Patent Application
20240148778
This disclosure relates to a method for obtaining a composition of yeast cell wall polysaccharides and to its use for the treatment of gastrointestinal pathologies or for the preparation of food supplements aimed at improving intestinal comfort.
An imbalance in pro-inflammatory and anti-inflammatory bacterial species of the microbiota, as well as the predominance of certain families of bacteria (Enterobacteriaceae, Fusobacteria) or the rarity of other species (Clostridia, Faecalibacterium), have been described in people who have chronic inflammatory bowel disease (CIBD). Specialized strains of Escherichia coli (E. coli) are also responsible for diarrhea, food poisoning, urinary tract infections, septicemia, and meningitis (Kaper J. B., Nataro J. P., Mobley H. L. —Pathogenic Escherichia coli, Nature Review Microbiology, 2004, 2, 123-140). In addition, many studies now support the concept in which the gut microbiota is a major environmental factor that can modulate the risk of colorectal cancer. Commensal bacteria of the microbiota could thus be directly pro-oncogenic. This is the case, for example, for certain strains of E. coli producing in vivo in the intestinal lumen a genotoxin called colibactin, which induces DNA damage to enterocytes 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).
Many microorganisms have already been described in the literature for their beneficial applications on the digestive tract in humans and for their nutritional value, as described for example in WO 2006/021965. These microorganisms are then commonly referred to by the term probiotics, which corresponds to living microorganisms capable of providing the host with health benefits when administered in sufficient quantity (Joint FAO/WHO Expert Consultation, Probiotics in food, FAO Food and nutrition paper No. 85, ISBN 92-5-105513-0).
More particularly, the use of Saccharomyces cerevisiae (S. cerevisiae) yeasts to treat pathologies associated with enterobacteriaceae is known. Thus, the earlier application FR 2 928 652 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 colonization and/or intestinal invasion by those microorganisms. In particular, administration of this yeast leads to a decrease in enterobacteriaceae in the colon. The results show that fractions rich in mannoproteins extracted from yeast strains (ScProl and/or SCB1) are able to inhibit in vitro the adhesion and invasion of human epithelial cells by strains of E. coli associated with Crohn's disease, one of the main CIBD (AIEC (Adherent Invasive E. Coli) strains). The study by Sivignon et al. (Inflamm Bowel Dis. 2015; 21(2):276-286) also teaches that cell wall compounds derived from the CNCM I-3856 strain reduce intestinal colonization by AIEC bacteria as well as the associated colitis symptoms in a mouse model mimicking Crohn's disease. Inhibiting the adhesion of AIECs to the intestinal wall results in a significant reduction in the invasion of AIECs into intestinal tissues and therefore their infectivity.
In recent years, increasing attention has been paid to glucans isolated from the cell wall of yeasts. Yeast cell wall glucans have thus been used to enhance or prime the immune response in humans and animals who have normal or diminished immunological function (G. Hetland. Curr. Med. Chem.-Anti-infective Agents 2003, 2:135; P. J. Rice, B. E. Lockhart, L. A. Barker, E. L. Adams, H. E. Ensley, D. L. Williams. Int. Immunopharmacol. 2004, 33:829). Patent application WO 2009/103884 teaches more particularly that yeast cell wall beta-glucans (in particular the CNCM I-3856 strain) limit intestinal inflammation. Jawahara et al. (PLoS One 2012; 7(7):e40648) further discloses that the beta-glucan fractions of S. cerevisiae yeasts (in particular the LYSC 318.2 strain) are capable of inhibiting the pathogenic action of Candida albicans. Finally, Pengkumsri et al. (Food Sci Technol, Campinas 2017, 31(1):124-130) discloses that beta-glucan fractions have immunomodulation properties that could be useful in the treatment of human colitis.
