CN118251136A - Physiologically acceptable yeast compositions and uses thereof - Google Patents

Abstract

A physiologically acceptable composition comprising (i) at least one component selected from the group consisting of inactivated saccharomyces cerevisiae, saccharomyces boulardii lysate, saccharomyces boulardii cell wall component and saccharomyces boulardii extract, further comprising (ii) at least one component selected from the group consisting of inactivated saccharomyces cerevisiae, saccharomyces cerevisiae lysate, saccharomyces cerevisiae cell wall component and saccharomyces cerevisiae extract, and further comprising (iii) at least one component selected from the group consisting of inactivated kluyveromyces marxianus, kluyveromyces marxianus lysate, kluyveromyces marxianus cell wall component and kluyveromyces marxianus extract. The invention further relates to the use of the composition as a medicament, as a food additive or functional ingredient for nutraceuticals, special medical use foods, cosmeceuticals and functional foods, and as a feed additive for functional ingredients in animal nutrition, as an ingredient for topical application.

Description

Physiologically acceptable yeast compositions and uses thereof

Technical Field

The present invention relates to a physiologically acceptable composition comprising yeast. The invention further relates to a process for preparing the composition. The invention further relates to the use of the composition as a medicament, as a food additive or functional ingredient for nutritional health products (nutraceuticals), special medical use foods, cosmeceuticals and functional foods, as a feed additive for functional ingredients in animal nutrition, as a component for topical application. In particular, the composition according to the invention is suitable for maintaining and improving intestinal health, for the treatment of medical disorders (medical disorders). In a preferred embodiment, the disorder is defined by a pro-inflammatory marker. In a preferred embodiment, the disorder is a gastrointestinal disorder (gastro-INTESTINAL DISORDER, gastrointestinal disorder, gastrointestinal disease). The treatment of a medical disorder according to the invention may be prophylactic treatment or treatment of an individual suffering from said disorder.

Background

Probiotics are living microorganisms that, when administered in sufficient amounts, can bring health benefits to the host. For centuries, humans have benefited from microorganisms in foods (e.g., microorganisms in fermented milk and yogurt) as early as the potential for health benefits of microorganisms is realized. Modern probiotics-containing nutrients and pharmaceuticals are direct derivatives of early fermented foods. To date, the most common probiotics are from the genera Lactobacillus and Bifidobacterium, but yeasts are also increasingly considered to be effective probiotic organisms.

Some international guidelines recommend the use of yeast-based probiotics for the treatment of acute gastrointestinal disorders such as diarrhea or chronic disorders such as Inflammatory Bowel Disease (IBD) and Irritable Bowel Syndrome (IBS). The probiotic activity of these yeasts is believed to be multifactorial and includes improving intestinal barrier function, pathogen competitive exclusion, production of antimicrobial peptides, immunomodulation, regulation of microbiota and nutritional effects. Yeast probiotics have many advantages over bacterial probiotics, such as better tolerance to the extreme environment of the stomach to the intestinal tract and insensitivity to antibiotics, thus providing the possibility of producing probiotic action during antibiotic treatment. A variety of yeast species have been shown to have probiotic action. A variety of yeasts have been proposed as probiotics, such as some strains belonging to Chrysonilia, debaryomyces, hansenula (Hanseniaspora), kluyveromyces (Kluyveromyces), LACHANENCEA, meyezoensis (Metschnikowia), pichia (Pichia), saccharomyces (Saccharomyces), torulaspora (Torulaspora) and Yarrowia (Yarrowia) (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;Dufosséet al.,2021.J Fungi 7(3):177).

To address the need for novel microbial strains that can produce prophylactic and/or therapeutic health beneficial effects on specific pathologies or dysfunctions or on both physical and mental general health conditions, US2010/303778 provides one specific Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain (deposited with the national center for culture of microorganisms (Collection Nationalede Cultures de Microorganismes), accession number CNCM I-3856) and one specific Saccharomyces boulardii variant (Saccharomyces var. Boulardii) yeast strain (deposited with the national center for culture of microorganisms, accession number CNCM I-3799). The yeasts may, for example, be used in a composition for the treatment of intestinal disorders.

There is a continuing need to provide alternative therapeutic possibilities for medical disorders, in particular gastrointestinal disorders such as diarrhea, IBD or IBS, or disorders in which inflammatory factors play a role, such as rheumatoid arthritis, osteoarthritis, local dermatitis, psoriasis, allergies or obesity. There is also a continuing need to provide alternative products suitable for improving intestinal health or improving gastrointestinal function. In particular, it is an object to provide a composition suitable for providing an improved protection or anti-inflammatory effect of the intestinal barrier function compared to known probiotic compositions, which composition comprises yeasts such as described in the prior art discussed above. One or more further objects that may be solved will appear from the description below.

Disclosure of Invention

We have found that one or more of these objects are achieved by providing a specific composition comprising at least three different yeasts.

Accordingly, the present invention relates to a physiologically acceptable composition comprising (i) at least one component selected from the group consisting of saccharomyces cerevisiae (s.boulardii) yeast (yeast), saccharomyces boulardii lysate, saccharomyces boulardii cell wall component and saccharomyces boulardii extract, further comprising (ii) at least one component selected from the group consisting of saccharomyces cerevisiae (s.cerevisiae yeast), saccharomyces cerevisiae lysate, saccharomyces cerevisiae cell wall component and saccharomyces cerevisiae extract, and further comprising (iii) at least one component selected from the group consisting of kluyveromyces marxianus (k.marxianus) yeast, kluyveromyces marxianus lysate, kluyveromyces marxianus cell wall component and kluyveromyces marxianus extract. As shown in the examples, particularly good results have been achieved with physiologically acceptable compositions comprising inactivated saccharomyces boulardii, inactivated saccharomyces cerevisiae and inactivated kluyveromyces marxianus.

