Yeast glycoside inhibitors

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 a method for obtaining a composition of yeast cell wall polysaccharides and its use for the preparation of food supplements aimed at treating gastrointestinal pathologies or improving intestinal comfort.

Background Art

[0002] An imbalance between pro-inflammatory and anti-inflammatory bacterial species of the microbiota, as well as the predominance of certain bacterial families (Enterobacteriaceae, Fusobacterium) or the scarcity of other species (Clostridia, Faecalibacterium), has been described in people with chronic inflammatory bowel disease (CIBD). Specialized strains of Escherichia coli (E. coli) are also responsible for diarrhea, food poisoning, urinary tract infections, sepsis, and meningitis (Kaper J.B., Nataro J.P., Mobley H.L. - Pathogenic Escherichia coli, Nature Review Microbiology, 2004, 2, 123-140). Furthermore, currently, many studies support the concept that the gut microbiota is a major environmental factor that can regulate the risk of colorectal cancer. Thus, the symbiotic bacteria of the microbiota can potentially 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] For example, as described in International Publication No. 2006 / 021965, many microorganisms have already been documented in the literature for their beneficial applications in the human digestive tract and their nutritional value. These microorganisms are then generally referred to as probiotics, which are living microorganisms that can provide health benefits to the host when administered in sufficient quantities (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 Saccharomyces cerevisiae (S. cerevisiae) yeast for treating Enterobacteriaceae-related conditions is known. Therefore, the prior French Patent No. 2928652 teaches that the yeast strain S. cerevisiae (CNCM I-3856, ScProl) is of therapeutic and prophylactic interest for the treatment of pathogenic microorganism-related gastrointestinal conditions, particularly by limiting colonization and / or intrusion into the intestine by these microorganisms. In particular, administration of this yeast leads to a reduction in Enterobacteriaceae in the colon. These results demonstrate that mannoprotein-rich fractions extracted from the yeast strains (ScProl and / or SCB1) can inhibit in vitro adhesion and invasion of human epithelial cells by Escherichia coli strains (AIEC (Adhesive Invasive Escherichia coli) strains) associated with Crohn's disease, one of the major CIBDs. Furthermore, a study by Sivignon et al. (Inflamm Bowel Dis. 2015; 21(2):276-286) also taught that a cell wall compound derived from the CNCM I-3856 strain reduces intestinal colonization (intestinal colonization) and associated colitis symptoms caused by AIEC bacteria in a mouse model mimicking Crohn's disease. Inhibiting AIEC adhesion to the intestinal wall significantly reduces AIEC entry into intestinal tissue, and therefore significantly reduces its infectivity.

[0005] In recent years, glucans isolated from yeast cell walls have attracted increasing attention. Therefore, yeast cell wall glucans are being used to enhance or stimulate immune responses in humans and animals with normal or impaired 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). International publication of a patent application, 2009 / 103884, more specifically teaches that yeast cell wall β-glucans (particularly strain CNCM I-3856) suppress colitis. Jawahara et al. (PLoS One 2012; 7(7):e40648) further disclosed that the β-glucan fraction of S. cerevisiae yeast (particularly strain LYSC 318.2) can inhibit the pathogenicity of Candida albicans. Finally, Pengkumsri et al. (Food Sci Technol, Campinas 2017, 31(1):124-130) disclosed 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, there is currently no satisfactory treatment for CIBD. Surgical removal of damaged portions of the small intestine is sometimes considered, but this is a frequently recurrent and disfiguring surgery. 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 progressive disease, physicians quickly initiate 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 carry significant side effects in the medium to long term. Therefore, the use of corticosteroids is becoming increasingly limited. Thus, it is essential to develop and improve existing prophylactic treatments, enabling improvements in bowel comfort in healthy subjects, and to prevent the onset of disease in at-risk subjects, thereby developing effective and tolerable treatments for patients suffering from gastrointestinal conditions such as CIBD. [Overview of the Initiative]

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

[0008] A composition of yeast polysaccharides comprising β-glucan and / or α-glucan and / or mannan is proposed, characterized in that the yeast polysaccharide is extracted from the yeast cell wall. Typically, the β-glucan is β(1,6)-glucan. In some embodiments, the composition further comprises mannan and α-glucan. In particular, this patent application proposes a composition comprising 5-25%, particularly 10-20%, of α-glucan, 30-50%, particularly 35-45%, of β-glucan (particularly β6-glucan), and 30-55%, particularly 40-50%, of mannan. Typically, the yeast is selected from yeasts of the genus Saccharomyces. Therefore, they may be selected from a group that includes strains deposited with the Collection Nationale de Cultures de Microorganismes (National Collection of Microorganism Cultures) as 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 strain DBVPG 6763 deposited with the Collection dei Lieviti Industriali (Industrial Yeasts Collection).

