Physiologically acceptable yeast compositions and uses thereof

Abstract

A physiologically acceptable composition comprising: (i) at least one component selected from the group consisting of inactivated S. boulardii yeast, S. boulardii lysate, S. boulardii cell wall components, and S. boulardii extract; and (ii) at least one component selected from the group consisting of inactivated S. cerevisiae yeast, S. cerevisiae lysate, S. boulardii cell wall components, and S. boulardii extract. and (iii) at least one component selected from the group consisting of inactivated K. marxianus yeast, K. marxianus lysate, K. marxianus cell wall component, and K. marxianus extract. The present invention further relates to the composition for use as a pharmaceutical; as a food additive or functional ingredient for dietary supplements, foods for special medical purposes, cosmetics, and functional foods; as a feed additive for functional ingredients in animal nutrition; and as an ingredient for topical application.

Description

[Technical field]

[0001] The present invention relates to a physiologically acceptable composition comprising yeast. The present invention further relates to a method for preparing said composition. The present invention further relates to a composition for use as a medicine; as a food additive or functional ingredient for dietary supplements, foods with special medical purposes, cosmetics and functional foods; as a feed additive for functional ingredients in animal nutrition; as an ingredient for topical application. In particular, the composition according to the present invention is suitable for use in the treatment of a medical disease, for maintaining and improving the health of the digestive tract. In a preferred embodiment, the disease is defined by proinflammatory markers. In a preferred embodiment, the disease is a gastrointestinal disease. The treatment of a medical disease according to the present invention can be a preventive treatment or a treatment of an individual having the disease. [Background technology]

[0002] Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Humans have already benefited from microorganisms in food, for example in fermented milk and yogurt, for centuries, long before the potential health benefits of probiotics became known. Modern probiotic-containing nutrients and medicines are direct descendants of early fermented foods. To date, the most common probiotics are bacteria of the genera Lactobacillus and Bifidobacterium, although yeasts are also increasingly being considered as effective probiotic organisms.

[0003] Yeast-based probiotics are recommended by several international guidelines to treat acute gastrointestinal disorders, such as diarrhea, or chronic diseases, such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). The probiotic activity of these yeasts is believed to be multifactorial and includes improvement of gastrointestinal barrier function, competitive exclusion of pathogens, production of antimicrobial peptides, immunomodulation, modulation of the microbiota, and nutritional effects. Yeast-based probiotics have many advantages over bacterial probiotics, such as surviving the extreme environment of the stomach to reach the intestine and being less susceptible to antibiotics, thus offering the possibility of a probiotic effect during antibiotic treatment. Several yeast species have been shown to have probiotic effects. For example, several strains belonging to the genera Chrysonilia, Debaromyces, Hanseniaspora, Kluyveromyces, Lachanencea, Metschnikowia, Pichia, Saccharomyces, Torulaspora, and Yarrowia have been suggested to act as probiotics (Ogunremi et al., 2015. J appl microbiol 117:797-808; Sugiharto et al., 2018. J adv vet 5(3):332-342; Agarbati et al., 2020. Foods 9(3):287; Dufosse et al., 2019. Foods 9(3):287; et al.,2021.J Fungi 7(3):177).

[0004] To address the need for new strains of microorganisms that can exert preventive and / or therapeutic health benefits against specific pathologies or dysfunctions, or against general physical and mental health conditions, US 2010 / 303778 provides specific Saccharomyces cerevisiae strains (deposited at the Collection Nationale de Cultures de Microorganismes under No. CNCM I-3856) and specific Saccharomyces var. boulardii yeast strains (deposited at the Collection Nationale de Cultures de Microorganismes under No. CNCM I-3799), which can be used in compositions for treating, for example, intestinal diseases. Summary of the Invention [Problem to be solved by the invention]

[0005] There continues to be a need to provide alternative treatment possibilities for medical disorders, particularly gastrointestinal disorders, such as diarrhea, IBD or IBS, or disorders in which inflammatory producers play a role, such as rheumatoid arthritis, osteoarthritis, topical dermatitis, psoriasis, allergies or obesity. There also continues to be a need to provide alternative products suitable for improving gastrointestinal health or for improving gastrointestinal function. In particular, it is an object to provide a composition suitable for providing improved protection of gastrointestinal barrier function or anti-inflammatory effects, compared to known probiotic compositions containing yeast, such as yeast as described in the prior art above. One or more further objects that can be addressed will become clear from the following description herein. [Means for solving the problem]

[0006] We have now discovered that one or more of several of the above objectives may be achieved by providing a particular composition comprising at least three different yeast species.

[0007] Accordingly, the present invention provides a physiologically acceptable composition comprising (i) at least one component selected from the group consisting of S. boulardii yeast, S. boulardii lysate, S. boulardii cell wall components, and S. boulardii extracts, and (ii) at least one component selected from the group consisting of S. cerevisiae yeast, S. cerevisiae lysate, S. boulardii cell wall components, and S. boulardii extracts. The present invention relates to a composition as described above, further comprising at least one component selected from the group consisting of S. cerevisiae cell wall components, and S. cerevisiae extracts, and further comprising at least one component selected from the group consisting of K. marxianus yeast, K. marxianus lysate, K. marxianus cell wall components, and K. marxianus extracts. As shown in the examples, particularly good results have been achieved with physiologically acceptable compositions comprising inactivated S. boulardii yeast, inactivated S. cerevisiae yeast, and inactivated K. marxianus yeast.

[0008] Furthermore, the present invention relates to said physiologically acceptable composition for use as a medicament.

[0009] Moreover, the present invention relates to said physiologically acceptable compositions for use in the treatment of humans or animals by therapy.

[0010] Furthermore, the present invention relates to a physiologically acceptable composition according to the invention for use in maintaining or improving gastrointestinal health or function.

[0011] Moreover, the present invention relates to a physiologically acceptable composition according to the present invention for use in treating an individual with a gastrointestinal disorder.Preferred gastrointestinal disorders to be treated are diseases associated with impaired gastrointestinal barrier function.In a particularly preferred embodiment, the composition according to the present invention is for use in treating diarrhea, IBD or IBS.

[0012] Moreover, the present invention relates to a physiologically acceptable composition according to the invention for use in the treatment of an individual having a disease defined by one or more proinflammatory markers, in particular one or more markers selected from the group consisting of IL-8, IP-10, MCP-1, TNFα and TNFα / IL-10. With regard to IL-10, it is observed that this is a regulatory / anti-inflammatory compound and its concentration is reduced and can also result in an increased TNFα / IL-10 ratio when TNFα itself is not increased. This ratio is also a relevant marker for a disease defined by one or more proinflammatory markers.

[0013] Preferably, the disease defined by one or more pro-inflammatory markers to be treated according to the present invention is selected from the group consisting of rheumatoid arthritis, osteoarthritis, topical dermatitis, psoriasis, allergies and obesity.

[0014] Moreover, the present invention relates to the use of the physiologically acceptable composition as a food additive, a feed additive, a functional food in human nutrition, a functional food in animal nutrition, a food additive or a functional ingredient for a dietary supplement.

[0015] Moreover, the present invention relates to methods of using the physiologically acceptable compositions as probiotics, postbiotics, paraprobiotics, prebiotics, symbiotics, or probiotic-substitutes.

[0016] Additionally, the present invention relates to medical devices comprising a physiologically acceptable composition.

[0017] In uses (non-medical or medical) according to the present invention, the (i) S. boulardii components, (ii) S. cerevisiae components, and (iii) K. marxianus components, in particular the inactivated S. boulardii yeast, the inactivated S. cerevisiae yeast, and the inactivated K. marxianus yeast, are typically the active ingredients of the composition.

[0018] The (medical) use according to the invention can be a preventive treatment (prophylactic) or treatment of an individual, particularly a human, having a medical condition to be treated. Treating an individual having a medical condition can include curing the medical condition, relieving the pain, or alleviating or relieving one or more symptoms associated with said medical disorder.