Further, the invention relates to said physiologically acceptable composition for use as a medicament.

Furthermore, the invention relates to said physiologically acceptable composition for use in the treatment of humans or animals by therapy.

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

Furthermore, the present invention relates to a physiologically acceptable composition according to the invention for use in the treatment of an individual suffering from a gastrointestinal disorder. Preferred gastrointestinal disorders to be treated are disorders associated with impaired intestinal barrier function. In a particularly preferred embodiment, the composition according to the invention is used for the treatment of diarrhea, IBD or IBS.

Furthermore, the present invention relates to a physiologically acceptable composition according to the present invention for use in the treatment of an individual suffering from a disorder defined by one or more pro-inflammatory markers, in particular one or more markers selected from the group consisting of IL-8, IP-10, MCP-1, TNF alpha and TNF alpha/IL-10. With respect to IL-10, it was observed that this is a modulating/anti-inflammatory compound, the concentration of which may decrease and result in an increase in the tnfα/IL-10 ratio, even though tnfα itself does not increase; the ratio is also a relevant marker for disorders defined by one or more pro-inflammatory markers.

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

Furthermore, the invention relates to the use of a physiologically acceptable composition as a food additive, feed additive, functional food in human nutrition, functional food in animal nutrition, food additive for nutraceutical or functional ingredient.

Furthermore, the invention relates to the use of a physiologically acceptable composition as a probiotic, metazoan (postbiotic), by-probiotic (paraprobiotic), prebiotic, symbiotic (symbiotic) or probiotic substitute.

Furthermore, the invention relates to a medical device (MEDICAL DEVICE) comprising a physiologically acceptable composition.

In the use according to the invention (non-medical or medical), the (i) saccharomyces boulardii, (ii) saccharomyces cerevisiae and (iii) kluyveromyces marxianus components, and in particular the inactivated saccharomyces boulardii, the inactivated saccharomyces cerevisiae and the inactivated kluyveromyces marxianus are typically active ingredients of the composition.

The (medical) use according to the invention may be prophylactic treatment (prophylactic) or treatment of an individual (in particular a human) suffering from a medical disorder to be treated. Treatment of an individual with a medical disorder may include curing the medical disorder, reducing pain, alleviating or alleviating one or more symptoms associated with the medical disorder.

The effectiveness of prophylactic treatment can be routinely determined, for example, by comparing the incidence of disease to a group or test animal treated with a composition used according to the invention and a reference product (placebo). The effect of treating an individual suffering from the disorder may be complete cure, symptom relief, pain relief, etc.

As shown in the examples below, compositions according to the present invention have been found to be effective in improving one or more relevant markers of intestinal health, such as decreasing production of pro-inflammatory cytokines by intestinal epithelial cells and immune cells (i.e., indicating anti-inflammatory effects) and increasing transepithelial resistance (i.e., indicating improved protection of the intestinal barrier function). The impact on these markers suggests that there is a positive impact on the treatment of gastrointestinal disorders characterized by inflammation and/or loss of epithelial integrity (such as diarrhea, IBD or IBS). In addition, the results support that the composition is also effective in treating or preventing gastrointestinal disorders, such as diarrhea or gastrointestinal infections, because multiple markers of intestinal health are improved by using pro-inflammatory stimuli to mimic acute gastrointestinal infections or to infect epithelial cells with bacteria known to induce diarrhea, such as the E.coli (E.coli) strain typically causing diarrhea. In particular, a surprising effect is obtained by combining three yeasts: saccharomyces boulardii, saccharomyces cerevisiae, and Kluyveromyces marxianus. In the examples, it is illustrated how intestinal health is improved by such yeast (in non-viable form) in combination with multiple markers, whereas individual yeasts (in non-viable form) are ineffective, less effective or even have adverse effects. Among other things, synergy is illustrated for MCP-1 (FIG. 2), IL-8 (FIG. 3), IP-10 and MCP-1 (FIGS. 4 and 5) following a TNF- α/INF- γ challenge, and the ratio TNF- α/IL-10 (FIG. 7). Furthermore, it is also illustrated how the yeast combination improves intestinal epithelial integrity, as measured by an increase in transepithelial resistance (TEER) on the intestinal cell monolayer (fig. 8,9 and 10).

The physiological composition according to the invention may contain live yeast cells selected from the group of saccharomyces boulardii, saccharomyces cerevisiae and kluyveromyces marxianus. However, as shown in the examples, the presence of live yeasts is not required. Thus, the inventors further concluded that one, two or each of the Saccharomyces cerevisiae, saccharomyces cerevisiae and Kluyveromyces marxianus in the composition may also be replaced completely or partially by a yeast lysate, cell wall material or yeast extract of Saccharomyces boulardii, saccharomyces cerevisiae and Kluyveromyces marxianus, respectively.

Drawings

FIG. 1 depicts in vitro IP-10 chemokine production by Caco-2 cells after incubation with 3 yeasts and combinations thereof in the absence of a pro-inflammatory stimulus.

FIG. 2 depicts in vitro MCP-1 chemokine production by Caco-2 cells after incubation with 3 yeasts and combinations thereof in the absence of pro-inflammatory stimuli.

FIG. 3 depicts in vitro IL-8 chemokine production by Caco-2 cells after incubation with 3 yeasts and combinations thereof in the absence of a pro-inflammatory stimulus.