[0009] In another embodiment, a method for obtaining a yeast composition, a) at least one step of fractionating the composition of the yeast cell wall (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 (referred to as "soluble fraction b") from an insoluble fraction a), wherein the fraction contains β-glucan (particularly β6-glucan), preferably also mannan and generally α-glucan, and this step typically includes at least one step of incubating soluble fraction a) in a weak acid solution to collect (extract) the soluble fraction ("soluble fraction b"). A method is provided that includes this.

[0010] Typically, this method includes a preliminary step to obtain an insoluble yeast cell wall fraction.

[0011] This method preferably includes step a1), incubating the insoluble fraction a) in a strong base solution, and then collecting the insoluble fraction (referred to as insoluble fraction a1). Once this intermediate step is carried out, the fraction (or composition) subsequently incubated in the weak acid solution of step b) above becomes "insoluble fraction a1".

[0012] This disclosure also relates to yeast polysaccharide compositions comprising β-glucan, mannan, and α-glucan as described in this patent application, for use in the treatment and / or prevention of gastrointestinal conditions, and in particular to yeast polysaccharide compositions obtained according to the methods described herein.

[0013] The present invention last relates to the non-therapeutic use of the compositions defined herein, in particular the compositions obtained according to the methods described herein, for preparing food compositions aimed at improving gastrointestinal comfort and / or improving and maintaining the homeostasis of the gut microbiota.

[0014] The features described in the following paragraphs can be implemented at will. They can be implemented independently of each other or in combination. [Brief explanation of the drawing]

[0015] Other features, details, and advantages will become clear by reading the detailed description below and analyzing the attached drawings.

[0016] [Figure 1] This figure illustrates various protocols that can be implemented. Figure (A) above illustrates the extraction of different yeast polysaccharide fractions from whole yeast or yeast cell walls. Figure (B) below corresponds to the method of the present invention in which different yeast polysaccharide fractions are extracted from yeast cell walls. [Figure 2] This diagram shows the pre-incubation protocol for T84 and Caco-2 intestinal epithelial cells. The fraction extracted from yeast is first incubated with AIEC bacteria and then added to a culture of intestinal epithelial cells organized into epithelium. Next, the residual adhesion level of the bacterial strain AIEC LF82 is estimated by decreasing the concentration of the yeast fraction (1; 0.5; 0.25; 0.1 mg / mL). [Figure 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 the entire 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 the entire yeast strain CNCM I-3856 (pre-incubation protocol) (mean ± SEM; **:p) < 0.01, t-test). [Figure 5] Residual adhesion levels (percentage) of strain AIEC LF82 to T84 cells in the presence of the entire yeast strain CNCM I-3856 or "soluble fraction b" (fraction 3) obtained from the cell wall of CNCM I-3856 yeast (pre-incubation 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 (pre-incubation 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 (pre-incubation 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 the entire yeast strain CNCM I-3856 or yeast strain LV04, or "soluble fraction b" (fraction 3) obtained from the entire yeast strain CNCM I-3856 or yeast cell wall of CNCM I-5268 (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 the cell wall of CNCM I-5268 yeast (pre-incubation protocol). [Figure 10] This graph shows the in vivo administration protocol for yeast fraction (YF: yeast fraction) in mice (a mouse model of AIEC colonization). The yeast fraction is administered orally at a dose of 5 mg / mouse. The fractions tested are: "Soluble a" (fraction 1) obtained from the entire yeast strain CNCM I-3856, or "Soluble b" (fraction 3) obtained from the entire yeast strain CNCM I-3856 or from the cell wall of CNCM I-5268 yeast. [Figure 11] Estimation of the number of AIEC LF82 bacteria in mouse feces 2 or 3 days after animal infection, as a function of administered yeast fraction. Results are shown as AIEC bacterial count per gram of feces (box plot, minimum to maximum) (WF: cell wall fraction). [Figure 12] Quantification of AIEC LF82 bacteria associated with the intestinal mucosa of treated and untreated mice, using yeast fractions, 4 days after infection. Results are shown as the number of AIEC bacteria per gram of tissue (box plot, minimum to maximum) (WF: cell wall fraction).

Best Mode for Carrying Out the Invention

[0017] Most of the drawings and the following description contain elements that are essentially certain. Therefore, they can not only help to provide a better understanding of the present disclosure, but can also contribute to its definition if necessary.

[0018] In the present application, "about" is understood to mean within 20%, particularly within 15%, 10%, or 5% variation with respect to the indicated numerical value. As an example, the expression "a temperature of about 100 °C" should be understood as a temperature of 80 °C to 120 °C, particularly 85 °C to 115 °C, more particularly 90 °C to 110 °C, preferably 95 °C to 105 °C.

