B (corrosive)] 1.77g sodium hydroxide + 175g potassium sodium tartrate. 2. Add H2O to 500mL.

[0076] Reference: Method for Fingerprinting Yeast Cell Wall Mannan (1969) Journal of Bacteriology, v.100, p1175.

[0077] [Obtaining "soluble fraction b": Separation of β1,3 glucan and β1,6 glucan (including glycogen, i.e., α-glucan) using a hot dilute acetic acid solution] The pellet obtained from 1.50 g of whole yeast or yeast cell wall fraction (the insoluble fraction after extraction with hot citrate buffer, also called "insoluble fraction a") is collected in 1 L of 1 N NaOH solution and then stirred at room temperature for 24 h to remove phosphopeptidemannan residues. 2. Collect the pellet (insoluble fraction a1) after centrifugation at 7000 rpm and 4° C. for 30 minutes. Wash the pellet with 1 L of water, centrifuge again and save the pellet. 3. The pellet is extracted with 800 mL of 0.5 N acetic acid and stirred at a temperature of 90 ° C for 3 hours. After cooling, it is centrifuged at 7000 rpm and 4 ° C for 20 minutes. The supernatant and the pellet are saved. The pellet is extracted again with 800 mL of 0.5 N acetic acid with stirring at 90 ° C for 3 hours, followed by a new centrifugation. Repeat step 3 at least five times until 4.5 L of supernatant (consisting mainly of glycogen and β6-glucan, also called "soluble fraction b") is collected. The supernatant is neutralized with NaOH solution and concentrated by water evaporator. It is then dialyzed (MWCO 3500) overnight at 4°C against water. The resulting fraction constitutes soluble fraction b. Analysis of this fraction demonstrates that it contains β-6 glucan, α-glucan (glycogen), and mannan tightly bound to glucan polymers (i.e., not dissociated by the heat extraction step). 5. The pellet (consisting mainly of β3 glucan) is dialyzed overnight at 4° C. against water. 6. Freeze-dry the dialyzed sample.

[0078] [References] 1.The structure of b(1->3)-D-glucan from yeast cell walls (1973) Biochem J,v.135,p19. 2.Refinement of the structures of cell-wall glucan of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy (2004) Carbohydrate Res.,v.339,p2255.

[0079] [Separation of glycogen and β6-glucan] 1. Add 500 mg of the composition "soluble fraction b" to 50 mL of working solution. Stir and then leave to stand at room temperature for 30 minutes. Red-brown granules are formed (glycogen-iodine precipitate). 2. Centrifuge at 5000 rpm for 30 minutes at room temperature. Save the supernatant and pellet. Add 50 mL of working solution to the supernatant, then add 100 mL of ethanol and stir at room temperature for 30 minutes. 3. Centrifuge again. Combine the granules (pellet 1, mainly glycogen) into a single fraction. Add another 600 mL of ethanol to the supernatant and stir at room temperature for 30 minutes. Pale yellow granules are formed. 4. Centrifuge to collect granules (pellet 2, mainly β6-glucan). 5. To dissociate iodine from glycogen, add 100 mL of 3N HCl to pellet 1 and stir until pellet 1 dissolves (brown solution). Then add 300 mL of ethanol and stir at room temperature for 30 minutes. Black-purple granules then form. Centrifuge and save pellet. Then add another 100 mL of 3N HCl to dissolve the tablets. Add another 300 mL of ethanol and stir at room temperature for 30 minutes, white spheres then form. Centrifuge and save granules (pellet 3, glycogen). 6. Dissolve pellets 2 and 3 in 50 mL of water each. Then dialyze overnight at 4° C. against water. 7. Freeze-dry the dialyzed sample.

[0080] [Preparation of working solution (iodine)] 1. Dissolve 4.35 g of iodine and 43.5 g of potassium iodide in 166.5 mL of water. 2. Add 10.5 mL of saturated CaCl2 solution.

[0081] Reference: Methodologies of tissue preservation and analysis of the glycogen content of the Broiler chicken liver (2007) Poultry Science,v.86,p.2653.

[0082] [Yeast used] CNCM I-3856: Live Saccharomyces cerevisiae yeast deposited under the number CNCM I-3856 LV04 Saccharomyces cerevisiae Brewer's yeast whole (Lesaffre internal collection) Cell wall fraction (shell) of yeast CNCM I-3856 (FP I-3856) Cell wall fraction (shell) of yeast CNCM I-5268 (FP I-5268).

[0083] [Test fractions derived from whole yeast or yeast cell wall (cell wall fraction) compositions] soluble b β6-glucan glycogen β3-glucan phosphate Phosphopeptidemannan (also called Fehling phosphopeptidemannan or "mannan").