FIG. 4 depicts in vitro IP-10 chemokine production by Caco-2 cells after incubation with 3 yeasts and combinations thereof in the presence of pro-inflammatory stimuli (TNF- α/INF- γ) mimicking inflamed intestinal epithelium.

FIG. 5 depicts in vitro MCP-1 chemokine production by Caco-2 cells after incubation with 3 yeasts and combinations thereof in the presence of pro-inflammatory stimuli (TNF- α/INF- γ) mimicking inflamed intestinal epithelium.

FIG. 6 depicts in vitro IL-8 chemokine production by Caco-2 cells after incubation with 3 yeasts and combinations thereof in the presence of pro-inflammatory stimuli (TNF- α/INF- γ) mimicking inflamed intestinal epithelium.

FIG. 7 shows the in vitro reduction in TNFα/IL-10 ratio observed in human THP-1 cells (macrophages).

Figure 8 depicts the protective effect of different yeasts and combinations thereof on intestinal epithelium. The yeast is shown compared to the negative control when incubated for 1 hour (left bar) or 2 hours (right bar) with an infectious agent known to disrupt the epithelial monolayer (E.coli ETEC H10407). Intestinal epithelial integrity was measured by an increase in transepithelial electrical resistance (TEER) of the monolayer.

FIG. 9 shows differentiation of intestinal epithelium as measured by increase in TEER when Caco-2 cell monolayers were incubated in the presence of yeast (upper panel) and combinations thereof (lower panel). Note that some error bars are too small to see.

FIG. 10 compares the effects observed in FIG. 9 (differentiation of intestinal epithelium as measured by increase in TEER when a Caco-2 cell monolayer is incubated in the presence of yeast and combinations thereof), expressed as% increase compared to control. The regression formula for the trend line is as follows, saccharomyces boulardii: y= -0.0374x+1.1848; saccharomyces cerevisiae: y=0.01dx+0.9844; kluyveromyces marxianus: y= -0.0031x+1.0408; saccharomyces boulardii + Saccharomyces cerevisiae: y=0.0081x+0.9871; and ABB C22: y=0.0142x+0.9384.

Detailed Description

For purposes of clarity and conciseness of description, features are described herein as part of the same or separate embodiments, however, it should be understood that the scope of the invention may include embodiments having combinations of all or some of the described features.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well. The term "or" includes any and all combinations of one or more of the associated listed items unless the context clearly indicates otherwise (e.g., if a "either" or "structure is used). It should be understood that the terms "comprises" and "comprising" specify the presence of stated features, but do not preclude 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 being "following" another step, it can be one or more intervening steps may be performed immediately following the other step or before the particular step may be performed, unless otherwise specified.

The term "at least substantially" is generally utilized herein to refer to the general features or functions described. When referring to a quantifiable feature, the term is generally used to denote more than 50%, particularly at least 75%, more particularly at least 90%, even more particularly at least 95% of the maximum value of the feature.

The term "substantially free" is generally used herein to mean that a feature is absent or present in an amount that is so low that it does not significantly affect the performance of the product.

In the context of the present application, the term "about" generally means a deviation from a given value of 15% or less, in particular a deviation of 10% or less, more in particular a deviation of 5% or less.

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

As used herein, the term "probiotic" refers to a live microorganism that, when administered in sufficient amounts, imparts a health benefit to an individual. Probiotics include a variety of microorganisms including bacteria and yeasts.

As used herein, the term "inactivated" or "dead" or "non-viable" refers to organisms, such as yeasts, that are unable to reproduce or colonize. The inactivated organism may have a whole or broken cell membrane. The skilled person will be able to obtain inactivated organism yeasts based on common general knowledge and the information disclosed herein. Possible methods include irradiation, heat inactivation, sonication, lyophilization and chemical inactivation.

As used herein, the term "heat killed" refers to organisms that are inactivated by heat treatment and therefore are not capable of metabolic activity or colonization. Heat treatment methods for inactivating organisms are known to those skilled in the art and include tyndall sterilization, pasteurization, ultra High Temperature (UHT) heating, ohmic heating (or joule heating), blanching, drying, boiling, and sterilization.

As used herein, the term "tyndall sterilization" refers to a sterilization process commonly used to heat kill probiotic microorganisms. The tyndall sterilization method involves repeating the step of heat sterilization for successive days, for example as described by Ronald m. Thus, the term "tyndallized (tyndallized)" refers to organisms that have been heat killed by tyndall sterilization and therefore are not capable of metabolic activity or colonization.

As used herein, the term "cell lysis" refers to any type of cell destruction that results in the release of intercellular biological components naturally contained in an organism's cells. Thus, the term "lysate" refers to the product obtained after cell lysis. As used herein, "lysate" refers in particular to substantially all lysates obtained by lysing organisms, and thus comprises macromolecules such as DNA, RNA, proteins, peptides and lipids from the lysed cells; and cell debris, including cell wall material and cell membrane components from lysed cells. Methods for obtaining lysates are known to those skilled in the art and include enzymatic, physical, and chemical methods. The cell wall component may be separated from the liquid portion of the lysate, for example, by centrifugation.

As used herein, the term "extract" of yeast refers to a fluid portion or fraction of a yeast cell or lysate, in particular a liquid content of a yeast cell, obtainable in particular by filtration or centrifugation, or a fraction thereof obtainable by extraction from a cell or lysate using an extraction phase.