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

[0020] More specifically, the inventors have demonstrated in a quite surprising way that a composition of yeast polysaccharides extracted from yeast cell wall fragments (also called yeast wall or cell wall) exhibits significantly better adhesion and bacterial invasion inhibitory activities than a yeast polysaccharide composition obtained from the whole yeast. Also surprisingly, the yeast polysaccharide composition according to the present invention containing particularly β1,6-glucan and, if necessary, mannan and α-glucan also exhibits higher activity than the mannan fraction previously described. This result is unexpected because the mannosidic structures exposed by glycoproteins expressed on the surface of intestinal cells were thought to be involved only in the attachment to bacterial cells via the fimbrial structure (FimH) exposed on the surface of type 1 pili (Barnich et al. JCI, 2007).

[0021] Accordingly, this disclosure relates to compositions 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 examples). Typically, the composition further comprises mannan and / or α-glucan.

[0022] Yeast cells are generally composed of an envelope, also called a shell or wall, and its contents. Therefore, in this application, the terms “wall,” “shell,” or “wall” modifying polysaccharides or yeast fragments described herein are used synonymously. Thus, the term “cell wall polysaccharide” means the polysaccharides that constitute the wall (or shell) of the yeast.

[0023] Three main groups of polysaccharides form the yeast cell wall, particularly in Saccharomyces cerevisiae yeast: mannose (or mannan) polymers, which account for approximately 40% of the dry mass of the cell wall; glucose polymers (β-glucan and α-glucan), which account for approximately 60% of the dry mass of the cell wall; and N-acetylglucosamine (chitin) polymers, which account for approximately 2% of the dry mass of the cell wall.

[0024] Glucans are polysaccharides composed of glucose monomers that may or may not be branched. They can be classified into different subtypes depending on the linkage of glucose molecules. α-Glucans are polymers of glucose monomers that are primarily linked by α-linkages (1-4).

[0025] Yeast β-glucans are polysaccharides composed of glucose monomers and can be classified into two subtypes depending on the glucose linkage: long chains of approximately 1500 β-1,3-glucose units, which account for about 85% of yeast β-glucans, and short chains of approximately 150 β-1,6-glucose units, which account for about 15% of yeast β-glucans (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 bonding with β-1,3-glucan, mannoprotein, 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 via an N-glycan core. In particular, Saccharomyces cerevisiae yeast mannans are polymers of α1,6 mannose branched at α1,2 with terminal α1,3 (see Sendid et al., Med Sci(Paris).2009;25(5):473-482). Mannan-rich fractions according to this patent application contain at least 30% mannan, and in particular at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% mannan.

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

[0029] The β3-glucan fraction is understood to mean a fraction rich in β1,3-glucan. Typically, such fractions contain at least 30% β-1,3-glucan polymer, and more particularly, at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% β-1,3-glucan polymer.

[0030] The α-glucan (or glycogen) fraction is understood to mean a fraction rich in α1,4-α1,6 glucans. Typically, such fractions contain at least 60% α1,4-α1,6 glucan polymers, and more particularly at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 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 polysaccharide compositions obtained from yeast cell wall fractions are rich in β-glucan, typically β6-glucan, and preferably mannan. While not wishing to be bound by any theory, the inventors believe that compositions obtained from yeast cell wall fractions, as described above, contain strong, non-dissociable, typically covalently linked heterogeneous polymers composed of α-glucan, β-glucan (particularly β-6-glucan), and mannan.

[0033] Therefore, 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, and especially 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 mannan-rich and may contain 30-55% mannan, particularly 40-50% mannan.

[0035] Preferably, the yeast polysaccharide composition according to this disclosure contains α-glucan, particularly 5-25%, and especially 10-20% α-glucan.

[0036] In a particular embodiment, the composition comprises polysaccharides in the following proportions. 5-25% α-glucan, preferably 10-20% α-glucan, 30-50% β-glucan, preferably 35-45% β-glucan, especially β(1,6)-glucan, It contains 30-55% mannan, preferably 40-50% mannan.

[0037] The yeast wall (or shell) used in this application may be derived from one or more types of yeast. Yeast is a single-celled eukaryotic microorganism belonging to the fungi kingdom. Yeasts of the genus Saccharomyces are particularly suitable for carrying out this disclosure. These yeasts constitute a taxonomic genus of non-mycelial ascomycetes and include several species used as fermenting agents in the food industry. Conventionally, yeasts belonging to the genus Saccharomyces may be selected from 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 baker's yeast, S. cerevisiae var. boulardii). For example, Saccharomyces cerevisiae yeast strains deposited with the Collection Nationale de Cultures de Microorganismes as 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 to this disclosure.

[0038] The compositions of this disclosure can be obtained from one or more yeasts of different strains or species.