[0084] [Preincubation protocol (see Figure 2)] Adhesion test: The test was performed using 1.5 × 10 5 This was performed on T84 or Caco-2 / TC7 cells seeded at 1.2 × 10 cells / well and incubated for 48 hours at 37 °C in an atmosphere containing 5% CO2. 7CFU / mL of AIEC LF82 bacteria were incubated with increasing concentrations of yeast samples (1:1 ratio) for 1 h at room temperature on a Stuart® orbital shaker. After 48 h of incubation, cells were infected with the bacteria / yeast extract mixture for 3 h at 37° C. in an atmosphere containing 5% CO2. Cells were infected at a multiplicity of infection of 10 bacteria / cell. The average adhesion rate (obtained from at least three independent experiments) is expressed as the residual adhesion percentage, which is the ratio of bacterial adhesion in the presence of yeast to adhesion in the absence of yeast, which is considered as 100%. Error bars correspond to the standard error of the mean or SEM.

[0085] Invasion test: The protocol is the same as that for the adhesion test. However, after a 3 h incubation period, the cells are incubated with gentamicin (100 µg / mL) for 1 h to remove extracellular bacteria and count only the invaded bacteria.

[0086] 〔result〕 All fractions were first tested in the adhesion protocol to T84 cells, then additional tests were performed in the invasion protocol on Caco-2 / TC7 cells.

[0087] As shown in Figure 3, the "soluble a" (phosphopeptide mannan) fraction from CNCM I-3856 whole yeast significantly inhibits AIEC bacterial adhesion to T84 epithelial cells. However, surprisingly, the "soluble b" fraction from this same whole yeast also exhibits significant inhibitory activity against AIEC bacterial adhesion.

[0088] FIG. 4 shows that the phosphorylated β3-glucan fraction from CNCM I-3856 whole yeast also significantly inhibited adhesion of AIEC bacteria to T84 epithelial cells, albeit to a more moderate extent.

[0089] Figure 5 shows a comparison of the inhibitory activity of the "soluble b" fraction obtained from whole yeast (CNCM I-3856) or from the cell wall fraction of this same yeast against the adhesion of AIEC bacteria to T84 epithelial cells. Both show significant inhibitory activity, but the fraction obtained from the cell wall fraction shows a higher activity than that obtained from whole yeast (residual adhesion at a dose of 1 mg / mL is 17% and 42%, respectively). These different activities correlate with the different composition of the "soluble b" fraction.

[0090] To verify that the results obtained were not related to the cytotoxic activity of the samples, cytotoxicity tests were performed with increasing doses of soluble fraction b. No cytotoxic effects were observed.

[0091] Figure 6 shows a comparison of the inhibitory activity of the "soluble b" fraction obtained from two yeast cell wall fractions: CNCM I-5268 or CNCM I-3856, against the adhesion of AIEC bacteria to T84 epithelial cells. A very pronounced inhibitory activity is observed for these two fractions. Since the residual adhesion percentages for equal doses are of the same order of magnitude, this result strongly suggests that the increase in inhibitory activity is not directly related to the strain used (CNCM I-5268 vs. CNCM I-3856), but rather to the production process. In other words, the "soluble b" fraction obtained from the yeast cell wall fraction shows a much stronger inhibitory activity against the adhesion of AIEC bacteria to epithelial cells than that observed for the "soluble b" fraction obtained from whole yeast.

[0092] Figure 7 shows the residual adhesion of AIEC LF82 bacteria to T84 epithelial cells in the presence of the "soluble b" fraction, glycogen or β6-glucan obtained from the CNCM I-5268 yeast cell wall fraction. The results show that β6-glucan is less effective than the "soluble b" fraction, and the glycogen fraction has intermediate activity. The "soluble b" fraction is the most effective fraction.

[0093] Figure 8 shows the residual adhesion (as a percentage) of AIEC LF82 bacteria to TC7 / Caco-2 cells in the presence of mannan fractions (Fehling mannan) obtained from whole yeast (CNCM I-3856, or LV04), the "soluble b" fraction obtained from CNCM I3856 whole yeast or the CNCM I-5268 yeast cell wall fraction. These new results confirm those previously obtained with T84 cells, i.e. the "soluble b" fraction obtained from the yeast cell wall fraction has the greatest inhibitory activity.

[0094] FIG. 9 shows results demonstrating strong inhibition of invasion of TC7 / Caco-2 cells by AIEC LF82 bacteria preincubated with increasing doses of the “soluble b” fraction obtained from the cell wall fraction of CNCM I-5268 yeast.

[0095] In vivo study of the activity of yeast fractions in CEABAC10 transgenic mice infected with AIEC LF82 bacterial strain. Intestinal colonization with the AIEC LF82 strain was performed in CEABAC10 transgenic mice expressing the human protein CEACAM6, which functions as a receptor for AIEC bacteria in the context of Crohn's disease.