[0019] The efficacy of prophylactic treatment can be routinely determined, for example, by comparing a cohort or test animals treated with a composition for use according to the present invention with a reference product (placebo) having a reduced incidence. The effect of treating an individual with a disease can be a complete cure, relief of symptoms, relief of suffering, etc. Effect of the Invention

[0020] As shown in the examples below, the composition according to the present invention has been found to be effective in improving one or more relevant markers of gut health, such as a reduction in inflammatory cytokine production by gut epithelial cells and immune cells (i.e., indicating an anti-inflammatory effect) and an increase in transepithelial electrical resistance (i.e., indicating improved protection of gut barrier function). The effect on these markers indicates a positive effect for use in the treatment of gastrointestinal diseases characterized by inflammation and / or loss of epithelial integrity, such as diarrhea, IBD or IBS. Furthermore, the results support that the composition is also effective for the treatment or prevention of gastrointestinal disorders, such as diarrhea or gastrointestinal infection, because several markers of gut health are improved by using pro-inflammatory stimuli or by simulating acute gastrointestinal infection by infecting epithelial cells with bacteria known to induce diarrhea, such as typical diarrhea-causing E. coli strains. In particular, a surprising effect is obtained by combining three yeasts: S. boulardii, S. cerevisiae and K. marxianus. In the examples, it is shown how several markers of gut health are improved by such a combination of yeasts (in non-viable form), while the individual yeasts (in non-viable form) have no effect, less effect or a negative effect. Synergistic effects are shown, inter alia, for MCP-1 (Figure 2), for IL-8 (Figure 3), for IP-10 and MCP-1 after TNF-alpha / IFN-gamma challenge (Figures 4 and 5), and for the TNF-alpha / IL-10 ratio (Figure 7). Furthermore, we show how the yeast combination improves the integrity of the gut epithelium, as measured by an increase in trans epithelial electrical resistance (TEER) across gut cell monolayers (Figures 8, 9 and 10).

[0021] Physiological compositions according to the present invention may include viable yeast cells selected from the group of S. boulardii, S. cerevisiae, and K. marxianus, however, as illustrated by the examples, viable yeasts need not be present. Therefore, the inventors have further concluded that one, two or each of S. boulardii, S. cerevisiae and K. marxianus in the composition may also be replaced, in whole or in part, by yeast lysate, cell wall material or yeast extract of S. boulardii, S. cerevisiae and K. marxianus, respectively. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 illustrates in vitro IP-10 chemokine production by Caco-2 cells after incubation with the three yeasts and their combinations in the absence of inflammatory stimuli. [Diagram 2] FIG. 2 describes the in vitro production of the MCP-1 chemokine by Caco-2 cells after incubation with the three yeasts and their combinations in the absence of inflammatory stimuli. [Diagram 3] FIG. 3 describes the in vitro production of IL-8 chemokine by Caco-2 cells after incubation with the three yeasts and their combinations in the absence of inflammatory stimuli. [Figure 4]Figure 4 illustrates in vitro IP-10 chemokine production by Caco-2 cells after incubation with the three yeasts and their combinations in the presence of pro-inflammatory stimuli (TNF-α / INF-γ) simulating inflamed gastrointestinal epithelium. [Diagram 5] Figure 5 describes the in vitro production of the MCP-1 chemokine by Caco-2 cells after incubation with the three yeasts and their combinations in the presence of pro-inflammatory stimuli (TNF-α / INF-γ) simulating an inflamed gastrointestinal epithelium. [Figure 6] Figure 6 describes the in vitro production of IL-8 chemokine by Caco-2 cells after incubation with the three yeasts and their combinations in the presence of pro-inflammatory stimuli (TNF-α / INF-γ) simulating an inflamed gastrointestinal epithelium. [Figure 7] FIG. 7 shows the in vitro decrease in the TNFα / IL-10 ratio observed in human THP-1 cells (macrophages). [Figure 8] Figure 8 illustrates the protective effect of different yeasts and their combinations on the gastrointestinal epithelium. A comparison of yeasts with a negative control is shown after 1 hour (left bar) or 2 hours (right bar) of incubation with an infectious agent known to disrupt the epithelial monolayer (E. coli ETEC H10407). The integrity of the gastrointestinal epithelium was measured by the increase in the trans epithelial electric resistance (TEER) of the monolayer. [Figure 9a] Figure 9 shows differentiation of the gut epithelium as measured by the increase in TEER upon incubation of Caco-2 cell monolayers in the presence of yeast (Figure 9a) and in combination (Figure 9b). Some error bars are too small to be visible. [Figure 9b]Figure 9 shows differentiation of the gut epithelium as measured by the increase in TEER upon incubation of Caco-2 cell monolayers in the presence of yeast (Figure 9a) and in combination (Figure 9b). Some error bars are too small to be visible. [Figure 10] FIG. 10 compares the effects observed in FIG. 9 (gut epithelial differentiation as measured by increase in TEER upon incubation of Caco-2 cell monolayers in the presence of yeast and combinations thereof) as a % increase versus control. The regression equations of the trend lines are as follows: S. boulardii: y=-0.0374x+1.1848; S. cerevisiae: y=0.011x+0.9844; K. marxianus: y=-0.0031x+1.0408; S. boulardii + S. cerevisiae: y=0.0081x+0.9871; and ABB C22: y=0.0142x+0.9384. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] For clarity and conciseness of description, features may be described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include embodiments having all or a partial combination of the described features.

[0024] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well. The word "or" includes any and all combinations of one or more of the associated listed items unless the context clearly indicates otherwise (e.g., when the "either... or" construction is used). It will be understood that the words "comprises" and "comprising" specify the presence of stated features but do not exclude the presence or addition of one or more other features. It will be further understood that when a particular step of a method is referred to as following another step, it may directly follow the other step, or one or more intermediate steps may be performed prior to performing the particular step, unless otherwise specified.

[0025] In this specification, the term "(at least) substantially" is generally used to indicate having the general property or function of something specified. When referring to a quantifiable characteristic, the term is generally used to indicate more than 50% of the maximum value of that characteristic, particularly at least 75% or more, more particularly at least 90% or more, and even more particularly at least 95%.

[0026] As used herein, the term "essentially free" is used generally to indicate that a characteristic is absent or present in such low amounts that it does not significantly affect the properties of the product.

[0027] In the context of this application, the term "about" generally means no more than a 15% deviation from a given value, particularly no more than a 10% deviation, and more particularly no more than a 5% deviation.

[0028] As used herein, the term "physiologically acceptable composition" refers to a composition that is suitable for administration to an individual, such as an animal or a human.

[0029] As used herein, the term "probiotics" refers to live microorganisms that, when administered in appropriate amounts, confer a health benefit to an individual. Probiotics include all types of microorganisms, such as those listed above, including bacteria and yeasts.

[0030] As used herein, the term "inactivated" or "dead" or "non-viable" refers to an organism, such as yeast, that is unable to reproduce or form colonies. An inactivated organism may have an intact or broken cell membrane. A person skilled in the art would be able to obtain an inactivated organism yeast based on general knowledge and the information disclosed herein. Possible means include irradiation, heat inactivation, sonication, freeze-drying and chemical inactivation.

[0031] As used herein, the term "heat-killed" refers to organisms that have been inactivated by heat treatment and are incapable of metabolic activity or colonization. Means of heat treatment to inactivate organisms are known to those of skill in the art and include tyndallization, pasteurization, ultra-high temperature (UHT) heating, Ohmic heating (or Joule heating), blanching, drying, boiling, and sterilization.

[0032] As used herein, the term "tyndallization" refers to a sterilization process that is often used to heat-kill probiotic microorganisms. For example, as described on page 11 in "Handbook of Microbiological Media" by Ronald M. Atlas (3rd ed., 2004, CRC Press), tyndallization involves repeating a heat-killing step for a certain number of consecutive days. Thus, the term "tyndallized" refers to organisms that have been heat-killed by tyndallization and are incapable of metabolic activity or colonization.