As used herein, the term "metabolite" refers to any substance derived from the growth or maintenance of yeast that persists in a medium and does not require preservation by a particular technique. Examples of metabolites are organic and inorganic acids, proteins, (poly) peptides, amino acids, (co) enzymes, fatty acids, (esterified) lipids, carbohydrates (including monosaccharides, disaccharides and polysaccharides), lipoproteins, glycolipids, glycoproteins, phosphates of sugars, vitamins, salts, metals or nucleic acids.

As used herein, the term "living" or "surviving" refers to organisms capable of propagating or colonizing.

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

As used herein, the term "nutritional product" refers to a composition intended to be ingested by an individual to provide the individual with at least one nutrient. Nutritional products typically comprise one or more components selected from the group consisting of proteins, fats, carbohydrates and micronutrients.

As used herein, the term "nutritional" refers to any nutritional product that provides additional health benefits in addition to the basic nutritional value found in foods.

As used herein, the term "cosmeceutical" refers to any cosmetic containing a biologically active ingredient that is said to have a health benefit.

As used herein, the term "Oral Rehydration Salts (ORS)" refers to sugar-based salt solutions suitable for use in oral rehydration therapy. ORS is recommended for preventing diarrhea and dehydration of individuals of any age due to any cause. ORS is further suggested for use in treating dehydrated individuals of any age.

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

As used herein, the term "metazoan" refers to any formulation of inactivated microorganisms and/or components thereof that imparts a health benefit to an individual. The health benefit imparting component may be a mixture of metabolites secreted by the probiotics in the cell-free supernatant, such as enzymes, secreted proteins, short chain fatty acids, vitamins, secreted biosurfactants, amino acids, peptides, organic acids, etc.

As used herein, the term "paraprobiotic" refers to an inactivated microorganism and/or cell fraction that imparts a health benefit to an individual.

As used herein, the term "synbiotics" or "symbiotics" refers to any formulation comprising a mixture of probiotics and prebiotics.

Hereinafter, the term "yeast material" or "yeast-based fraction (yeast based fraction)" is used as genus of living yeast cells, inactivated yeast cells, yeast lysates, yeast cell wall components and yeast extracts. Also, the term "microbial material" is used as a genus of living microorganisms, inactivated microorganisms, microbial lysates, microbial cell wall components, and microbial extracts.

Components of physiologically acceptable compositions

In the present invention, saccharomyces cerevisiae may be any strain classified or classifiable as Saccharomyces cerevisiae variant (S.cerevisiae. Boulardii), in particular any such strain of probiotic bacteria in live or inactivated form. In the physiologically acceptable compositions according to the invention, the yeasts Brevibacterium are generally inactivated. In one embodiment, the composition comprises at least one saccharomyces boulardii selected from the group consisting of: saccharomyces cerevisiae DSM 33954, 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, and Saccharomyces boulardii CNCM I-3799. In particular, good results have been achieved with a composition comprising inactivated saccharomyces boulardii DSM 33954.

In the present invention, saccharomyces cerevisiae may be any strain belonging to the species Saccharomyces cerevisiae, in particular any strain of probiotic bacteria in live or inactivated form, except strains classified or classifiable as Saccharomyces cerevisiae variants. The species Saccharomyces cerevisiae is also known in the art as Saccharomyces cerevisiae, or Candida robusta. In the physiologically acceptable compositions according to the invention, saccharomyces cerevisiae is generally inactivated. In a preferred embodiment, the composition comprises at least one Saccharomyces cerevisiae selected from the group of Saccharomyces cerevisiae Y1529 (deposited under ATCC), saccharomyces cerevisiae CNCM I-3856, saccharomyces cerevisiae S288C, and Saccharomyces cerevisiae UFMG 905, with Saccharomyces cerevisiae S288C and Saccharomyces cerevisiae Y1529 being particularly preferred. In particular, good results have been achieved with the use of mineral-rich s.cerevisiae (e.g. zinc-rich s.cerevisiae).

In the present invention, kluyveromyces marxianus can be any strain belonging to the species Kluyveromyces marxianus. This species is also known in the art as kluyveromyces marxianus (Saccharomyces marxianus), candida lactis (CANDIDA KEFYR), candida pseudotropicalis (Candida pseudotropicalis), kluyveromyces fragilis (Kluyveromyces fragilis), kluyveromyces chickpea (Kluyveromyces cicerisporus). In the physiologically acceptable compositions according to the invention, kluyveromyces marxianus is generally 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 V21/012335, and kluyveromyces marxianus Z17. In particular, good results are achieved with the composition according to the invention comprising kluyveromyces marxianus V21/012335 cells. Accordingly, in a preferred composition of the invention, kluyveromyces marxianus V21/012335 cells, an extract of Kluyveromyces marxianus V21/012335 cells, a lysate of Kluyveromyces marxianus V21/012335 cells or cell wall material of Kluyveromyces marxianus V21/012335 cells thereof are present.

By using standard references, such as, for example "The yeasts,a taxonomic study"(CP Kurtzman,JW Fell and T Boekhout),5^th edition,2011,Elsevier,, the person skilled in the art will be able to determine whether a given yeast strain is classified or classifiable as a saccharomyces cerevisiae variant, as a species saccharomyces cerevisiae or as kluyveromyces marxianus. Furthermore, based on standard references, such as, for example 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,, the person skilled in the art will be able to distinguish Saccharomyces cerevisiae variants from other yeast strains belonging to the species Saccharomyces cerevisiae.