To the knowledge of the inventors, currently there is still no satisfactory curative treatment for CIBD. Surgery is sometimes considered, with excision of the injured portions of the small intestine, but this is a disabling operation with frequent relapses. Therefore, generally only the symptoms are treated, such as inflammation (through steroidal or non-steroidal anti-inflammatories and antibodies that target inflammatory cytokines) and chronic pain (typically through analgesics such as cannabinoids). In patients whose disease is progressive, doctors quickly institute immunomodulatory therapy to stop the attacks, prevent the appearance of new lesions, and prevent the risk of tumor development linked to chronic inflammatory conditions. However, these treatments also involve significant side effects in the medium and long term. Corticosteroids are thus being used less and less. It is therefore essential on the one hand to develop and improve existing prophylactic treatments, making it possible to improve intestinal comfort in healthy subjects, and on the other hand to prevent the development of pathologies in subjects at risk as well as to develop effective and better-tolerated therapies for patients suffering from gastrointestinal pathologies such as CIBD.
It therefore remains important to continue with innovative approaches in order to identify new solutions having increased effectiveness in improving the quality of life of patients suffering from gastrointestinal pathologies, in particular pathologies involving infectious agents. In particular, it would be quite relevant to identify new active ingredients or compositions having greater effectiveness in terms of an anti-adhesion and/or anti-invasive action.
A composition of yeast polysaccharides comprising β-glucans and/or α-glucans and/or mannans is proposed, characterized in that said yeast polysaccharides are extracted from yeast cell walls. Typically, the β-glucans are β(1,6)-glucans. In some embodiments, the composition further comprises mannans and α-glucans. In particular, this patent application proposes a composition comprising from 5 to 25%, in particular from 10 to 20%, of α-glucans; from 30 to 50%, in particular from 35 to 45%, of β-glucans (in particular β6-glucans); and from 30 to 55%, in particular from 40 to 50%, of mannans. Typically, the yeasts are chosen among yeasts of the Saccharomyces genus. They can thus be chosen from the group comprising the strains deposited with 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, and strain DBVPG 6763 deposited with the Collezione dei Lieviti Industriali (Industrial Yeasts Collection).
According to another aspect, a method is provided for obtaining a yeast composition comprising:
Typically, the method comprises a preliminary step of obtaining an insoluble yeast cell wall fraction.
The method preferably comprises a step al) of incubating the insoluble fraction a) in a strong base solution, after which the insoluble fraction (referred to as insoluble fraction a1) is collected. When this intermediate step is implemented, the fraction (or composition) incubated in the weak acid solution of step b) described above is then “insoluble fraction a1”.
This disclosure also relates to a composition of yeast polysaccharides comprising β-glucans, mannans, and α-glucans as described in this patent application, in particular as obtained according to the method described herein, for its use in the treatment and/or prevention of gastrointestinal pathologies.
This invention lastly relates to a non-therapeutic use of a composition as defined in this disclosure, and in particular obtained according to the method described herein, for the preparation of a food composition intended to improve gastrointestinal comfort and/or to improve and maintain the homeostasis of the intestinal microbiota.
The features discussed in the following paragraphs may optionally be implemented. They may be implemented independently of each other or in combination with each other.
Other features, details, and advantages will become apparent upon reading the detailed description below, and upon analyzing the appended drawings, in which:
The drawings and the following description for the most part contain elements that are certain in nature. Therefore not only can they serve to provide a better understanding of this disclosure, but where appropriate they also contribute to its definition.
“Approximately” in this application is understood to mean within a variation of 20%, in particular 15%, 10%, or 5%, relative to the numerical value indicated. As an example, the expression “a temperature of approximately 100° C.” should be understood as a temperature of between 80 and 120° C., in particular between 85 and 115° C., more particularly between 90° C. and 110° C., and preferably between 95° C. and 105° C.
The inventors of this application have developed a new method which makes it possible to obtain a composition of yeast cell wall fragments, of which the effectiveness, in particular in inhibiting bacterial adhesion and invasion, particularly of adherent and invasive E coli bacteria (AIEC), is significantly increased in comparison to the whole yeasts or derivatives described in the prior art.
More particularly, the inventors have demonstrated, in a completely surprising manner, that a composition of yeast polysaccharides extracted from yeast cell wall fragments (also called yeast walls or cell walls) exhibits an adhesion- and bacterial invasion-inhibiting activity that is vastly 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 in particular contain 01,6-glucans, and optionally mannans and α-glucans, also exhibit higher activity than the mannan fractions previously described. This result is unexpected, as it had been considered that the mannosidic structures exposed by glycoproteins expressed on the surface of enterocytes were exclusively responsible for the attachment to bacterial cells, via fimbrial structures (FimH) exposed on the surface of type 1 pili (Barnich et al. JCI, 2007).