[0033] As used herein, the term "cell lysis" refers to any type of cell disruption that results in the release of intercellular biological components naturally contained within the cells of an organism. Hence, the term "lysate" refers to the product obtained after cell lysis. As used herein, "lysate" specifically refers to essentially the entire lysate obtained by lysing an organism, and therefore includes macromolecules such as DNA, RNA, proteins, peptides and lipids from the lysed cells, as well as cell debris, such as the above-mentioned cell debris including cell wall material and cell membrane components from the lysed cells. Methods for obtaining lysates are known to those skilled in the art and include enzymatic, physical and chemical methods. Cell wall components can be separated from the liquid portion of the lysate, for example by centrifugation or filtration.

[0034] As used herein, the term "extract" of yeast refers to a fluid part or fraction of a yeast cell or lysate, in particular the liquid content of a yeast cell, in particular said liquid content or a fraction thereof obtained by filtration or by centrifugation, or said liquid content obtained by extraction from the cell or lysate with an extraction phase.

[0035] As used herein, the term "metabolites" refers to any substance resulting from the growth or maintenance of yeast and remaining in the medium, but which does not need to be preserved by special techniques. Examples of metabolites are organic and inorganic acids, proteins, (poly)peptides, amino acids, (co)enzymes, fatty acids, (esterified) lipids, carbohydrates (including monosaccharides, disaccharides, polysaccharides), lipoproteins, glycolipids, glycoproteins, sugar phosphates, vitamins, salts, metals, or nucleic acids.

[0036] As used herein, the terms "alive" or "viable" refer to an organism capable of reproduction or colonization.

[0037] As used herein, the term "individual" refers to any organism, such as an animal or a human, that can benefit from the administration of a physiologically acceptable composition of the present invention. As used herein, the term "animal" refers in particular to vertebrates, such as those mentioned above, including fish, birds, mammals, reptiles and amphibians. The animals can be farm animals, livestock or laboratory animals. Individuals that can be treated according to the present invention can in particular be selected from humans, non-human primates and monkeys, cows, sheep, pigs, goats, horses, dogs, cats, mice, rodents, such as rats and guinea pigs, poultry, such as chickens, hens, turkeys, ducks and geese, and aquatic animals, such as fish and shrimp. The term "individual" does not denote a particular age or sex (e.g., male / female). Thus, humans of any age group can be treated according to the present invention, including adults (18 years and older) and children (0-17 years), such as neonatal individuals (0-12 months old) or infants (12-36 months old). In a preferred embodiment, the composition is for use in the treatment of humans, and the examples specifically illustrate the beneficial, even synergistic, effects on intestinal cells. In another preferred embodiment, the composition is for use in the treatment of non-human mammals.

[0038] As used herein, the term "nutritional product" refers to a composition intended for ingestion by an individual that provides at least one nutrient to the individual. Nutritional products generally include one or more components selected from the group consisting of proteins, fats, carbohydrates, and micronutrients.

[0039] As used herein, the term "nutraceutical" refers to a nutritional product that provides additional health benefits beyond the basic nutritional value found in food.

[0040] As used herein, the term "cosmeceutical" refers to any cosmetic product that has bioactive ingredients that purportedly have health benefits.

[0041] As used herein, the term "oral rehydration salts" (ORS) refers to a sugar-based salt solution suitable for use in oral rehydration therapy. ORS is recommended for the prevention of dehydration from diarrhea of ​​any cause in individuals of any age. ORS is also recommended for treating dehydrated individuals of any age.

[0042] As used herein, the term "prebiotic" refers to any substance that is selectively utilized by microorganisms to confer a health benefit to an individual. Prebiotics are in particular non-digestible food ingredients that stimulate the growth and / or activity of said microorganisms.

[0043] As used herein, the term "postbiotic" refers to any preparation of inactivated microorganisms and / or their components that provide a health benefit to an individual. The components that provide a health benefit can be a combination of metabolites secreted by the probiotics, such as enzymes, secreted proteins, short chain fatty acids, vitamins, secreted biosurfactants, amino acids, peptides, organic acids, etc., in the cell-free supernatant.

[0044] As used herein, the term "paraprobiotic" refers to inactivated microorganisms and / or cell fractions that confer a health benefit to an individual.

[0045] As used herein, the term "synbiotic" or "symbiotic" refers to any preparation consisting of a combination of a probiotic and a prebiotic.

[0046] Hereinafter, the term "yeast material" or "yeast-based fraction" is used as a generic term for live yeast cells, inactivated yeast cells, yeast lysates, yeast cell wall components and yeast extracts. Similarly, the term "microbiological material" is used as a generic term for live microorganisms, inactivated microorganisms, lysates of microorganisms, cell wall components of microorganisms and extracts of microorganisms.

[0047] Physiologically acceptable components of the composition

[0048] In the present invention, the S. boulardii can be any strain classified or classifiable as S. cerevisiae var. boulardii, in particular any such strain that is probiotic in live or inactivated form. In the physiologically acceptable composition according to the present invention, the S. boulardii is typically inactivated. In one embodiment, the composition comprises S. boulardii DSM 33954, S. boulardii CNCM I-745, S. boulardii Hansen CBS 5926, S. boulardii BLD-3, S. boulardii CCTCC M2012116, S. boulardii CNCM I-1079, S. boulardii ATCC 11161, S. boulardii CNCM I-1079, S. boulardii ATCC 11162, S. boulardii DSM 33954, S. boulardii CNCM I-745, S. boulardii Hansen CBS 5926, S. boulardii BLD-3, S. boulardii CCTCC M2012116, S. boulardii CNCM I-1079, S. boulardii ATCC 11162 MYA-796, S. boulardii Unique 28, S. boulardii Kirkman, S. boulardii Unisankyo, and S. boulardii CNCM I-3799. In particular, good results have been achieved with a composition comprising inactivated S. boulardii DSM 33954.

[0049] In the present invention, the S. cerevisiae can be any strain belonging to the species S. cerevisiae, in particular any strain that is a probiotic in live or inactivated form, except for strains classified or classifiable as S. cerevisiae var. boulardii. The species S. cerevisiae is also called baker's yeast, brewer's yeast, or Candida robusta in the art. In the physiologically acceptable composition according to the present invention, the S. cerevisiae is typically inactivated. In a preferred embodiment, the composition comprises at least one S. cerevisiae selected from the group of S. cerevisiae Y1529 (deposited under ATCC), S. cerevisiae CNCM I-3856, S. cerevisiae S288C, and S. cerevisiae UFMG 905, of which S. cerevisiae S288C and S. cerevisiae Y1529 are particularly preferred. In particular, good results have been achieved with mineral-enriched S. cerevisiae, for example zinc-enriched S. cerevisiae.

[0050] In the present invention, the K. marxianus can be any strain belonging to the species K. marxianus. This species is also referred to in the art as Saccharomyces marxianus, Candida Kefyr, Candida pseudotropicalis, Kluyveromyces fragilis, and Kluyveromyces cicerisporus. In the physiologically acceptable composition according to the present invention, the K. marxianus is typically inactivated. In one embodiment, the composition comprises one or more strains selected from the group consisting of Kluyveromyces marxianus AS41, Kluyveromyces marxianus B0399, Kluyveromyces marxianus CIDCA 8154, Kluyveromyces marxianus CBS1553, Kluyveromyces marxianus M3, Kluyveromyces marxianus V21 / 012435, and Kluyveromyces marxianus Z17. Good results have been achieved with compositions according to the invention comprising Kluyveromyces marxianus V21 / 012435 cells. Thus, Kluyveromyces marxianus V21 / 012435 cells, an extract of Kluyveromyces marxianus V21 / 012435 cells, a lysate of Kluyveromyces marxianus V21 / 012435 cells, or cell wall material of Kluyveromyces marxianus V21 / 012435 cells are present in preferred compositions of the invention.