The relative amounts of (i) at least one component selected from the group consisting of saccharomyces cerevisiae, saccharomyces cerevisiae lysate, saccharomyces boulardii cell wall component, and saccharomyces boulardii extract (also referred to herein as a saccharomyces boulardii-based fraction), (ii) at least one component selected from the group consisting of saccharomyces cerevisiae, saccharomyces cerevisiae lysate, saccharomyces cerevisiae cell wall component, and saccharomyces cerevisiae extract (also referred to herein as a saccharomyces cerevisiae-based fraction), and (iii) at least one component selected from the group consisting of kluyveromyces marxianus, kluyveromyces marxianus lysate, kluyveromyces marxianus cell wall component, and kluyveromyces marxianus extract (also referred to herein as a kluyveromyces marxianus-based fraction) can vary within wide ranges.

Typically, the saccharomyces boulardii-based fraction of the composition according to the invention is: based on the total yeast component, 0.05-99.95wt.%, preferably 5-95wt.%, more preferably 15-80wt.%, particularly 20-60wt.%, more particularly 25-50wt.%.

Typically, the saccharomyces cerevisiae-based fraction of the composition according to the invention is: based on total yeast component, 0.05-90wt.%, preferably 5-90wt.%, more preferably 10-90wt.%, more preferably 15-80wt.%, particularly 20-80wt.%, more particularly 20-60wt.%, more particularly 25-50wt.%.

Generally, the kluyveromyces marxianus-based fraction of the composition according to the invention is: based on the total yeast component, 0.05-99.95wt.%, preferably 5-90wt.%, more preferably 10-90wt.%, more preferably 15-80wt.%, particularly 20-80wt.%, more particularly 20-60wt.%, more particularly 25-50wt.%.

Typically, the saccharomyces boulardii-based fraction, the saccharomyces cerevisiae-based fraction and the kluyveromyces marxianus-based fraction together form at least 10wt.%, preferably at least 25wt.%, more preferably at least 50wt.%, in particular at least 75wt.% of the total microbiological material (e.g., bacterial, algal or fungal cells, lysates thereof, extracts thereof, cell wall parts thereof). Other microbial materials, particularly probiotic microorganisms or cellular materials of probiotic microorganisms, may be present in the composition if desired. Thus, the sum of the saccharomyces boulardii-based fraction, the saccharomyces cerevisiae-based fraction and the kluyveromyces marxianus-based fraction is 100% or less of the total microbiological material, for example 99wt.% or less based on the total microbiological material.

In an advantageous embodiment, the physiological composition comprises (all based on total yeast components and preferably on total microbial material) 5-95wt.% of a fraction based on saccharomyces cerevisiae (typically inactivated saccharomyces boulardii), 10-80wt.% of a fraction based on saccharomyces cerevisiae (typically inactivated saccharomyces cerevisiae) and 5-95wt.% of a fraction based on kluyveromyces marxianus (typically inactivated kluyveromyces marxianus). In particular, a composition having a total microbial material based on the composition, a fraction content based on saccharomyces boulardii in the range of 15-50wt.%, a fraction content based on saccharomyces cerevisiae in the range of 15-50wt.% and a fraction content based on kluyveromyces marxianus in the range of 15-50wt.% achieves good results, for example in terms of various pro-inflammatory markers or TEERs, provided that the total amount of the three fractions is 100wt.% or less. In this context, the yeast-based fraction advantageously at least essentially consists of inactivated yeast cells. In this context, the total microbiological material advantageously consists at least substantially of inactivated saccharomycete cells. As will be appreciated by those skilled in the art, based on the information and common general knowledge disclosed herein, different levels may be employed to achieve satisfactory results.

The interest in using non-viable inactivated probiotics has increased due to the safety issues with the use of viable microorganisms, for example in fragile or immunocompromised patient populations or newborns. Various methods of inactivation are known to those skilled in the art and include radiation, heat inactivation, sonication, lyophilization and chemical inactivation. The tyndall sterilization method is a sterilization process commonly used for heat sterilization of probiotic microorganisms. Interestingly, according to the present invention, tyndallized yeasts have been shown to exert related biological responses, such as restoration of normal intestinal homeostasis.

Thus, typically, the physiologically acceptable compositions of the invention comprise non-viable Saccharomyces cerevisiae, and non-viable Kluyveromyces marxianus. If one or more of the yeasts are mineral-enriched yeasts, such as zinc-enriched yeasts, it is particularly preferred that the mineral-enriched yeasts are inactivated, i.e., non-viable. Preferably, the inactivated yeast is heat killed. More preferably, the inactivated yeast is tyndallized. The tyndall sterilization method may be based on tyndall sterilization methods generally known in the art. In particular, good results have been achieved with a composition comprising a tyndallized saccharomyces cerevisiae, a further tyndallized saccharomyces cerevisiae and a further tyndallized kluyveromyces marxianus.

Typically, at least substantially all of the yeast cells present in the physiologically acceptable composition according to the invention are non-viable. Thus, typically the yeast material is substantially free of viable yeast cells. However, in a specific embodiment, the physiologically acceptable composition of the invention comprises at least one live yeast selected from the group consisting of saccharomyces boulardii, saccharomyces cerevisiae and kluyveromyces marxianus. If at least one of the yeasts is present in a live form, it is preferred that at least Kluyveromyces marxianus is present in a live form. The content of live yeast material, if present, is typically a small fraction of the total yeast material, particularly 10wt.% or less, more particularly 1wt.% or less.