The present disclosure therefore relates to a composition of yeast cell wall polysaccharides comprising β-glucans. Typically, the β-glucans comprise β(1,6)-glucans (also called 06-glucans below, particularly in the examples). Typically, said composition further comprises mannans and/or α-glucans.
A yeast cell is schematically composed of an envelope, also called shell or wall, and content. In the present application, the terms “parietal”, “shell” or “wall” qualifying the polysaccharides or the yeast fragments described herein are thus used synonymously. The term “cell wall polysaccharides” thus means the polysaccharides that compose the wall (or shell) of the yeast.
Three main groups of polysaccharides form the yeast cell wall, in particular in Saccharomyces cerevisiae yeast: polymers of mannose (or mannans), representing about 40% of the dry mass of the yeast cell wall; polymers of glucose (β-glucan and α-glucan), representing about 60% of the dry mass of the cell wall; and polymers of N-acetylglucosamine (chitin), representing about 2% of the dry mass of the cell wall.
Glucans are polysaccharides composed of glucose monomers that may or may not be branched. They can be separated into different subtypes according to the mode of glucose binding. □-glucans are polymers of glucose monomers that are mainly linked together by □ □bonds (1-4).
Yeast β-glucans are polysaccharides composed of glucose monomers and can be divided into two subtypes according to the mode of glucose binding: long chains of about 1500 β-1,3-glucose units which represent about 85% of the yeast β-glucans, and short chains of about 150 β-1,6-glucose units which represent about 15% of the 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).
The short chains of β-1,6-glucans are involved in covalent bonds with β-1,3-glucan, mannoproteins, and chitin. These cross-links can 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).
The mannans are polymers or oligomers of mannoses which are associated via an N-glycan core. In particular, the Saccharomyces cerevisiae yeast mannans are polymers of α1,6 mannoses branched at α1,2 with terminal α1,3 (see in particular Sendid et al., Med Sci (Paris). 2009; 25(5):473-482). A fraction enriched in mannans according to this patent application comprises at least 30% mannans, in particular at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% mannans.
β6-glucan fraction is understood to mean a fraction enriched in β-1,6-glucan polysaccharides. Typically, such a fraction comprises at least 30% β-1,6-glucans, in particular at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% β-1,6-glucan polymers.
β3-glucan fraction is understood to mean a fraction enriched in 01,3-glucans. Typically, such a fraction comprises at least 30% β-1,3-glucan polymers, in particular at least at least 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% β-1,3-glucan polymers.
α-glucan (or glycogen) fraction is understood to mean a fraction enriched in α1,4 α1,6 glucans. Typically, such a fraction comprises 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.
Yeast polysaccharide compositions described in the prior art and typically obtained from whole yeast typically comprise 60-80% α-glucans, conventionally 65-75% α-glucans, 5-25% β-glucans, conventionally 10-20% β-glucans, and 5-25% mannans, conventionally 10 to 20% mannans.
The inventors have shown that the polysaccharide compositions obtained from yeast cell wall fractions are enriched in β-glucans, typically in 06 glucans, and preferably also in mannans. Without wishing to be tied to any theory, the inventors believe that the compositions obtained from a yeast cell wall fraction comprise heterogeneous polymers associated by strong, non-dissociable and typically covalent bonds, composed of alpha-glucans, beta-glucans (in particular beta-6 glucans), and mannans, as described above.
Thus, the yeast polysaccharide compositions of the invention typically comprise at least 30% β-glucans, in particular 30 to 50% β-glucans, preferably 35 to 45% β-glucans, in particular β(1,6)-glucans.
Preferably, the yeast polysaccharide compositions of this disclosure further comprise mannans. Typically, the compositions of this patent application are enriched in mannans and can comprise from 30 to 55% mannans, in particular from 40 to 50% mannans.
Preferably, the yeast polysaccharide compositions according to this disclosure comprise α-glucans, in particular from 5 to 25%, particularly from 10 to 20% α-glucans.