[0051] By using standard references such as "The yeasts, a taxonomic study" (CP Kurtzman, JW Fell and T Boekhout), 5th Edition, 2011, Elsevier, the skilled artisan will be able to determine if a given yeast strain is classified or classifiable as S. cerevisiae var. boulardii, as the species S. cerevisiae, or as K. marxianus. Moreover, a person skilled in the art will be able to distinguish Saccharomyces cerevisiae var. boulardii from other yeast strains belonging to the species Saccharomyces cerevisiae based on standard literature, e.g. Edwards-Ingram L, Gitsham P, Burton P, et al., "Genotypic and physiological characterization of Saccharomyces boulardii, the probiotic strain of Saccharomyces cerevisiae", Appl Environ Microbiol 2007;73:2458-67.

[0052] (i) at least one component selected from the group consisting of S. boulardii yeast, S. boulardii lysate, S. boulardii cell wall components, and S. boulardii extracts (also referred to herein as S. boulardii-based fractions); (ii) S. cerevisiae yeast, S. cerevisiae lysate, S. cerevisiae cell wall components, and S. cerevisiae extracts (also referred to herein as S. boulardii-based fractions); The relative amounts of (i) at least one component selected from the group consisting of Kluyveromyces marxianus yeast, Kluyveromyces marxianus lysate, Kluyveromyces marxianus cell wall component, and Kluyveromyces marxianus extract (also referred to herein as Kluyveromyces marxianus-based fraction), can vary within wide ranges.

[0053] Typically, the S. boulardii based fraction of the composition according to the invention is between 0.05 and 99.95% by weight, preferably between 5 and 95% by weight, more preferably between 15 and 80% by weight, in particular between 20 and 60% by weight, more in particular between 25 and 50% by weight, based on the total yeast component.

[0054] Typically the S. cerevisiae based fraction of the composition according to the invention is between 0.05 and 90% by weight, preferably between 5 and 90% by weight, more preferably between 10 and 90% by weight, more preferably between 15 and 80% by weight, in particular between 20 and 80% by weight, more in particular between 20 and 60% by weight, more in particular between 25 and 50% by weight, based on the total yeast component.

[0055] Typically, the K. marxianus based fraction of the composition according to the invention is 0.05-99.95% by weight, preferably 5-90% by weight, more preferably 10-90% by weight, more preferably 15-80% by weight, in particular 20-80% by weight, more in particular 20-60% by weight, more in particular 25-50% by weight, based on the total yeast component.

[0056] Typically, the S. boulardii based fraction, the S. cerevisiae based fraction and the K. marxianus based fraction together form at least 10% by weight, preferably at least 25% by weight, more preferably at least 50% by weight, especially at least 75% by weight of the total microbiological material (e.g. bacterial, algal or fungal cells, their lysates, their extracts, their cell wall parts). Optionally, other microbiological material, especially probiotic microorganisms or cellular material of probiotic microorganisms, may be present in the composition. Thus, the sum of the S. boulardii based fraction, the S. cerevisiae based fraction and the K. marxianus based fraction is based on total microbiological material and is less than 100% by weight of the total microbiological material, for example less than 99% by weight.

[0057] In an advantageous embodiment, the physiological composition comprises 5-95% by weight of a S. boulardii based fraction (typically inactivated S. boulardii), 10-80% by weight of a S. cerevisiae based fraction (typically inactivated S. cerevisiae) and 5-95% by weight of a K. marxianus based fraction (typically inactivated K. marxianus) (all based on total yeast components, preferably based on total microbiological material). In particular, good results are achieved, for example with respect to some proinflammatory markers or TEER, with compositions having a content of S. boulardii based fraction in the range of 15-50% by weight, a content of S. cerevisiae based fraction in the range of 15-50% by weight, and a content of K. marxianus based fraction in the range of 15-50% by weight (all ranges based on the total microbiological material of the composition, provided that the sum of the three fractions is not more than 100% by weight). Advantageously herein, the yeast based fraction consists at least substantially of inactivated yeast cells. Advantageously herein, the total microbiological material consists at least substantially of inactivated yeast cells. As will be appreciated by those skilled in the art, different contents may be applied to obtain satisfactory results, based on the information disclosed herein and on general general knowledge.

[0058] Due to safety concerns regarding the use of live microorganisms, for example in vulnerable or immunocompromised patient groups or neonates, there is growing interest in using non-viable inactivated probiotics.Several inactivation methods are known to those skilled in the art and include irradiation, heat inactivation, sonication, freeze-drying and chemical inactivation.Tindalization is a sterilization process that is often used to heat-kill probiotic microorganisms.Interestingly, according to the present invention, tindalized yeast has been shown to exert relevant biological responses, such as restoring normal intestinal homeostasis.

[0059] Thus, typically, the physiologically acceptable composition of the present invention comprises non-viable S. boulardii, non-viable S. cerevisiae and non-viable K. marxianus. When one or more of the yeasts is a mineral-rich yeast, such as a zinc-rich yeast, it is particularly preferred that the mineral-rich yeast is inactivated, i.e., non-viable. Preferably, the inactivated yeast is heat-killed. More preferably, the inactivated yeast is tindalized. The tindalization may be based on the tindalization process commonly known in the art. In particular, good results have been achieved with compositions comprising tindalized S. boulardii, as well as tindalized S. cerevisiae and as well as tindalized K. marxianus.

[0060] Usually, at least substantially all of the yeast cells present in the physiologically acceptable composition according to the invention are non-viable. Thus, usually, the yeast material is essentially free of live yeast cells. However, in a particular embodiment, the physiologically acceptable composition according to the invention comprises at least one live yeast selected from S. boulardii, S. cerevisiae and K. marxianus. When at least one of the yeasts is present in a live form, preferably at least K. marxianus is present in a live form. When present, the content of live yeast material is typically a small portion of the total yeast material, in particular not more than 10% by weight, more in particular not more than 1% by weight.

[0061] The S. boulardii contained in the physiologically acceptable composition of the present invention for administration into the digestive tract is usually present in an amount of 10 6 Cells (based on the total weight of the yeast component) ~10 per gram 11 Cells (based on the total weight of the yeast component) 10 per gram 7 Cells (based on the total weight of the yeast component) ~10 per gram 11 cells (based on the total weight of the yeast component), more preferably 10 per gram 9 Cells (based on the total weight of the yeast component) ~2x10 per gram 10 K. marxianus in physiologically acceptable compositions of the present invention for administration into the gastrointestinal tract is typically present at a concentration ranging from 10 to 10 cells per gram (based on the total weight of the yeast component). 6 Cells (based on the total weight of the yeast component) ~10 per gram 10 cells (based on the total weight of the yeast component), preferably 10 per gram 7 Cells (based on the total weight of the yeast component) ~5x10 per gram 9 The yeast cells (based on the total weight of the yeast component) are present in a range of concentrations.

[0062] For other modes of administration, such as topical administration, the S. boulardii contained in the physiologically acceptable composition of the present invention is typically present in an amount of 10 4 Cells (based on the total weight of the yeast component) ~10 per gram 11 cells (based on the total weight of the yeast component), preferably 10 per gram 6 Cells (based on the total weight of the yeast component) ~10 per gram 10 For alternative modes of administration, such as topical administration, the S. cerevisiae contained in the physiologically acceptable compositions of the present invention are typically present in concentrations ranging from 10 to 10 per gram. 4 Cells (based on the total weight of the yeast component) ~10 per gram 11 cells (based on the total weight of the yeast component), preferably 10 per gram 6 Cells (based on the total weight of the yeast component) ~10 per gram 10 For alternative modes of administration, such as topical administration, K. marxianus in physiologically acceptable compositions of the present invention is typically present in a concentration ranging from 10 to 10 per gram. 4 Cells (based on the total weight of the yeast component) ~10 per gram 11 cells (based on the total weight of the yeast component), preferably 10 per gram 6 Cells (based on the total weight of the yeast component) ~10 per gram 10 The yeast components are present in a range of concentrations of cells (based on the total weight of the yeast components).

[0063] The concentrations of live and inactivated yeast are measured and expressed as cells per gram of total yeast component. For live yeast, the concentration in "cells per gram" is equivalent to colony forming units (CFU) per gram of total yeast component. When a combination of live and inactivated (non-colony forming) cells is present, the total cells will usually be in the normal ranges described above, preferably in the preferred ranges described above, or in the more preferred ranges. For yeast lysates, extracts, or cell wall components, suitable concentrations are usually 10 per gram. 4 Cells (based on the total weight of the yeast component) ~10 per gram 11 It corresponds to the amount of lysate, extract, and cell wall components obtained from the cells (based on the total weight of the yeast components).