[0096] It has previously been shown that CNCM I-3856 S. cerevisiae yeast extract reduces colonic colonization by AIEC LF82 bacteria, thus leading to a reduction in colitis symptoms (Sivignon et al. IBD, 2015).

[0097] Preliminary results are now available for the new fractions tested in vitro.

[0098] The protocol applied is shown in FIG.

[0099] Briefly, yeast fractions were orally administered once a day from day -7 to day 0 and twice a day (5 h apart) from days 1 to 3. The fractions were solubilized in PBS at a concentration of 25 mg / mL (daily) and administered orally by gavage (5 mg / mouse) in a volume of 0.2 mL / mouse. From day -3 to day +4, mice received 0.5% DSS in drinking water. On day -1, mice were orally treated with streptomycin (5 mg / mouse).

[0100] At the same time, LB (Luria Bertani) medium was inoculated with AIEC LF82 bacterial strain (at 1 / 100) and incubated at 37°C with stirring until logarithmic growth phase. After centrifugation, the bacteria were cultured at 2.5 × 10 10 The bacteria were then concentrated to 0.2 mL, i.e., 5 × 10 9 Bacteria / mL were gavaged into mice.

[0101] Mice were monitored for weight and symptoms of colitis for 4 days post-infection. Feces were collected on days 1, 2, 3, and 4 post-infection to assess bacterial colonization.

[0102] Mice were euthanized 4 days after infection and the intestines were removed to assess mucosa (ileum + colon) associated bacterial colonization. The fractions tested were as follows: A: untreated batch (n=10); B: CNCM I-3856 whole yeast soluble fraction a) (n=8); C: CNCM I-3856 whole yeast soluble fraction b) (n=8); D: Soluble fraction obtained from CNCM I-5268 yeast cell wall fraction b) (n=8).

[0103] Figure 11 shows the estimated bacterial count in mouse feces 2 and 3 days after infection as a function of the yeast fraction administered. The results show that the amount of bacteria in the feces is reduced by 25 in response to treatment with the soluble fraction b) obtained from the CNCM I-5268 yeast cell wall fraction compared to the untreated group, confirming the excellent anti-adhesive properties of this fraction demonstrated in vitro in preclinical models.

[0104] Figure 12 shows the quantification of AIEC LF82 bacteria associated with the intestinal mucosa in mice treated with yeast fractions or untreated 4 days after infection. The results show the absence of bacteria in 100% of mice treated with soluble fraction b) obtained from the CNCM I-5268 yeast cell wall fraction, thus confirming the results obtained in the feces.

Claims

1. A method for obtaining a composition of yeast cell wall polysaccharides containing β(1,6)-glucan, comprising the steps of: a) obtaining an insoluble composition of yeast cell walls; b) at least one step of fractionating the insoluble yeast cell wall composition obtained at the end of step a) and collecting the insoluble fraction a); c) optionally incubating the insoluble fraction a obtained at the end of step b) in a strong base solution and collecting the insoluble fraction a1; d) at least one step of incubating the insoluble fraction a obtained at the end of step b) or the insoluble fraction a1 obtained at the end of step c) in a weak acid solution and recovering the soluble fraction b; wherein said soluble fraction b comprises a composition of yeast cell wall polysaccharides including β(1,6)-glucan.

2. The method according to claim 1, characterized in that it comprises a step of incubating the insoluble fraction a obtained at the end of step b) in a strong basic solution, and a step of collecting insoluble fraction a1.

3. The method according to claim 1 or claim 2, characterized in that the yeast is selected from yeasts of the genus Saccharomyces.

4. The method according to claim 1 or 2, characterized in that the yeast is selected from yeasts of the species Saccharomyces cerevisiae, in particular obtained from any of the strains deposited at the Collection Nationale de Culture de Microorganismes under the numbers CNCM I-3799, CNCM I-3856, CNCM I-4407, CNCM I-4563, CNCM I-4812, CNCM I-4978, CNCM I-5128, CNCM I-5129, CNCM I-5268 and CNCM I-5269.

5. A composition of yeast cell wall polysaccharides comprising β(1,6)-glucan, characterized in that the composition is obtainable by the method of claim 1 or claim 2, optionally comprising: 5 to 25%, particularly 10 to 20%, of α-glucan; 30 to 50%, particularly 35 to 45%, of β-glucan; 30-55%, especially 40-50% mannan A composition comprising:

6. 6. The composition of claim 5 for use as a medicament.

7. 7. A composition according to any one of claims 6 for the treatment of gastrointestinal conditions associated with pathogenic microorganisms.

8. The composition for use according to claim 6, wherein the composition is for veterinary use.

9. The composition for use according to claim 7, wherein the pathogenic microorganism is E. coli associated with the intestinal mucosa (mucosa-associated E. coli).

10. Non-therapeutic use of the composition described in claim 5 for preparing a food composition intended to improve gastrointestinal comfort and / or improve the intestinal flora in humans or animals.

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