In certain embodiments, said compositions comprise the following proportions of polysaccharides:
The walls (or shells) of yeast used in this application can be derived from one or more types of yeast. Yeasts are unicellular eukaryotic microorganisms belonging to the fungi kingdom. Yeasts particularly suitable for implementing this disclosure are yeasts of the Saccharomyces genus. These yeasts constitute a taxonomic genus of ascomycetes which do not form mycelium and include several species used in the food industry as fermentation agents. Conventionally, the yeasts belonging to the Saccharomyces genus can be chosen 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 or distiller's or baker's yeast, S. cerevisiae var. boulardii). For example, the Saccharomyces cerevisiae yeast strains deposited with the Collection Nationale de Cultures 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, are particularly well suited for this disclosure.
The compositions of this disclosure can be obtained from one or more yeasts of different strains or of different species.
The compositions of this disclosure are typically obtained from yeast wall preparations or yeast wall fragments separated from the cellular contents (cytoplasm), in particular by extraction of cell wall polysaccharides from yeast wall fragments. The methods for obtaining yeast walls or yeast wall fragments are well known in the technical field of this disclosure. Concerning this, one can consult the reference work “Yeast Technology”, 2nd edition, 1991, G. Reed and T. W. Nagodawawithana, published by Van Nostrand Reinhold, New York, ISBN 0-444-31892-8.
Briefly, yeast cell wall fragments or yeast shell fragments can be obtained by lysis of the yeast cells that is chemical (solvents, acids, bases), physical (sonication, high pressure), enzymatic (typically the use of proteases and nucleases), or autolytic (endogenous enzymes), followed by separation of the soluble and insoluble parts, for example by physical means such as centrifugation and collection of the insoluble fraction (or part). 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, and nucleotides resulting from nucleic acid (RNA, DNA) degradation. The centrifugation pellet contains the intact or partially degraded yeast walls in the form of yeast cells emptied of their contents.
Yeast autolysis is hydrolysis of the yeast's cell contents by its own enzymes. It is typically obtained by placing a suspension of yeast cells under certain physical medium conditions and/or in contact with activators causing apoptosis of the yeast cells and the release of its enzymes into the cell body. Hydrolysis of the cell contents produces soluble compounds. The insoluble fraction collected after the separation step constitutes the product called the yeast cell walls and includes the yeast cytoskeleton and the membranes and components not solubilized by autolysis or heterolysis. This insoluble fraction is often recovered in the form of an aqueous yeast cell wall suspension (or composition).
The yeast cell walls can be in liquid form (15-20% dry matter), dry form (more than 85% dry matter), or paste form (25-85% dry matter). The yeast cell walls are preferably presented in dry form. In some embodiments, wall fragments of CNCM I-5268 yeast are used as examples in the compositions and/or methods described herein.
For implementing the invention described herein, one can use yeast cell walls or shells or any composition containing them, originating from the same type of yeast or yeasts of different types (strains) or different species.
This disclosure also relates to a method for obtaining a composition of yeast cell wall polysaccharides as described above. Preferably, said method comprises in particular:
Typically, said method comprises 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 hydrolyzate (in particular a yeast autolysate), as described above. This step aims in particular to eliminate all or part (at least 60%, in particular at least 75%, more particularly at least 80%, at least 85%, at least 90%, and even more particularly at least 95%) of the compounds not associated or weakly associated with the wall (in this regard see the method for obtaining a yeast cell wall fraction described above).
Step a) of hot extraction (or hot hydrolysis) can be carried out by hot incubation of the yeast cell wall fragments in a solution, typically buffered to neutral pH, at a temperature above 75° C., in particular between 80 and 180° C., more particularly between 90 and 150° C., more particularly between 95 and 145° C. Typically at a temperature of approximately 120° C. Neutral pH is understood to mean a pH of approximately 7, in particular a pH of between 6 and 8. Hot incubation is typically maintained for a period of at least 30 minutes, in particular at least 1 hour, typically between 1 hour and 4 hours, or between 1 hour and 3 hours. Preferably, incubation is maintained for approximately 1.5 hours. The buffer solution is typically a solution based on citrate buffer (approximately 20 mM) or any other equivalent buffer solution in the field. After incubation, the soluble and insoluble fractions are separated, typically by centrifugation, and the insoluble fraction (insoluble a) is collected. Typically the centrifugation can be carried out at a speed of 4000 to 6000 rpm, preferably approximately 5000 rpm, for a duration of at least 20 min, in particular between 20 min and 40 min, preferably approximately 30 minutes. This step can be repeated between 2 and 4 times, preferably 2 times. The soluble fraction, collected at the end of this hot hydrolysis step, typically comprises phosphopeptidomannans, not strongly associated (typically non-covalently associated) with the cell wall polysaccharides.