[0064] The physiologically acceptable composition of the present invention may comprise a yeast rich in minerals, particularly Saccharomyces cerevisiae. Preferably, the physiologically acceptable composition of the present invention comprises a yeast rich in zinc, more preferably Saccharomyces cerevisiae rich in zinc. The physiologically acceptable composition of the present invention may comprise a mineral salt, preferably a zinc salt, most preferably zinc sulfate.

[0065] Zinc-rich yeast provides a natural source of zinc with high bioavailability. Zinc is considered an important nutrient for immunity and diarrhea management. Interestingly, supplementation with zinc organically bound or mixed through yeast organisms has been shown to result in better bioavailability compared to inorganic zinc. Zinc as well as other minerals such as selenium, chromium, iron, copper, magnesium, manganese, potassium, calcium and iodine have also been shown to be beneficial in restoring mineral balance in individuals and can be concentrated to yeast for better bioavailability. Mineral-rich yeast can be obtained by culturing yeast in a medium supplemented with one or more of, for example, zinc, selenium, chromium, iron, copper magnesium, manganese, potassium, calcium or iodine. Minerals are typically organically bound or otherwise absorbed by yeast proteins, resulting in one or more minerals being absorbed into the yeast cells. The minerals can be added to the medium before, during or after cultivation. As used herein, the term "mineral-rich yeast strain" refers in particular to yeast strains fermented in the presence of mineral salts or to yeast strains to which mineral salts have been added after fermentation, with a final concentration of such minerals being up to 12% by weight, in particular up to 5% by weight, more in particular up to 2% by weight, for example up to 1% by weight, based on the dry weight of the total product. In the case of zinc addition, a final concentration in the range of 1-12% by weight, in particular in the range of about 4% to about 10% by weight, is preferred.

[0066] Modes of administration and dosage forms of physiologically acceptable compositions

[0067] The physiologically acceptable compositions of the present invention are preferably administered intra-alimentary canal or topically. The administration into the alimentary canal is preferably oral. In a specific embodiment, the compositions are administered by tube feeding or as a suppository. Topical administration specifically includes administration into the mucus or into the skin. Specific examples include administration into the eye and into the vagina. Formulations of the compositions according to the present invention suitable for oral ingestion include, but are not limited to, capsules, coated capsules, tablets, sachets, pills, pearls, soft gels, vials, powders, granules, solutions, suspensions, emulsions, elixirs, syrups, powders, lozenges, gums, hard candies and gels. Formulations of compositions according to the invention suitable for topical administration include creams (e.g., for vaginal application), capsules (e.g., for vaginal application), tablets (e.g., for vaginal application), ointments, pastes, foams, gels, lotions, shampoos, mousses, sprays (e.g., for application on the skin, eyes or mucous membranes), suppositories, solutions (e.g., for vaginal application), bandages, bioadhesives, liquids (e.g., for application to the skin, for application to the eyes, e.g. eye drops, or for application to mucous membranes) and suspensions.

[0068] Formulations of compositions according to the present invention suitable for local administration include compositions suitable for application to parts of the gastrointestinal tract where the effect of the yeast component is required, such as suppositories or gastrointestinal medical devices.

[0069] Topical administration in the treatment of gastrointestinal disorders generally involves administration to the mucosa or epithelium of the gastrointestinal tract. The medical product comprising the composition (for use) according to the invention can be a product suitable for forming a protective biofilm or the like on the surface of the gastrointestinal epithelium. Such a product can be based, for example, on known products for the treatment of IBS.

[0070] In a specific embodiment, the composition is to be administered as a sustained release product.

[0071] The physiologically acceptable composition of the present invention can be administered using a medical device. Such a medical device for topically administering the physiologically acceptable composition can be a bandage, a (transdermal) patch.

[0072] The composition according to the invention may be a food product. The food product may be fermented or non-fermented. Examples of particularly suitable food products include dairy products such as yoghurt, yoghurt drinks, cheese, milk, milk powder, infant formula, cream, ice cream, cream powder and butter; fruit-based products such as fruit juice, compote or fruit jelly; solid foods such as flour, cereals, snacks, biscuits; and liquid preparations such as vegetable drinks, smoothies, isotonic drinks, salt solutions and enteral nutrition recipes. The physiologically acceptable composition according to the invention for consumption by animals may be any suitable food product for animals and, in addition to the foods listed above, includes tablets, coated tablets, granules, cereals, (dried) meat, (dried) fish, oilseeds, cakes, cookies, sugar cane, and roughage such as grass, hay, silage, root crops, straw and stalks.

[0073] The nutritional products according to the invention may be in the form of a dietary supplement, a food additive, a feed additive, a functional food in human nutrition, a functional food in animal nutrition, a food additive or functional ingredient for nutraceuticals, or a food for special medical purposes.

[0074] The physiologically acceptable composition according to the invention can be a pharmaceutical, cosmetic or nutraceutical product. The pharmaceutical can further comprise pharmaceutical acceptable adjuvants and / or additives. Adjuvants and additives are well known to those skilled in the art.

[0075] Oral hydration salts (ORS) are a preferred example of a product according to the invention. ORS can be classified as a medicine (e.g. WHO) or as a food supplement, depending on national regulations. Particularly suitable inactivated yeast cells for ORS have been found to be S. boulardii DSM 33954 (available as ABB1 from ABBiotek-Spain, https: / / www.abbiotek.com), S. cerevisiae Y1529 (deposited at ATTC, available as ABB6 from ABBiotek), and K. marxianus V21 / 012435 (deposited at National Measurement Institute, Port Melbourne Vic 3207, Australia, available as ABB7 from ABBiotek). A mixture of these three yeast strains is available from ABBiotek as ABB C22.

[0076] The physiologically acceptable composition may further comprise a bulking agent, in particular a carbohydrate, such as maltodextrin.

[0077] An example of an ORS formulation in liquid form contains S. boulardii, S. cerevisiae, and K. marxianus (ABB C22) and further contains water, glucose, sodium citrate, sodium chloride, maltodextrin, zinc sulfate, silicon dioxide, flavoring, a sweetener (acesulfame K), and an acidulant (citric acid).

[0078] One example of an ORS formulation in powder form contains S. boulardii, K. marxianus, and S. cerevisiae (ABB C22), and further contains dextrose, lemon, flavoring, citric acid, magnesium citrate, malic acid, sodium citrate, sodium chloride, potassium phosphate, calcium ascorbate, sucralose, and riboflavin.

[0079] Preparation of Physiologically Acceptable Compositions

[0080] The physiologically acceptable composition can be prepared by combining the different components according to methodologies known per se for producing yeast preparations.

[0081] For example, all yeasts may be produced from non-GMO yeast strains. Fermentation processes known per se for the yeast of interest are used to produce a primary growth yeast that grows under sterile aerobic conditions. The resulting product or yeast cream may be stored refrigerated to maintain cell viability, if desired.

[0082] The yeast cream can be subjected to an inactivation treatment, such as pasteurization or tindalization, to obtain a heat-treated version of the yeast. If desired, the yeast can be dried, for example by spray drying, which is preferably carried out after inactivation (if non-viable yeast is to be used for the composition).

[0083] Maltodextrin or other bulking agents may be used as support to standardize the consistency. The three yeasts may be mixed and then subjected to a homogenization step.