[0039] The compositions of this disclosure are typically obtained from yeast wall preparations or yeast wall fragments isolated from cellular contents (cytoplasm), particularly 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 this disclosure. For this, see the reference “Yeast Technology”, 2nd edition, 1991, published by G. Reed and TWNagodawawithana, Van Nostrand Reinhold, New York, ISBN 0-444-31892-8.

[0040] In short, yeast cell wall fragments or yeast shell fragments can be obtained by lysing yeast cells, which are chemical (solvents, acids, bases), physical (sonication, high pressure), enzymatic (typically using proteases and nucleases), or autodegradable (endogenous enzymes), followed by separation of soluble and insoluble parts by physical means, such as centrifugation and collection of the insoluble fraction (or portion). Typically, centrifugation of the lysed yeast cell biomass yields a supernatant and a centrifugation pellet. The supernatant consists mainly of free amino acids and peptides resulting from protein degradation, as well as nucleotides resulting from nucleic acid (RNA, DNA) degradation. The centrifugation pellet contains intact or partially degraded yeast walls in the form of empty yeast cells.

[0041] Yeast autolysis is the hydrolysis of the cellular contents of yeast by the yeast's own enzymes. This is typically obtained by placing a suspension of yeast cells under specific physical culture medium conditions and / or by contacting it with activators that induce apoptosis of yeast cells and the release of their enzymes into the cell body. Hydrolysis of the cellular contents produces soluble compounds. The insoluble fraction collected after the separation step constitutes a product called the yeast cell wall and includes 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] Yeast cell walls may be in liquid form (15–20% dry material), dry form (over 85% dry material), or paste form (25–85% dry material). Yeast cell walls are preferably present in dry form. In some embodiments, CNCM I-5268 yeast wall fragments are used as examples of the compositions and / or methods described herein.

[0043] To carry out the present invention as described herein, yeast cell walls or shells, or any composition containing them, derived from the same type of yeast or different types (strains) or different species of yeast, may be used.

[0044] This disclosure also relates to a method for obtaining the above-mentioned yeast cell wall polysaccharide composition. Preferably, the method includes, in particular, the following: (a) at least one step of fractionating the composition of yeast cell walls and collecting the insoluble fraction, in particular at least one step of thermal extraction of the insoluble fraction from the composition of yeast cell walls (also referred to as “insoluble fraction a” in the examples of this patent application), and (b) at least one step of extraction in a weak acid solution, typically the step of incubating “insoluble fraction a” in a weak acid solution and then collecting a soluble fraction (referred to as “soluble fraction b”).

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

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

[0047] This method preferably includes step a1) of extracting an insoluble fraction in a strong base solution, and typically includes incubating the insoluble fraction obtained at the end of step a) (referred to as “insoluble fraction a”) in a strong base solution, and finally collecting the insoluble fraction (referred to as “insoluble fraction a1”). Once this intermediate step is carried out, the fraction (or composition) incubated in the weak acid solution of step b) above then becomes “insoluble fraction a1”.

[0048] Step a1), extraction in a strong base solution, typically involves 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. Incubation in the strong base solution is typically carried out at room temperature, preferably 16–24°C, and particularly at about 20°C. It is carried out with stirring, ideally for at least 16 hours, typically 16–32 hours, and 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, particularly 20–40 minutes, and preferably about 30 minutes. A high-temperature incubation step follows centrifugation. The inventors believe that this step can remove residual phosphopeptide mannan.

[0049] Step b) Extraction in a weak acid solution typically involves incubating “insoluble fraction a” or “a1” (if an intermediate step of extraction in a strong base has been performed) 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. Incubation in the weak acid solution is typically carried out at a temperature of at least 70°C, particularly 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 (referred to as 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, particularly 20 to 40 minutes, preferably about 30 minutes. This step should be repeated at least twice, especially at least three, four, five, six, seven, or eight times, and especially three to eight times.

[0050] Typically, the centrifugal separation step of the method of this patent application is carried out at room temperature, preferably 16–24°C, and particularly at about 20°C.

[0051] Typically, the “soluble fraction b” obtained according to the protocol described herein is a fraction rich in β-glucan, particularly 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 compositions such as those described above, and has the advantageous effects claimed herein.

[0052] It should be noted that the β3-glucan fraction (i.e., typically rich in β-3 glucans as described above) can be obtained from yeast cell wall fragments (shells) in accordance with this disclosure, provided that the insoluble fraction (also called "insoluble fraction b") is collected at the end of step b). Similarly, this step b) can be repeated at least twice, and especially at least three, four, five, six, seven, eight times, and especially three to eight times.