The method preferably comprises a step a1) of extracting an insoluble fraction in a strong base solution, typically a step of incubating the insoluble fraction obtained at the end of step a) (referred to as “insoluble fraction a”) in a strong base solution, at the end of which the insoluble fraction (referred to as “insoluble fraction a1” is collected. When this intermediate step is implemented, the fraction (or composition) incubated in the weak acid solution of step b) described above is then “insoluble fraction a1”.
Step a1) of extracting in a strong base solution typically includes a step of incubating 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 between 16 and 24° C., in particular at about 20° C. It is ideally carried out while stirring for a period of at least 16 hours, typically between 16 hours and 32 hours and preferably 24 hours. The soluble and insoluble fractions are then separated, typically by centrifugation, and insoluble fraction a1) is recovered. Typically the centrifugation can be carried out at a speed of 5000 to 8000 rpm, preferably approximately 7000 rpm, for a duration of at least 20 min, in particular between 20 min and 40 min, preferably approximately 30 minutes. The hot incubation steps follow the centrifugation. The inventors believe that this step allows eliminating residual phosphopeptidomannans.
Step b) of extracting in a weak acid solution typically includes a step of incubating “insoluble fraction a” or “a1” (if the intermediate step of extracting in a strong base has been implemented) in a weak acid solution. The weak acid solution is typically a solution of acetic acid or any other equivalent, preferably concentrated to approximately 0.5 N. The incubation in the weak acid solution is typically carried out at a temperature of at least 70° C, in particular between 75 and 130, more particularly between 75 and 115° C, more particularly at approximately 90° C. It is ideally carried out, preferably with stirring, for a period of at least 1 hour, typically between 2 and 4 hours and preferably 3 hours. The soluble and insoluble fractions are then separated, preferably by centrifugation, and the soluble fraction (referred to as soluble fraction b) is collected. Typically the centrifugation can be carried out at a speed of 4000 to 6000 rpm, preferably approximately 5000 rpm, for a duration of at least 20 min, in particular between 20 min and 40 min, preferably approximately 30 minutes. This step is repeated at least twice, in particular at least 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, in particular between 3 and 8 times.
Typically the centrifugation steps of the method of this patent application are carried out at room temperature, preferably between 16 and 24° C., in particular at approximately 20° C.
Typically the “soluble fraction b” obtained according to the protocol described herein is a fraction enriched in β-glucans, in particular β-6 glucans, and preferably also in mannans (as defined above). Such a fraction also typically contains α-glucans. The method of the invention thus makes it possible to obtain a composition in particular as described above, and having the advantageous effects as claimed herein.
It should be noted that a β3-glucan fraction (i.e. typically enriched in beta-3 glucans as described above) can be obtained from yeast cell wall fragments (shells) according to this disclosure, when the insoluble fraction (also referred to as “insoluble fraction b”) is collected at the end of step b). Similarly, this step b) can be repeated at least twice, in particular at least 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, in particular between 3 and 8 times.