[0084] Methods of Treating an Individual and Improving Gastrointestinal Health or Gastrointestinal Function in an Individual

[0085] According to the invention, the physiologically acceptable composition is advantageously used to treat gastrointestinal disorders, such as irritable bowel syndrome (IBS); inflammatory bowel disorder (IBD), such as Crohn's disease or ulcerative colitis; functional constipation; diarrhea, such as antibiotic-associated diarrhea, traveller's diarrhea, acute gastroenteritis, pediatric diarrhea, dysbiosis diarrhea or chronic diarrhea, in particular in immunocompromised patients; functional abdominal pain; functional abdominal bloating, postprandial distress syndrome. Syndrome, gastrointestinal allergies or intolerance; necrotizing enterocolitis; gastrointestinal infections caused by bacteria, such as Escherichia, Salmonella, Shigella, Staphylococcus, Vibrio, Campylobacter, Yersina, Clostridium, or Helicobacter; viruses, such as norovirus, adenovirus, cytomegalovirus, enterovirus, astrovirus, hepatitis The present invention is used in the treatment of individuals with gastrointestinal infections caused by Norovirus, rotavirus, or parasites such as Giardia, Entamoeba, Cryptosporidium, Cyclospora, or Ascaris; and combinations thereof. Preferably, a gastrointestinal disorder selected from the group consisting of IBD, IBS, and diarrhea is treated. In a specific embodiment, the diarrhea treated is bacterially induced diarrhea, such as diarrhea induced by E. coli.

[0086] Furthermore, the physiologically acceptable compositions are advantageously used in the treatment of individuals having a disease defined by one or more proinflammatory markers, wherein said markers include IL-8, IP-10, MCP-1, TNFα / IL-10 and TNFα. Diseases defined by proinflammatory markers that may be treated according to the present invention include rheumatoid arthritis, osteoarthritis, topical dermatitis, psoriasis, allergies and obesity. In particular, the physiologically acceptable compositions of the present invention are suitable for the treatment of inflammatory diseases defined by one or more of said proinflammatory markers in the absence of infection.

[0087] Moreover, the physiologically acceptable compositions according to the invention are particularly suitable for maintaining or improving gut health or function, in particular in states of gut health or function involving impaired or weakened gut barrier function or alterations in inflammatory cytokine release. The term "gut health" as described herein means the health of the gut. The gut health of an individual may be affected, for example, by infectious causes or by non-infectious causes, such as suboptimal diet. The term "gastrointestinal function" refers to the operation of all organs and structures associated with the gastrointestinal system. Markers for determining gut health or function are known to those skilled in the art and include, for example, transepithelial electrical resistance as an indicator of epithelial barrier integrity, as well as markers of inflammation and injury. Examples of markers are described in Celi et al. (2019) "Biomarkers of gastrointestinal functionality in animal nutrition and health", Animal Feed Science and Technology 250:9-31.

[0088] The dosage, duration and frequency of administration can be selected within a wide range depending on the intended purpose and the subject to which the composition is to be administered.The duration of treatment can be relatively short, for example, one week or less, or one day or less, for example, in the case of acute symptoms of a disease or disease, such as diarrhea.The duration of treatment can also be extended, for example, one week or more, one month or more, or one year or more, for example, in the case of chronic diseases, such as IBD.

[0089] The physiologically acceptable composition for use according to the present invention can be administered as a single dose for complete treatment, depending on the application. If multiple administrations are intended, the number of administrations is generally not more than 10 times per day. For example, in the case of using ORS, administration can be performed three or more times per day, but typically not more than about three times per day, preferably not more than about two times per day, and particularly not more than about once per day. In one embodiment, the composition is administered at least about once per week (on average). Preferably, the composition is administered at least once per three-day period (on average), more preferably at least once per two-day period (on average).

[0090] Physiologically acceptable compositions may also include or be co-administered with (other) probiotics, prebiotics, postbiotics, antibiotics, analgesics, anti-inflammatory agents, anti-diarrheal agents, e.g., motility or secretion inhibitors, (other) oral rehydration salts, laxatives, or combinations thereof.

[0091] Those skilled in the art will be able to determine the appropriate dosage of physiologically acceptable composition for administration to an individual based on common general knowledge and the information disclosed herein without undue experimentation. As will be understood by those skilled in the art, the actual preferred dosage will depend on various factors, such as the activity of the specific yeast used, the metabolic stability and duration of action of the yeast, the individual's age, weight, general health, sex and species, diet, mode and time of administration, excretion rate, drug combination, severity of the specific disorder, and the various factors mentioned above, including the individual. The dosages disclosed herein are indicative of the average case for a human individual. Of course, there may be individual cases where higher or lower dosages are appropriate. For oral administration or other administration to the digestive tract, the usual effective daily dosage, especially for humans, is about 100 mg (of yeast components) to about 1000 mg (of yeast components), preferably about 200 mg (of yeast components) to about 900 mg (of yeast components), more preferably about 200 mg (of yeast components) to about 650 mg (of yeast components). Typically, the effective daily amount for topical administration, particularly for humans, is from about 1 mg (of the yeast component) to about 1000 mg (of the yeast component), preferably from about 5 mg (of the yeast component) to about 500 mg (of the yeast component).

[0092] The present invention provides physiologically acceptable compositions for use as pharmaceuticals.

[0093] The present invention provides a method of treating an individual in need of improved gut health or improved gastrointestinal function, comprising administering an effective amount of a physiologically acceptable composition according to the invention, thereby improving gut health or gastrointestinal function. The present invention has been found to be particularly effective in improving gut barrier function and in promoting an anti-inflammatory state, as well as protecting against viral, bacterial and yeast infections, while modulating the microbiota towards a eubiotic state.

[0094] The present invention provides a method for treating an individual having a gastrointestinal disorder, such as diarrhea, IBD, IBS, or (other) gastrointestinal disorder associated with impaired gut barrier function, comprising administering to said individual an effective amount of a physiologically acceptable composition according to the present invention.

[0095] The present invention provides a method of treating an individual having a disease defined by pro-inflammatory markers, such as rheumatoid arthritis, osteoarthritis, localized dermatitis, psoriasis, allergies, or obesity, comprising administering to said individual an effective amount of a physiologically acceptable composition according to the present invention.

[0096] The present invention provides a method of use of a physiologically acceptable composition according to the present invention for use in the preparation of a product, which may be a pharmaceutical or a food (e.g. a medical food or clinical food), for use in the treatment of a gastrointestinal disorder, preferably a gastrointestinal disorder selected from the group consisting of diarrhea, IBD and IBS, or (another) gastrointestinal disorder associated with impaired gastrointestinal barrier function.

[0097] The invention provides a method of using a physiologically acceptable composition according to the invention for use in the preparation of a product, which may be a pharmaceutical or a food (e.g. a medical food or a clinical food), for use in the treatment of diseases defined by proinflammatory markers, such as rheumatoid arthritis, osteoarthritis, topical dermatitis, psoriasis, allergies and obesity.

[0098] The invention provides the use of a physiologically acceptable composition according to the invention for the preparation of a product for maintaining or improving gastrointestinal health or gastrointestinal function.

[0099] The invention will now be described by the following examples, which are provided by way of illustration, it being understood that many variations are possible in the methods described and in the amounts indicated without departing from the spirit of the invention and the scope of the appended claims.

[0100] Working Example

[0101] Example 1: Anti-inflammatory effect on gastrointestinal epithelial cells Materials and Methods All yeasts were produced from non-GMO yeast strains. The yeasts included in the experiments were S. boulardii DSM 33954 (deposited at DSMZ, German Collection of Microorganisms and Cell Cultures, Germany) (available from ABB1); S. cerevisiae Y1529 (deposited as ATTC) (available from ABB6, zinc-rich form); and K. marxianus V21 / 012435 (deposited at National Measurement Institute, Port Melbourne Vic 3207, Australia) (available from ABB7). As used herein, these yeast combinations are also available from ABB22.

[0102] In the fermentation process, a primary growth yeast was generated where growth occurred under sterile aerobic conditions. During fermentation, temperature, pH and growth rate were strictly controlled. In the case of S. cerevisiae, zinc sulfate was added to the yeast cream at the end of the fermentation process to a concentration of about 10% based on dry weight. The resulting product, i.e., yeast cream, was stored refrigerated to maintain cell viability. Prior to spray drying, the cooled yeast cream was treated in a high temperature pasteurization system to obtain a tindalized version of the yeast. Maltodextrin or other bulking agents can be used as support agents to standardize the concentration.