The branchia yeast contained in the physiologically acceptable composition of the invention for administration into the gastrointestinal tract is typically present in a concentration in the range of 10 ⁶ cells/gram (based on the total weight of the yeast component) to 10 ¹¹ cells/gram (based on the total weight of the yeast component), preferably 10 ⁷ cells/gram (based on the total weight of the yeast component) to 10 ¹¹ cells/gram (based on the total weight of the yeast component), More preferably from 10 ⁹ cells/gram (based on total weight of yeast component) to 2x10 ¹⁰ cells/gram (based on total weight of yeast component). Saccharomyces cerevisiae included in the physiologically acceptable compositions of the present invention for administration into the gastrointestinal tract is typically present at a concentration in the range of 10 ⁶ cells/gram (based on the total weight of the yeast component) to 10 ¹¹ cells/gram (based on the total weight of the yeast component), preferably 10 ⁸ cells/gram (based on the total weight of the yeast component) to 5x10 ⁹ cells/gram (based on the total weight of the yeast component). The kluyveromyces marxianus contained in the physiologically acceptable compositions of the invention for administration into the gastrointestinal tract is typically present at a concentration in the range of 10 ⁶ cells/gram (based on the total weight of the yeast component) to 10 ¹⁰ cells/gram (based on the total weight of the yeast component), preferably 10 ⁷ cells/gram (based on the total weight of the yeast component) to 5x10 ⁹ cells/gram (based on the total weight of the yeast component).

The branchia yeast contained in the physiologically acceptable compositions of the invention for another mode of administration (e.g., topical administration) is typically present at a concentration in the range of 10 ⁴ cells/gram (based on the total weight of the yeast component) to 10 ¹¹ cells/gram (based on the total weight of the yeast component), preferably 10 ⁶ cells/gram (based on the total weight of the yeast component) to 10 ¹⁰ cells/gram (based on the total weight of the yeast component). The saccharomyces cerevisiae included in the physiologically acceptable composition of the invention for another mode of administration, such as topical administration, is typically present in a concentration ranging from 10 ⁴ cells/gram (based on the total weight of the yeast component) to 10 ¹¹ cells/gram (based on the total weight of the yeast component), preferably from 10 ⁶ cells/gram (based on the total weight of the yeast component) to 10 ¹⁰ cells/gram (based on the total weight of the yeast component). The kluyveromyces marxianus contained in the physiologically acceptable compositions of the present invention for another mode of administration, such as topical administration, is typically present at a concentration ranging from 10 ⁴ cells/gram (based on the total weight of the yeast component) to 10 ¹¹ cells/gram (based on the total weight of the yeast component), preferably from 10 ⁶ cells/gram (based on the total weight of the yeast component) to 10 ¹⁰ cells/gram (based on the total weight of the yeast component).

The concentrations of live and inactivated yeasts are measured and expressed as the number of cells per gram of total yeast component. For live yeasts, the concentration in "cells/gram" corresponds to "Colony Forming Units (CFU) per gram of total yeast component". If a combination of living cells and inactivated cells (not forming colonies) is present, the total number of cells will generally be within the usual range described above, preferably within the preferred range described above or more preferably within the range described above. For a yeast lysate, extract or cell wall fraction, suitable concentrations generally correspond to amounts of lysate, extract and cell wall fraction available from 10 ⁴ cells/gram (based on total weight of yeast fraction) to 10 ¹¹ cells/gram (based on total weight of yeast fraction), respectively.

The physiologically acceptable composition of the invention may comprise a mineral-enriched yeast, in particular Saccharomyces cerevisiae. Preferably, the physiologically acceptable composition of the invention comprises a zinc-rich yeast, more preferably a zinc-rich saccharomyces cerevisiae. The physiologically acceptable composition of the invention may comprise a mineral salt, preferably a zinc salt, most preferably zinc sulphate.

Zinc-rich yeasts provide a natural source of high bioavailability zinc. Zinc is considered a key nutrient for immune and diarrhea management. Interestingly, studies have shown that supplementation of zinc by yeast organisms as organically bound or blended zinc results in better bioavailability than inorganic zinc. Not only zinc, but also several other minerals such as selenium, chromium, iron, copper, magnesium, manganese, potassium, calcium and iodine have also been shown to be beneficial in restoring the mineral balance of individuals and can be enriched in yeasts making them better bioavailable. The yeast enriched in minerals can be obtained by culturing the yeast in a medium supplemented with one or more minerals such as zinc, selenium, chromium, iron, copper, magnesium, manganese, potassium, calcium or iodine. Minerals are typically organically bound to or otherwise absorbed by the yeast protein, resulting in the absorption of one or more minerals in the cells of the yeast. Minerals may be added to the culture medium before, during or after the culturing. As used herein, a mineral-enriched yeast strain, in particular a yeast strain fermented in the presence of mineral salts or a yeast strain to which mineral salts are added after fermentation, contains such minerals in a final concentration of at most 12wt.%, in particular at most 5wt.%, more in particular at most 2wt.%, for example at most 1wt.%, based on the dry weight of the whole product. For zinc, it is preferably added to a final concentration in the range of 1-12wt.%, in particular about 4wt.% to about 10wt.%.

Modes of administration and formulations of physiologically acceptable compositions

The physiologically acceptable compositions of the invention are preferably administered into the gastrointestinal tract or topically. Oral administration to the gastrointestinal tract is preferred. In a specific embodiment, the composition is administered by tube feeding or as a suppository. Topical application includes in particular application to mucous or skin. Specific examples are ocular administration and vaginal administration. Formulations of compositions according to the present invention suitable for oral ingestion include, but are not limited to: capsules, coated capsules, tablets, sachets, pills, pearls, soft capsules, vials, powders, granules, solutions, suspensions, emulsions, elixirs, syrups, sprays, lozenges, troches, chewing gums, hard candies and gels. Formulations of compositions according to the invention suitable for topical administration include creams (e.g., for vaginal applications), capsules (e.g., for vaginal applications), tablets (e.g., for vaginal applications), ointments, pastes, foams, gels, lotions, shampoos, mousses, sprays (e.g., for application to the skin, eyes or mucous membranes), suppositories, solutions (e.g., for vaginal applications), plasters, bioadhesives, liquids (e.g., for application to the skin, eyes, such as eye drops or mucous membranes), and suspensions.