In certain embodiments, a fraction enriched with β6-glucans (preferably containing at least 60%, in particular at least 65, 70%, 75%, 80%, 85%, 90%, 95%, 99% β6-glucans) can be obtained from the soluble fraction obtained at the end of step b) (“soluble fraction b”). Thus, an isolated fraction of β6-glucans can for example be obtained by enzymatic treatment, typically with an amyloglucosidase (for example from Aspergillus niger). Preferably, however, the soluble fraction obtained at the end of step b) (“soluble fraction b”) can be treated with an iodine solution (as illustrated in the examples). Incubation in the iodine solution is typically carried out at room temperature (preferably between 16 and 24° C., in particular at approximately 20° C.) (it is ideally carried out with stirring, for a period of at least 15 min, typically between 20 and 45 min, and preferably 30 min). After treatment with the iodine solution, the soluble and insoluble fractions are then separated, typically by centrifugation, and the soluble fraction (also referred to as “soluble fraction c”) is collected. Typically the centrifugation can be carried out at a speed of 4000 to 6000 rpm, preferably approximately 5000 rpm, for a duration of at least 20 min, in particular between 20 min and 40 min, preferably approximately 30 minutes. An ethanol solution can then be added to the supernatant and the solution centrifuged under the same conditions as before. A concentrated solution of strong acid (typically a hydrochloric acid solution concentrated to at least 2N, preferably to about 3N) is added to the pellet to dissociate the β6-glucan and α-glucan complexes, then precipitated (in conventional manner with ethanol or any equivalent). This second method makes it possible to collect isolated β6-glucan and α-glucan enriched fractions (see also the methods described in the results).
As indicated above, the data obtained by the inventors have shown that the “soluble fraction b”, obtained from yeast cell walls, very significantly inhibits the adhesion of type AIEC pathogenic bacteria, with the result of significantly reducing the invasion of AIEC (adherent invasive E. coli) bacteria in the intestinal tissues and therefore their infectivity. AIEC have 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 very susceptible to AIEC infections although this pathotype is generally not very virulent against rodents. Treatment of mice infected with yeast strains or identified yeast derivatives has reduced colonization of the digestive tract by AIEC bacteria, thus validating the in vitro data.
Without wishing to be tied to any particular theory, the inventors believe that the “soluble fraction b”, as described herein, comprises alpha (1-4) and (1-6) glucosidic units as well as mannosidic units which seem important for the presentation or the 3D conformation of the binding structures, such that such a fraction makes it possible to effectively inhibit the adhesion of bacteria to the intestinal wall.
This patent application thus relates to the use of a composition exhibiting an advantageous activity, as described herein, and/or obtained according to the method of this disclosure, as a medication. In particular, a composition as described herein is particularly suitable for the treatment or prevention of gastrointestinal pathologies or diseases. Said composition is typically a composition obtained from yeast cell walls, preferably of the Saccharomyces genus and enriched in β-glucans (in particular 06 glucans) and advantageously also in mannans. Preferably, said composition comprises at least 30% β-glucans and advantageously at least 30% mannans.
The terms “treatment” or “treating” as used herein, are defined as the administration of a composition as described herein to a patient in need thereof, for the purpose of curing, alleviating, remedying, improving, and/or affecting disease, and/or any symptom of disease, in particular gastrointestinal disease, a bowel disorder, or a functional bowel disorder. In particular, the terms “treating” or “treatment” indicate the reduction or alleviation of at least one undesirable clinical symptom associated with the disease, for example pain, inflammation, diarrhea, nausea or vomiting, loss of appetite, or fatigue. The terms “prevent” or “prevention” as used herein are defined as the administration of a composition as described herein to a patient in need thereof, with the aim of preventing the onset of a disease or of at least one of its symptoms and/or of reducing the severity of at least one of its symptoms.
In certain embodiments, the compositions of the invention can be used in clinical nutrition for the treatment or prevention of the pathologies described herein.
Patient is typically understood to mean a mammal and in particular a human being. In some cases, the patient may be in remission and the administration of a composition according to this patent application may be aimed at preventing or limiting relapses, in particular at reducing the severity of at least one of the symptoms of the disease or of the functional disorder in the event of relapse.
In certain embodiments, the composition of the invention is intended for veterinary use, typically for animal health. In such embodiments the patient is a non-human mammal, typically chosen among domestic or companion animals (such as cats or dogs) or livestock (ruminants, pigs, goats, sheep, horses, donkeys, etc.).
The gastrointestinal pathologies covered by this patent application may or may not be chronic, possibly associated with diarrhea or constipation. They typically include functional bowel disorders, infectious bowel diseases, colorectal cancer, and/or inflammatory bowel diseases.
Functional bowel disorders include in particular irritable bowel syndrome, functional abdominal distension, functional constipation, functional diarrhea, and any other unspecified functional bowel disorder (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, of viral, bacterial, or parasitic origin. Chronic inflammatory bowel disease (CIBD) typically includes Crohn's disease and ulcerative colitis.