[0103] The three heat-inactivated yeast species were premixed and subsequently homogenized. Stock solutions of each yeast species were mixed for the in vitro studies described below.

[0104] The yeast concentration of each yeast stock was determined by flow cytometry. For each yeast, 1x10 7 Standardized samples were prepared in terms of cells / mL. To obtain the combination, samples of each yeast strain were mixed in a homogenous ratio of each strain, i.e., 0.333x10 of each of the three strains in the mixture. 7 The cells were mixed to a concentration of 1000 cells / ml.

[0105] The in vitro immunomodulatory activity of yeasts and their combinations was examined by chemokine production by Caco-2 cells in the presence and absence of inflammatory stimuli. Caco-2 cells were grown to confluence in 96-well plates. At the start of the experiment, cells were washed once with antibiotic-free medium. Monolayers were incubated with the test components in antibiotic-free medium at 37°C for 1 h (triplicates). The cells were then incubated with medium containing the test components and 50 μg / ml gentamicin (Invitrogen) (duplicates). One of the duplicates was further stimulated with a mixture of recombinant TNFα (10 ng / ml) and recombinant IFNγ (5 ng / ml) (R&D systems) as a proinflammatory stimulus (Figures 4-6). As blank controls, only unstimulated medium was used in the cases of Figures 1 to 3, and medium under pro-inflammatory stimulation (a mixture of recombinant TNFα (10 ng / ml) and recombinant IFNγ (5 ng / ml)) was used in the cases of Figures 4 to 6.

[0106] Supernatants were collected 24 hours after stimulation and stored at 20° C. Bio Plex assays (BioRad) were used to measure IL-8, IP-10 and MCP-1 levels according to the manufacturer's protocol.

[0107] To confirm the absence of cytotoxicity of the test components, after harvesting the culture supernatants, the metabolic activity of the cells was analyzed by WST-1 assay (Roche) according to the manufacturer's protocol. The cells showed no metabolic activity, indicating that any observed effects were not due to metabolic alterations or cytotoxicity.

[0108] As in other examples, one-way ANOVA was performed, followed by calculation of statistical differences between control and test conditions using Dunnett's post hoc test. Significance thresholds used in the figures are as follows: *p<0.05, **p<0.01, and ***p<0.001.

[0109] result FIG. 1 illustrates in vitro IP-10 chemokine production by Caco-2 cells following incubation with the three yeasts and their combinations in the absence of proinflammatory stimuli.

[0110] A synergistic reduction in inflammatory cytokines in gastrointestinal epithelial cells was observed, especially for the yeast composition containing S. boulardii, S. cerevisiae, and K. marxianus (hereafter referred to as composition ABB C22) compared to the individual yeasts (Figures 2-3). A synergistic reduction in inflammatory cytokines in gastrointestinal epithelial cells after inflammatory challenge with TNFα / IFNγ was observed for yeast composition ABB C22 compared to the individual yeasts (Figures 4-5). Moreover, the ABB C22 composition was observed to be superior to compositions containing only the yeasts S. boulardii and S. cerevisiae (Figures 2-6), indicating the important role of K. marxianus for obtaining some of the marked positive effects, which are even synergistic at least in some respects.

[0111] Example 2: Anti-inflammatory effects on immune cells Materials and Methods Several yeasts and their combinations were prepared similarly to Example 1.

[0112] The in vitro immunomodulatory activity of several yeasts and their combinations was investigated by tracking cytokine production by the THP-1 cell line (macrophage).

[0113] Human THP-1 cell line, 1x10 in 96-well plate 5 Cells / well were cultured in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma) and incubated for 48 h to induce differentiation of THP-1 monocytes into macrophages. Cells were washed and incubated in culture medium for a further 72 h. After this, cells were incubated with the test compound for 1 h, after which the cells were incubated in the presence of the test compound with or without LPS (100 ng / ml, Sigma) for a further 16 h. All conditions were tested in triplicate.

[0114] Supernatants were collected after stimulation and stored at -20°C. ELISA assays (IL-10 Human Uncoated ELISA Kit, TNF-α Human Uncoated ELISA Kit, Life Technologies) were used to measure TNF-α and IL-10 levels according to the manufacturer's protocol. The TNF-α / IL-10 ratio was calculated as a measure of the anti-inflammatory effect of the tested components.

[0115] To confirm the non-cytotoxicity of the test components, the metabolic activity of the cells was analyzed by WST-1 assay (Roche) after harvesting the culture supernatants, according to the manufacturer's protocol. It was found that the cells did not show any metabolic activity.

[0116] result A decrease in the TNFα / IL-10 ratio, a measure of the anti-inflammatory effect in immune cells, was observed for the yeast composition ABB C22 compared to the individual yeasts and compared to compositions containing only the yeasts S. boulardii and S. cerevisiae (Figure 7).

[0117] Example 3: Intestinal barrier integrity after challenge Materials and Methods Several yeasts and their combinations were prepared similarly to Example 1.

[0118] The effects of yeasts and their combinations on gut barrier function upon challenge were investigated by tracking the transepithelial electrical resistance (TEER) on the cell layers of the gut.

[0119] Caco-2 cells have an average pore size of 0.4 μm and a diameter of 0.33 cm. 2 The cells were seeded (2 × 10) on Transwell polycarbonate cell culture inserts (Greiner Bio One) containing 4 cells / cm 2 ), and cultured to fully differentiated 1000 Ω. As an indicative measure of barrier integrity, TEER was measured using an EVOM2 epithelial volt ohmmeter (World Precision Instruments).

[0120] On the day of the experiment, cells were washed and incubated with antibiotic and serum-free medium containing the test components for 1 hour at 37° C. The wells were then exposed to ETEC H10407 (MOI 200:1) in the presence of the test components for 6 hours. TEER was measured before the start of the experiment (t=1), 1 hour after exposure to the test components, before the addition of ETEC (t=0), and 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours after exposure to ETEC (t=1, t=2, t=4, and 6 hours, respectively).

[0121] The TEER values ​​of individual conditions after exposure to a pathogen are related to their own TEER values ​​at t = 0, ΔTEER (Ω.cm 2 The results were expressed as mean ± SD (%). A negative control (ETEC H10407 only) and a positive control that was not exposed to either the pathogen or the test components were included. All conditions were tested in triplicate.

[0122] Transepithelial flux using FITC-dextran (Sigma) was measured at various time points after TEER measurements.

[0123] result Protection of the integrity of the gastrointestinal epithelium was measured after incubation with an infectious agent known to disrupt the epithelial monolayer (here, E. coli ETEC). The yeast combination ABB C22 had a higher increase in TEER relative to the negative control after 1 and 2 hours of incubation compared to the respective yeasts or the combination of S. cerevisiae and S. boulardii (Figure 8).

[0124] Example 4: Intestinal barrier integrity formation Materials and Methods Several yeasts and their combinations were prepared similarly to Example 1.

[0125] The effects of yeasts and their combinations on the proliferation and differentiation of intestinal epithelial cells were followed during the formation of a gastrointestinal cell monolayer.

[0126] Caco-2 cells have an average pore size of 0.4 μm and a diameter of 0.33 cm. 2 Cells (2 × 10) were seeded onto Transwell polycarbonate cell culture inserts (Greiner Bio One) containing 4 cells / cm 2 After allowing the cells to adhere overnight, test components were added to the apical side of the cells.

[0127] Test materials were prepared and stored in aliquots at 20° C. New aliquots were taken every two days to refresh the materials. To avoid overgrowth of epithelial cells by the tested yeasts (if they are capable of growing under aerobic conditions), 10% conditioned medium of yeasts and their combinations was used with heat-killed yeasts.

[0128] As an indicative measure of cell proliferation and barrier formation, TEER was measured every 2 days using an EVOM2 epithelial volt ohmmeter (World Precision Instruments).