[0053] In certain embodiments, a β6-glucan-rich fraction (preferably containing at least 60%, particularly at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% β6-glucan) can be obtained from the soluble fraction ("soluble fraction b") obtained at the end of step b). Thus, the isolated fraction of β6-glucan can be obtained, for example, by enzymatic treatment with amyloglucosidase (e.g., derived from Aspergillus niger). However, preferably, the soluble fraction ("soluble fraction b") obtained at the end of step b) can be treated with an iodine solution (as shown in the examples). Incubation in the iodine solution is typically carried out at room temperature (preferably 16–24°C, particularly about 20°C) (ideally with stirring for at least 15 minutes, typically 20–45 minutes, preferably 30 minutes). After treatment with iodine solution, the soluble and insoluble fractions are then separated, typically 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, particularly 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 a 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 complex, and then precipitate it (in conventional methods using 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 described above, the data obtained by the inventors showed that "soluble fraction b" obtained from yeast cell walls significantly inhibited the adhesion of AIEC-type pathogenic bacteria, resulting in a substantial reduction in the invasion of AIEC (adhesive invasive Escherichia coli) bacteria into intestinal tissue and thus a substantial reduction in their infectivity. AIEC has also been shown to adhere to intestinal cells by binding to a mannose-rich glycoprotein known as CEACAM6. Therefore, transgenic mice expressing the human CEACAM6 protein are highly susceptible to AIEC infection, although this pathogenic form is generally not very virulent to rodents. Treatment of mice infected with yeast strains or identified yeast derivatives reduced colonization of the gastrointestinal tract by AIEC bacteria, so the in vitro data were validated.

[0055] While we do not wish to be bound by any particular theory, the inventors believe that the “soluble fraction b” described herein contains alpha(1-4) and (1-6) glucoside units as well as mannoside units, which are important for the presentation of the binding structure or 3D stereostructure. Therefore, such a fraction can effectively inhibit bacterial adhesion to the intestinal wall.

[0056] Accordingly, this patent application relates to the pharmaceutically active use of compositions described herein and / or obtained according to the methods of this disclosure. In particular, the compositions described herein are particularly suitable for the treatment or prevention of gastrointestinal conditions or diseases. The compositions are typically, preferably, obtained from the cell walls of Saccharomyces yeast, and are rich in β-glucan (particularly β6-glucan), and preferably mannan. Preferably, the compositions contain at least 30% β-glucan, and preferably at least 30% mannan.

[0057] As used herein, the terms “to treat” or “to treat” are defined as the administration of a composition described herein to a patient in need of it, for the purpose of curing, alleviating, restoring, improving, and / or affecting a disease and / or any symptom of the disease, particularly gastrointestinal disease, intestinal disorder, or functional intestinal disorder. In particular, the terms “to treat” or “to treat” indicate the reduction or alleviation of at least one undesirable clinical symptom associated with the disease, e.g., pain, inflammation, diarrhea, nausea or vomiting, loss of appetite, or fatigue. As used herein, the terms “to prevent” or “prevent” are defined as the administration of a composition described herein to a patient in need of it, 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 medical conditions described herein.

[0059] The patient is understood to typically mean a mammal, particularly a human. In some cases, the patient may be in remission, and administration of the composition of this patent application may be aimed at preventing or suppressing relapse, in particular at reducing the severity of at least one of the symptoms of disease or functional impairment in a relapsed event.

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

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

[0062] Functional bowel disorders include, in particular, irritable bowel syndrome, functional abdominal distension, functional constipation, functional diarrhea, and any other unspecified functional bowel disorders (see, in particular, 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 composition of this patent application is particularly useful for inhibiting the colonization of the gastrointestinal tract by pathogenic microorganisms, reducing their adhesion to the intestinal mucosa, and further enhancing the intestinal barrier function against pathogenic microorganisms. Tests to inhibit the adhesion of pathogenic microorganisms to intestinal epithelial cultures such as strain T84, or to intestinal cells obtained from patient intestinal biopsies, with or without pre-incubation with the test composition, are described in particular in this application, as well as in the application International Publication No. 2009 / 103884 and the paper by Sivignon et al. (IBD, 2015, vol 21(2):276-286).

[0064] As described above, infectious gastrointestinal lesions, as well as CIBD and colorectal cancer, are generally associated with an imbalance in the gut microbiota (enterotoxosis) related to the presence and / or invasion of 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, this patent application demonstrates that the compositions described herein are particularly useful in reducing adhesion to and invasion of the gastric and / or intestinal mucosa, as well as associated inflammation, particularly for the conditions listed above.

[0066] The pathogenic microorganisms targeted by the compositions described herein are typically enteropathogens, and are typically bacterial species of the Enterobacteriaceae family (such as Salmonella spp., Klebsiella spp., Serratia spp., or Escherichia coli), Clostridioides difficile species, or Candida albicans species, and are advantageously bacteria in which type 1 ciliated adhesion (FimH) is involved in cell adhesion. E. coli associated with the colonic mucosa (mucosal-associated E. coli) are preferably targeted, and in particular the following types of E. coli: AIEC (adhesive invasive E. coli), ETEC (enterotoxotogenic E. coli), EIEC (enteroinvasive E. coli), EPEC (enteropathogenic E. coli), EHEC (enterohemorrhagic E. coli), EAEC (enteroaggregative E. coli), DAEC (diffuse adhesive E. coli), and UPEC (urinary tract pathogenic E. coli). In certain embodiments, colibactin-producing E. coli are of particular interest, especially in patients who have, have had, or are at risk of developing colorectal cancer.