The compositions of this patent application are particularly useful for combating gastrointestinal colonization by pathogenic microorganisms, reducing adhesion to the intestinal mucosa, and even strengthening the intestinal barrier function against pathogenic microorganisms. Tests for inhibiting the adhesion of pathogenic microorganisms to intestinal epithelial cultures such as the T84 line, or to enterocytes obtained from intestinal biopsies of patients, with or without preincubation with the tested composition, are described in particular in the results of this application as well as in application WO 2009/103884 and the article by Sivignon et al. (IBD, 2015, vol 21(2):276-286).
As indicated above, infectious gastrointestinal pathologies, as well as CIBD and colorectal cancer, are generally associated with the presence of pathogenic microorganisms and/or an imbalance of the intestinal microbiota (dysbiosis) linked to the invasive nature of a specific pathogenic microorganism (see Rahmouni O, Dubuquoy L, Desreumaux P, Neut C. “Enteric microflora in inflammatory bowel disease patients”. Med Sci (Paris). 2016 November; 32(11):968-973; Kaper J. B., Nataro J. P., Mobley H. L. —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).
In general, it has been shown in this patent application that the compositions described herein are particularly useful for reducing adhesion to and invasion of gastric and/or intestinal mucous membranes and the associated inflammation, in particular for the pathologies listed above.
The pathogenic microorganisms targeted by the compositions described herein are typically intestinal pathogens, typically bacterial species of the Enterobacteriaceae family (such as Salmonella spp., Klebsiella spp., Serratia spp., or Escherichia coli (E. coli), bacteria of the Clostridioides difficile species, or yeasts of the Candida albicans species, advantageously bacteria whose adhesion to cells involves the type 1 pili adheson FimH. E. coli bacteria associated with colonic mucosa (mucosa associated E. coli) are preferably targeted, and in particular E. coli bacteria of the following types: 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 certain embodiments, colibactin-producing E. coli bacteria are of particular interest, in particular in patients suffering from colorectal cancer, having suffered from colorectal cancer, or at risk of developing colorectal cancer.
This patent application also relates to the non-therapeutic use of a composition as described above in the form of a nutraceutical, a food supplement, or a functional food aimed at improving or maintaining intestinal comfort and/or improving the intestinal flora (typically by limiting the intestinal colonization by pathogenic commensal bacteria). Improving or maintaining intestinal comfort is understood in particular to mean limiting or preventing intestinal bloating, limiting or preventing aerophagia, and/or regulating digestion, in humans or animals.
A composition according to this patent application may comprise, in addition to the active fraction extracted from yeast cell walls (composed of the polysaccharide cell wall fraction as described above and preferably obtained according to the described method), any excipient, carrier, and/or adjuvant conventionally used in the pharmaceutical field, or for the formulation of a food supplement, nutraceutical, or functional food, and which is chemically compatible with said active fraction (i.e. β-glucans, mannans, and yeast cell wall α-glucans, in particular the “soluble fraction b” illustrated as an example). For example, a composition of the invention may comprise components chosen among vitamins, trace elements, amino acids, and other additives intended for nutrition and/or animal or human health.
Functional food or nutraceutical are understood to mean a food that contains ingredients having beneficial health effects or capable of improving physiological functions, in particular herein for digestive well-being. Food supplement is understood to mean a foodstuff intended to supplement the normal diet, in accordance with EU Directive 2002/46/EC. A food supplement constitutes a concentrated source of nutrients or other substances having a nutritional or physiological effect, when taken alone or in combination, in small quantities. Foodstuffs intended for specific nutrition is understood to mean a food with a specific nutritional objective, intended for a well-defined population group, such as infants, young children, or athletes.
Physiologically acceptable adjuvants, vehicles, and excipients are typically described in the “Handbook of Pharmaceutical Excipients”, second edition, American Pharmaceutical Association, 1994. To formulate a pharmaceutical composition 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 solutions, solvents, dispersing media, coatings, preservatives, antibacterial and antifungal agents, isotonic agents, and absorption-delaying agents.
The composition may be used as a drug or as an active ingredient, or in a context of non-therapeutic use, typically as a dietary supplement. The formulation and dosage of the active fraction are then adapted to the chosen use.