[0129] result The increase in TEER was used to measure the spontaneous remodeling of the gut epithelium over time. An increase in TEER was observed after 16 days for the three yeast species compared to the negative control and was maintained until day 20 only for S. cerevisiae (Figure 9a, top). In contrast, the increase in TEER from day 16 observed for the combination ABB C22 relative to the control was maintained and increased until day 22 (Figure 9b, bottom).

[0130] A larger slope of TEER increase is observed for the combination ABB C22, indicating a faster TEER increase, compared to the individual yeast strains as well as the combination of S. cerevisiae and S. boulardii (slope of the trendlines in FIG. 10).

Claims

1. A physiologically acceptable composition comprising: (i) at least one component selected from the group consisting of inactivated Saccharomyces boulardii yeast, Saccharomyces boulardii lysate, Saccharomyces boulardii cell wall components, and Saccharomyces boulardii extract; (ii) further comprising at least one component selected from the group consisting of inactivated Saccharomyces cerevisiae yeast, Saccharomyces cerevisiae lysate, Saccharomyces cerevisiae cell wall components, and Saccharomyces cerevisiae extract; and (iii) further comprising at least one component selected from the group consisting of inactivated Kluyveromyces marxianus yeast, Kluyveromyces marxianus lysate, Kluyveromyces marxianus cell wall components, and Kluyveromyces marxianus extract.

2. The physiologically acceptable composition according to claim 1, comprising inactivated Saccharomyces boulardii yeast, inactivated Saccharomyces cerevisiae yeast, and inactivated Kluyveromyces marxianus yeast.

3. The physiologically acceptable composition according to claim 2, wherein at least one of the inactivated yeasts is heat-killed.

4. The (inactivated) Saccharomyces boulardii is present at a concentration of at least 10 6 cells (based on the total weight of the yeast component), where the (inactivated) Saccharomyces cerevisiae is present at a concentration of at least 10 6 cells (based on the total weight of the yeast component), and where the (inactivated) Kluyveromyces marxianus is present at a concentration of at least 10 6 cells (based on the total weight of the yeast component). The physiologically acceptable composition according to claim 1. **Claim 5**: The (inactivated) Saccharomyces boulardii is present at a concentration of 10^9 cells per gram (based on the total weight of the yeast component) to 4x10^10 cells per gram (based on the total weight of the yeast component), where the (inactivated) Saccharomyces cerevisiae is present at a concentration of 10^8 cells per gram (based on the total weight of the yeast component) to 5x10^9 cells per gram (based on the total weight of the yeast component), and where the (inactivated) Kluyveromyces marxianus is present at a concentration of 10^6 cells per gram (based on the total weight of the yeast component) to 5x10^9 cells per gram (based on the total weight of the yeast component). The physiologically acceptable composition according to claim 1. **Claim 6** The physiologically acceptable composition according to claim 1, wherein the Saccharomyces cerevisiae is zinc-enriched Saccharomyces cerevisiae. **Claim 7** Saccharomyces boulardii is selected from the group consisting of Saccharomyces boulardii CNCM I-745, Saccharomyces boulardii Hansen CBS 5926, Saccharomyces boulardii BLD-3, Saccharomyces boulardii CCTCC M2012116, Saccharomyces boulardii CNCM I-1079, Saccharomyces boulardii ATCC MYA-796, Saccharomyces boulardii Unique28, Saccharomyces boulardii Kirkman, Saccharomyces boulardii Unisankyo, Saccharomyces boulardii DSM 33954, and Saccharomyces boulardii CNCM I-3799, and wherein Saccharomyces cerevisiae is selected from the group consisting of Saccharomyces cerevisiae CNCM I-3856, Saccharomyces cerevisiae Y1529, Saccharomyces cerevisiae S288C, and Saccharomyces cerevisiae UFMG 905, and wherein Kluyveromyces marxianus is selected from the group consisting of Kluyveromyces marxianus AS41, Kluyveromyces marxianus B0399, Kluyveromyces marxianus CIDCA 8154, Kluyveromyces marxianus CBS1553, Kluyveromyces marxianus M3, Kluyveromyces marxianus V21 / 012435, and Kluyveromyces marxianus Z17, the physiologically acceptable composition according to claim 1.

8. The composition according to claim 7, wherein Saccharomyces boulardii is Saccharomyces boulardii DSM 33954, wherein Saccharomyces cerevisiae is Saccharomyces cerevisiae S288C or Saccharomyces cerevisiae Y1529, and wherein Kluyveromyces marxianus is Kluyveromyces marxianus V21 / 012435.

9. The composition according to claim 1, wherein the Saccharomyces boulardii-based fraction of the composition is 5 to 95% by weight based on the total yeast component; the Saccharomyces cerevisiae-based fraction of the composition is 10 to 90% by weight based on the total yeast component; and the Kluyveromyces marxianus-based fraction of the composition is 5 to 95% by weight based on the total yeast component, provided that the sum of the three fractions is 100% by weight or less.

10. The composition according to claim 1, comprising 15 to 50% by weight of inactivated Saccharomyces boulardii cells based on the total microbiological substance of the composition, 15 to 50% by weight of inactivated Saccharomyces cerevisiae cells based on the total microbiological substance of the composition, and 15 to 50% by weight of inactivated Kluyveromyces marxianus cells based on the total microbiological substance of the composition, provided that the sum of the three fractions is 100% by weight or less.

11. The composition according to claim 1, wherein the composition is a pharmaceutical, a dietary supplement or a cosmetic.

12. The composition according to claim 1, wherein the composition is a nutritional product.

13. The composition according to claim 1, wherein the composition is an oral rehydration salt.

14. The composition according to claim 1 for use in the treatment of humans or animals by therapy.

15. The composition according to claim 1 for use in maintaining or improving the health of the digestive tract or gastrointestinal function in an individual.

16. The composition according to claim 1 for use in the treatment of gastrointestinal disorders.

17. The composition according to claim 16, wherein the gastrointestinal disorder is accompanied by a disorder of the gastrointestinal barrier function.

18. The composition according to claim 16, wherein the gastrointestinal disorder is selected from the group consisting of diarrhea, inflammatory bowel disease, and irritable bowel syndrome.

19. The composition according to claim 18, wherein the gastrointestinal disorder is bacterial-induced diarrhea.

20. The composition according to claim 1 for use in the treatment of a disease defined by an inflammation-inducing marker.

21. The composition according to claim 20, wherein the disease defined by an inflammation-inducing marker is selected from the group consisting of rheumatoid arthritis, osteoarthritis, atopic dermatitis, psoriasis, allergy, and obesity.

22. The composition according to claim 14, wherein the composition is to be administered into the digestive tract or the composition is to be administered topically.

23. The composition according to claim 15, wherein the composition is to be administered into the digestive tract or the composition is to be administered topically.

24. The composition according to claim 14, wherein the composition is for use in the treatment of humans.

25. The composition according to claim 15, wherein the composition is for use in the treatment of humans.

26. A method of using the composition according to any one of claims 1 to 13 as a food additive, a feed additive, a functional food in human nutrition, a functional food in animal nutrition, a food additive or a functional ingredient for dietary supplements.

27. A method of using the composition according to any one of claims 1 to 13 as a probiotic, a postbiotic, a paraprobiotic, a prebiotic, a synbiotic, or a probiotic alternative.

28. A medical device comprising the composition according to any one of claims 1 to 25. Claim 29 The medical device according to claim 28, wherein the medical device is selected from the group of patches and plasters. Claim 30 Saccharomyces boulardii yeast having the deposit number DSM 33954 (deposited with the German DSMZ, German Collection of Microorganisms and Cell Cultures). Claim 31 A Kluyveromyces marxianus yeast having the deposit number V21 / 012435 (deposited with the National Measurement Institute, Port Melbourne Vic 3207, Australia). Claim 32 A method for preparing the composition according to any one of claims 1 to 25, the method comprising inactivating Saccharomyces boulardii yeast, Saccharomyces cerevisiae yeast, and Kluyveromyces marxianus yeast. Claim 33 The method according to claim 32, wherein the inactivating comprises heat inactivation. Claim 34 The method according to claim 33, wherein the heat inactivation comprises tindalization.

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