[0067] This patent application also relates to the non-therapeutic use of the above composition in the form of a nutraceutical, dietary supplement, or functional food for the purpose of improving or maintaining intestinal comfort and / or improving the gut microbiota (typically by inhibiting colonization of the intestines by pathogenic commensal bacteria). Improving or maintaining intestinal comfort is understood to mean, in particular, suppressing or preventing intestinal bloating, suppressing or preventing aerophagia, and / or regulating digestion in humans or animals.

[0068] The compositions of this patent application may include, in addition to the active fraction extracted from yeast cell walls (composed of the polysaccharide cell wall fraction described above, preferably obtained according to the method described), any excipients, carriers, and / or adjuvants that have been conventionally used in the pharmaceutical field or for the formulation of nutritional supplements, nutritional supplements or functional foods, and that are chemically compatible with the active fraction (i.e., β-glucan, mannan, and α-glucan of the yeast cell wall, particularly "soluble fraction b" shown as an example). For example, the compositions of the present invention may include components selected from vitamins, trace elements, amino acids, and other additives intended for nutritional intake and / or animal or human health.

[0069] Functional foods or nutritional supplements are understood to mean foods that have beneficial health effects or contain ingredients that can improve physiological functions, particularly digestive health, as used herein. Dietary supplements are understood to mean foods intended to supplement a normal diet, in accordance with EU Directive 2002 / 46 / EC. Dietary supplements, when taken in small amounts alone or in combination, constitute a concentrated source of nutrients or other substances that have nutritional or physiological effects. Foods for specific nutritional purposes are understood to mean foods with specific nutritional purposes, intended for clearly defined population groups such as infants, toddlers, 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 may also advantageously refer to the latest editions of the European Pharmacopoeia or the United States Pharmacopoeia (USP). Commonly used carriers, excipients, and adjuvants include, but are not limited to, physiological salines, solvents, dispersions, coatings, preservatives, antimicrobial and antifungal agents, isotonic agents, and absorption retarders.

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

[0072] In therapeutic applications, the composition may be formulated for oral or intravenous administration.

[0073] In the context of pharmaceutical compositions or nutritional supplements, the food compositions according to the present invention may be in liquid form or in different dosage forms such as capsules, sugar-coated tablets, pills, powders, suppositories, or any other drug formulations. As functional foods, the food compositions according to the present invention may be provided in a wide variety of food and beverage forms, such as juices or milk-based preparations. [Examples]

[0074] 〔material and method〕 [Method for extracting phosphopeptide mannan from "soluble fraction a" using 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. Centrifuge at 2.5000 rpm at 4°C for 30 minutes. Collect the supernatant. 3. Resuspend the pellet in 300 mL of 0.02 M citrate buffer and autoclave again at 121°C for 90 minutes. Then, separate the supernatant and pellet by centrifugation again. 4. Combine the two supernatants (X mL). 5. Add an equal volume of Fehling's solution (X mL) to the supernatant and stir overnight at 4°C. A precipitate will form. The phosphopeptide mannan-copper complex is grayish-blue. Collect the precipitate by centrifugation at 5000 rpm for 30 minutes at 4°C. Add 6,100 mL of 3N HCl to the precipitate, then stir at 4°C until the precipitate dissolves. The copper complex will decompose, and the solution will turn green. Add 7,300 mL of ethanol to precipitate the phosphopeptide mannan, and stir overnight at 4°C. Centrifuge at 5,000 rpm at 4°C for 30 minutes and collect the precipitate (white). 8. Dissolve the phosphopeptide mannan precipitate in 50 mL of water. Dialyze it against water overnight at 4°C (MWCO3500). Then, dry the dialyzed mannan and freeze-dry it.