Formulations of compositions according to the invention suitable for topical administration include compositions suitable for application to the gastrointestinal tract portion where action of the yeast component is desired, such as suppositories or gastrointestinal medical devices.

Topical administration in the treatment of gastrointestinal disorders typically involves administration at the mucosa or epithelium of the gastrointestinal tract. The medical product comprising the composition (for use) according to the invention may be a product suitable for producing a protective biofilm or the like on the intestinal epithelial surface. Such products may be based on known products for example for the treatment of IBS.

In a specific embodiment, the composition is administered as a slow release product.

The physiologically acceptable compositions of the invention may be administered using medical devices. Such medical devices for topical application of physiologically acceptable compositions may be plasters, (transdermal) patches.

The composition according to the invention may be a food product. The food product may be a fermented food product or a non-fermented food product. Examples of particularly suitable food products include dairy products such as yoghurt, yoghurt drinks, cheese, milk powder, infant formula, cream, ice cream, cream powder and butter; fruit-based products such as fruit juices, preserves, or jellies; solid foods such as flour, cereals, snacks, biscuits, and liquid preparations such as vegetable beverages (vegetable beverage, vegetable beverages), smoothies, isotonic beverages (isotonic drinks), saline solutions and enteral nutrition recipes (enteral nutrition recipe, enteral nutrition meals). The physiologically acceptable composition according to the invention for ingestion by an animal may be any food suitable for an animal and includes, in addition to the foods listed above, tablets, coated tablets, granules, grains, (dry) meat, (dry) fish, meal, cakes, biscuits, sugar cane and roughages, such as grasses, hay, silage, root crops, straw and stalks.

The nutritional product 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 a functional ingredient for a nutraceutical or a food for special medical use.

The physiologically acceptable composition according to the invention may be a pharmaceutical product, a cosmeceutical product or a nutraceutical product (nutraceutical product ). The pharmaceutical product may further comprise pharmaceutically acceptable adjuvants and/or excipients. Adjuvants and excipients are well known to those skilled in the art.

Oral Rehydration Salts (ORS) are preferred examples of products according to the invention; ORS can be categorized as a pharmaceutical (e.g., WHO) or as a food supplement, depending on national regulations. Inactivated yeast cells that have been found to be particularly suitable for ORS are Saccharomyces boulardii DSM 33954 (available as ABB1 from ABBiotek-Spain, https:// www.abbiotek.com), saccharomyces cerevisiae Y1529 (deposited as ATTC, available as ABB6 from ABBiotek), and Kluyveromyces marxianus V21/012335 (deposited as Australian national institute of metrology (National Measurement Institute), port Melbourne Vic 3207, available as ABB7 from ABBiotek). A mixture of these three yeast strains is available from ABBiotek as ABB C22.

The physiologically acceptable composition may further comprise a filler, in particular a carbohydrate, such as maltodextrin.

Examples of ORS formulations in liquid form include Saccharomyces boulardii, saccharomyces cerevisiae, and Kluyveromyces marxianus (ABB C22); and further water, glucose, sodium citrate, sodium chloride, maltodextrin, zinc sulfate, silica, flavoring agents, sweeteners (potassium acesulfame) and acidulants (citric acid).

Examples of ORS formulations in powder form include Saccharomyces boulardii, kluyveromyces marxianus, and Saccharomyces cerevisiae (ABB C22); further dextrose, lemon, flavoring, citric acid, magnesium citrate, malic acid, sodium citrate, sodium chloride, potassium phosphate, calcium ascorbate, sucralose, and riboflavin.

Production of physiologically acceptable compositions

The physiologically acceptable compositions can be prepared by combining the different components based on methods known per se for preparing yeast preparations.

For example, all yeasts can be produced from non-GMO yeast strains. The primary growth yeasts can be produced using fermentation processes known per se for yeasts of interest, the growth taking place under sterile, aerobic conditions. The resulting product or yeast extract can be stored refrigerated to maintain cell viability, if desired.

The yeast extract can be subjected to an inactivation treatment, such as high temperature sterilization or tyndall sterilization, to obtain a heat treated version of the yeast. If desired, the yeasts can be dried, for example by spray drying, which is preferably carried out after inactivation (if non-viable yeasts are used in the composition).

Maltodextrin or other fillers may be used as a support to have standardized concentrations. The three yeasts are mixed and after mixing a homogenization step may be performed.