In the context of therapeutic use, the composition may be formulated for oral or enteral administration.
In the context of a pharmaceutical composition or a food supplement, the composition of the food according to this invention may be in different dosage forms, such as liquid form or in the form of a capsule, sugar-coated tablet, pill, powder, suppository, or any other dosage formulation. As a functional food, the composition of the foods according to this invention may be presented in a wide variety of food and drink forms, for example juices or milk-based preparations.
Materials and Methods
Method for the extraction of “soluble fraction a” phosphopeptidomannans by Fehling's solution:
Precipitates will form. The phosphopeptidomannan-copper complex is grey-blue. Collect the precipitate by centrifugation at 5000 rpm, 4° C., for 30 minutes.
The copper complex decomposes, and the solution turns green.
Collect the precipitate (white color) by centrifugation at 5000 rpm, 4° C., for 30 minutes.
Preparation of Fehling's solution (mix freshly used buffers A and B)
Buffer A
Buffer B (Corrosive)
Obtaining “soluble fraction b”: separation of β1,3 Glucans and β1,6 Glucans (with Glycogen i.e. α-glucans) by a hot dilute acetic acid solution:
Allow to cool then centrifuge at 7000 rpm at 4° C. for 20 min. Save the supernatant and the pellet. The pellet is extracted again with 800 mL of 0.5 N acetic acid, while stirring at 90° C. for
References:
Separation of glycogen and β6-glucans
Light yellow granules are formed.
Preparation of the working solution (iodine):
Reference: Methodologies of tissue preservation and analysis of the glycogen content of the Broiler chicken liver (2007) Poultry Science, v.86, p. 2653.
Yeasts used:
Fractions tested, obtained from compositions of whole yeast(s) or of yeast cell walls (cell wall fraction):
Protocols for preincubation (see
Results:
Initially, all fractions were tested in a protocol of adhesion to T84 cells. Additional tests were then performed on Caco-2/TC7 cells and in an invasion protocol.
As illustrated in
In order to verify that the obtained result was not linked to any cytotoxic activity of the sample, a cytotoxicity test was carried out with increasing doses of soluble fraction b. No cytotoxic effects were observed.
In vivo studies of the activity of yeast fractions in CEABAC10 transgenic mice infected with the AIEC LF82 bacterial strain.
Colonization of the intestinal tract by the AIEC LF82 strain was carried out in CEABAC10 transgenic mice expressing the human protein CEACAM6, acting as a receptor for AIEC bacteria in the context of Crohn's disease.
It has previously been shown (Sivignon et al. IBD, 2015) that CNCM I-3856 S. cerevisiae yeast extracts reduced colonization of the colon by the AIEC LF82 bacterium, thus leading to a reduction in colitis symptoms.
Preliminary results have now been obtained with the new fractions tested in vitro.
The protocol applied is illustrated in
Briefly, yeast fractions were administered orally once daily from day −7 to day 0, and twice daily (5 hours apart) from day 1 to day 3. The fractions were solubilized in PBS at a concentration of 25 mg/mL (every day) and an amount of 0.2 mL/mouse was administered by gavage (5 mg/mouse). From day −3 to day +4, the mice received 0.5% DSS in drinking water. On day −1, the mice were treated p.o. with streptomycin (5 mg/mouse).
At the same time, LB (Luria Bertani) culture medium was inoculated (at 1/100th) with the AIEC LF82 bacterial strain and incubated at 37° C. while stirring until the exponential growth phase. After centrifugation, the bacteria were concentrated to 2.5×1010 bacteria/mL. A quantity of 0.2 mL was then administered by gavage to the mice, i.e. 5×109 bacteria/mL.
The mice's weight and colitis symptoms were monitored for 4 days after infection. Feces were collected on days 1, 2, 3, and 4 post-infection in order to assess bacterial colonization.
The mice were euthanized on the fourth day after infection and the intestine was removed to assess the bacterial colonization associated with the mucosa (ileum+colon).
The fractions tested were as follows:
| Number | Date | Country | Kind |
|---|---|---|---|
| 2102618 | Mar 2021 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2022/050459 | 3/15/2022 | WO |