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

[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 (containing glycogen, i.e., α-glucan) using a high-temperature dilute acetic acid solution] A pellet obtained from 1.50 g of whole yeast or yeast cell wall fraction (insoluble fraction after extraction with high-temperature citrate buffer, also called "insoluble fraction a") is collected in 1 L of 1N NaOH solution and then stirred at room temperature for 24 hours to remove phosphopeptide mannan residue. After centrifuging at 2.7000 rpm at 4°C for 30 minutes, collect the pellet (insoluble fraction a1). Wash the pellet with 1 L of water, centrifuge again, and store the pellet. 3. Extract the pellet with 800 mL of 0.5 N acetic acid and stir at 90°C for 3 hours. After cooling, centrifuge at 7000 rpm at 4°C for 20 minutes. Store the supernatant and pellet. Extract the pellet again with 800 mL of 0.5 N acetic acid while stirring at 90°C for 3 hours, then centrifuge again. Step 3 is repeated at least five times until 4.5 L of supernatant (composed 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 overnight against water at 4°C (MWCO3500). The resulting fraction constitutes soluble fraction b. Analysis of this fraction demonstrates the presence of β-6-glucan, α-glucan (glycogen), and mannan strongly bound to the glucan polymer (i.e., not dissociated by the thermal extraction step). 5. Dialyze the pellet (mainly composed of β3-glucan) in water at 4°C overnight. 6. Freeze-dry the dialysis 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] [Isolation of glycogen and β6-glucan] Add 1,500 mg of the "soluble fraction b" composition to 50 mL of the working solution. After stirring, allow to stand at room temperature for 30 minutes. Reddish-brown granules will form (glycogen-iodine precipitate). 2. Centrifuge at room temperature at 5000 rpm for 30 minutes. 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 will form. 4. Centrifuge to collect granules (pellets 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 (a brown solution). Then add 300 mL of ethanol and stir at room temperature for 30 minutes. Blackish-purple granules will then form. Centrifuge and store the pellet. Next, add another 100 mL of 3N HCl to dissolve the tablet. Add another 300 mL of ethanol and stir at room temperature for 30 minutes until white spheres form. Centrifuge and store the granules (pellet 3, glycogen). 6. Dissolve pellets 2 and 3 in 50 mL of water each. Then dialyze them in water at 4°C overnight. 7. Freeze-dry the dialysis sample.

[0080] [Preparation of the working solution (iodine)] Dissolve 4.35 g of iodine and 43.5 g of potassium iodide in 1.166.5 mL of water. Add 2.10.5 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 (entire strain) (Collected internally at Lesaffre) 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 obtained from a composition of whole yeast or yeast cell walls (cell wall fraction)] soluble b β6-glucan glycogen β3-glucan phosphate Phosphopeptide mannan (also known as Fehling's phosphopeptide mannan or simply "mannan").

[0084] [Protocol for pre-incubation (see Figure 2)] Adhesion test: The test was conducted on a 48-well plate with 1.5 × 10 5 The procedure was performed on T84 or Caco-2 / TC7 cells seeded in cells / well and incubated at 37°C for 48 hours in an atmosphere containing 5% CO2. 1.2 × 10 7CFU / mL of AIEC LF82 bacteria were incubated in a Stuart® orbital shaker at room temperature for 1 hour, gradually increasing the concentration of yeast sample (1:1 ratio). After 48 hours of incubation, cells were infected with a bacterial / yeast extract mixture at 37°C in a 5% CO2 atmosphere for 3 hours. Cells were infected with an infection multiplicity 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, considered to be 100%. Error bars correspond to the standard error of the mean or SEM.

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

[0086] 〔result〕 First, all fractions were tested using the adhesion protocol to T84 cells. Next, additional testing was performed using the invasion protocol against Caco-2 / TC7 cells.

[0087] As shown in Figure 3, the "soluble a" (phosphopeptide mannan) fraction derived from the entire CNCM I-3856 yeast significantly inhibits the adhesion of AIEC bacteria to T84 epithelial cells. Surprisingly, however, the "soluble b" fraction derived from the same entire yeast also shows significant inhibitory activity against AIEC bacterial adhesion.

[0088] Figure 4 shows that the phosphorylated β3-glucan fraction derived from the entire CNCM I-3856 yeast also significantly inhibits the adhesion of AIEC bacteria to T84 epithelial cells, albeit to a more moderate degree.

[0089] Figure 5 shows a comparison of the inhibitory activity of the whole yeast (CNCM I-3856) or the “soluble b” fraction obtained from the cell wall fraction of the 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 higher activity than that obtained from the whole yeast (residual adhesion at a dose of 1 mg / mL is 17% and 42%, respectively). These different activities correlate with the different compositions of the “soluble b” fraction.

[0090] To verify that the obtained results were not related to the cytotoxic activity of the sample, a cytotoxicity test was performed by increasing the dose of soluble fraction b. No cytotoxic effect was observed.

[0091] Figure 6 shows a comparison of the inhibitory activity of "soluble b" fractions obtained from two yeast cell wall fractions: CNCM I-5268 or CNCM I-3856, against the adhesion of AIEC bacteria to T84 epithelial cells. Very significant inhibitory activity is observed in both fractions. Since the residual adhesion percentages for equal doses are similar, 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 yeast cell wall fractions exhibits much stronger inhibitory activity against the adhesion of AIEC bacteria to epithelial cells than 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 "soluble b" fraction obtained from the CNCM I-5268 yeast cell wall fraction, in the presence of glycogen or β6-glucan. The results indicate 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 a mannan fraction (Fehling's mannan) obtained from whole yeast (CNCM I-3856, or LV04), and a “soluble b” fraction obtained from whole CNCM I3856 yeast or CNCM I-5268 yeast cell wall fraction. These new results support the results previously obtained in T84 cells, namely that the “soluble b” fraction obtained from the yeast cell wall fraction has the greatest inhibitory activity.

[0094] Figure 9 shows the results demonstrating potent inhibition of TC7 / Caco-2 cell entry by AIEC LF82 bacteria pre-incubated with an increased dose of the "soluble b" fraction obtained from the cell wall fraction of CNCM I-5268 yeast.

[0095] [In vivo study on the activity of yeast fraction in CEABAC10 transgenic mice infected with the AIEC LF82 bacterial strain] Intestinal colonization by 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] Previous studies have shown that CNCM I-3856 S. cerevisiae yeast extract reduces colonization by AIEC LF82 bacteria, thus leading to a reduction in the symptoms of colitis (Sivignon et al. IBD, 2015).

[0097] Preliminary results are currently available for a new fraction tested in vitro.

[0098] The applicable protocol is shown in Figure 10.

[0099] In short, the yeast fraction was administered orally once daily from day -7 to day 0, and twice daily (5-hour intervals) from day 1 to day 3. The fraction was solubilized in PBS at a concentration of 25 mg / mL (daily), and 0.2 mL / mouse was force-administered orally (5 mg / mouse). From day -3 to day +4, the mice were given 0.5% DSS in drinking water. On day -1, the mice were orally treated with streptomycin (5 mg / mouse).

[0100] Simultaneously, the AIEC LF82 bacterial strain was inoculated (at a 1 / 100 ratio) into LB (Luria Bertani) medium and incubated at 37°C with stirring until the logarithmic growth phase. After centrifugation, the bacteria were divided into 2.5 × 10⁶ units. 10 The solution was concentrated to bacteria / mL. Then, a volume of 0.2 mL, i.e., 5 × 10⁶, was added. 9 Bacteria / mL were administered orally (gavage) to mice.

[0101] For four days after infection, the body weight and symptoms of colitis in the mice were monitored. To assess bacterial colonization, fecal samples were collected on days 1, 2, 3, and 4 post-infection.

[0102] Four days after infection, the mice were euthanized, and their intestines were removed to evaluate bacterial colonization in the mucous membranes (ileum + colon). The fractions tested were as follows: A: Untreated batch (n=10) B: Soluble fraction of the entire CNCM I-3856 yeast a) (n=8), C: CNCM I-3856 Total soluble fraction of yeast b) (n=8) D: Soluble fraction obtained from the cell wall fraction of CNCM I-5268 yeast (b) (n=8).

[0103] Figure 11 shows the estimated bacterial count in mouse feces at 2 and 3 days post-infection as a function of the administered yeast fraction. The results show a 25 reduction in the amount of bacteria in feces in response to treatment with the soluble fraction b) obtained from the CNCM I-5268 yeast cell wall fraction, compared to the untreated group, which supports the superior anti-adhesion properties of this fraction demonstrated in vitro in a preclinical model.

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

Claims

1. A method for obtaining a composition of yeast cell wall polysaccharides containing β(1,6)-glucan, a) A step of 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 a step of collecting the insoluble fraction a, c) The step of incubating the insoluble fraction a obtained at the end of step b) in a strong base solution, and the step of collecting the insoluble fraction a1, d) At least one step of incubating the insoluble fraction a1 obtained at the end of step c) in a weak acid solution, and a step of recovering the soluble fraction b, A method comprising the above, wherein the soluble fraction b comprises a composition of yeast cell wall polysaccharides containing β(1,6)-glucan.

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

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

4. The method according to claim 3, characterized in that the yeast of the species Saccharomyces cerevisiae is obtained from any of the strains deposited with the Collection Nationale de Culture de Microorganismes as 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 containing β(1,6)-glucan, characterized in that it is obtained by the method described in claim 1 or claim 2.

6. 5-25% α-glucan, 30-50% β-glucan, 30-55% mannan and The composition according to claim 5, characterized by containing the following:

7. The composition according to claim 5 for use as a drug.

8. The composition according to claim 7 for the treatment of gastrointestinal conditions related to pathogenic microorganisms.

9. The composition for use according to claim 7, wherein the composition is for veterinary use.

10. The composition for use according to claim 8, wherein the pathogenic microorganism is Escherichia coli associated with the intestinal mucosa (mucosa-associated Escherichia coli).

11. A non-therapeutic use of the composition according to claim 5, for the purpose of preparing a food composition for the purpose of improving gastrointestinal comfort and / or improving the intestinal flora in humans or animals.

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