Methods of treating individuals and improving intestinal health or gastrointestinal function in individuals

According to the invention, the physiologically acceptable composition is advantageously used for the treatment of individuals suffering from: gastrointestinal disorders such as Irritable Bowel Syndrome (IBS); inflammatory Bowel Disease (IBD), such as crohn's disease or ulcerative colitis; functional constipation; diarrhea, such as antibiotic-associated diarrhea, traveler's diarrhea, acute gastroenteritis, pediatric diarrhea, dysbacteriosis diarrhea, or chronic diarrhea, especially in immunocompromised patients; functional abdominal pain; functional abdominal distension, postprandial anxiety syndrome; gastrointestinal allergies or intolerance; necrotizing enterocolitis; gastrointestinal tract infections caused by bacteria such as Escherichia (Escherichia), salmonella (Salmonella), shigella (Shigella), staphylococcus (Staphylococcus), vibrio (Vibrio), campylobacter (Campylobacter), yersinia (Yersina), clostridium (Clostridium), or Helicobacter (Helicobacter); gastrointestinal infections caused by viruses, such as norovirus, adenovirus, cytomegalovirus, enterovirus, astrovirus, hepatitis virus or rotavirus; gastrointestinal tract infections caused by parasites, such as Giardia (Giardia), amoeba (Entamoeba), cryptosporidium (Cryptosporidium), cyclosporin (Cyclospora) or ascariasis (Ascaris); and combinations thereof. Preferably, the gastrointestinal disorder selected from the group consisting of IBD, IBS and diarrhea is treated. In a specific embodiment, the diarrhea treated is a bacterial-induced diarrhea, such as an E.coli-induced diarrhea.

Furthermore, the physiologically acceptable compositions are advantageously used to treat individuals suffering from disorders defined by one or more pro-inflammatory markers, including IL-8, IP-10, MCP-1, TNF alpha/IL-10 and TNF alpha. Disorders defined by pro-inflammatory markers for treatment by the present invention include rheumatoid arthritis, osteoarthritis, local dermatitis, psoriasis, allergies, and obesity. In particular, the physiologically acceptable compositions of the invention are suitable for treating inflammatory disorders defined by one or more of the pro-inflammatory markers in the absence of infection.

Furthermore, the physiologically acceptable compositions according to the invention are particularly suitable for maintaining or improving intestinal health or gastrointestinal function, especially those disorders involving impaired or diminished intestinal barrier function or altered inflammatory cytokine release. The term "intestinal health" as used herein refers to the health of the intestinal tract. The intestinal health of an individual may be affected by, for example, infectious or non-infectious causes (e.g., non-optimal diet). The term "gastrointestinal function" refers to the operation of all organs and structures associated with the gastrointestinal system. Markers for determining intestinal health or gastrointestinal function are known to those skilled in the art and include, for example, transepithelial resistance as an indication of epithelial barrier integrity and examples of markers described in markers .Celi et al.(2019)"Biomarkers of gastrointestinal functionality in animal nutrition and health"Animal Feed Science and Technology 250:9-31 of inflammation and injury.

The dosage, duration and frequency of administration can be selected within a wide range, depending on the intended purpose and the subject to whom the composition is to be administered. The duration of treatment may be a relatively short period of time, such as a week or less, or a day or less, such as for acute manifestations of a disorder or symptoms of a disorder, such as diarrhea. The duration of treatment may also be prolonged, for example, one week or more, one month or more, one year or more, for example if chronic disorders such as IBD are present.

The physiologically acceptable composition for use according to the invention may be administered as a single dose for complete treatment, depending on the application. If multiple dosing is intended, the number of dosing will typically be 10 or less per day. For example, in the case of ORS, more than 3 doses may be administered per day, but typically about 3 times per day or less, preferably about 2 times per day or less, and in particular about once per day or less. In one embodiment, the composition is administered (on average) at least about once a week. Preferably, the administration is (on average) at least once every three days, more preferably (on average) at least once every two days.

The physiologically acceptable composition may comprise or may be co-administered with (another) probiotic, prebiotic, metazoan, antibiotic, analgesic, anti-inflammatory agent, antidiarrheal agent such as an exercise or secretion inhibitor, (other) oral rehydration salts, laxatives or mixtures thereof.

The person skilled in the art will be able to determine, based on common general knowledge and the information disclosed herein, without undue experimentation, the appropriate dosage of a physiologically acceptable composition to be administered to an individual. As the skilled artisan will appreciate, the actual preferred dosage will depend on a variety of factors including the activity of the particular yeast used, the metabolic stability and length of action of that yeast, the age, body weight, general health, sex and type of individual diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disorder, and the individual. The dosages disclosed herein are indicative of the average condition of a human individual. Of course, there are individual cases where a higher or lower dosage range is required. The usual effective daily dose for oral administration or for other administration into the gastrointestinal tract, especially for humans, is from about 100mg (yeast component) to about 1000mg (yeast component), preferably from about 200mg (yeast component) to about 900mg (yeast component), more preferably from about 200mg (yeast component) to about 650mg (yeast component). The usual effective daily dosage for topical administration, especially for humans, is from about 1mg (yeast component) to about 1000mg (yeast component), preferably from about 5mg (yeast component) to about 500mg (yeast component).

The present invention provides a physiologically acceptable composition for use as a medicament.

The present invention provides a method of treating an individual in need of improving intestinal health or in need of improving gastrointestinal function comprising administering an effective amount of a physiologically acceptable composition according to the present invention, thereby improving intestinal health or gastrointestinal function. In particular, the invention has been found to be effective in improving intestinal barrier function and driving anti-inflammatory states, as well as preventing viral, bacterial and yeast infections, while regulating microbiota to a probiotic state.

The present invention provides a method of treating an individual suffering from a gastrointestinal disorder, such as diarrhea, IBD, IBS or (other) gastrointestinal disorders associated with impaired intestinal barrier function, comprising administering to said individual an effective amount of a physiologically acceptable composition according to the present invention.

The present invention provides a method of treating a subject suffering from a disorder defined by a pro-inflammatory marker (such as rheumatoid arthritis, osteoarthritis, local dermatitis, psoriasis, allergy or obesity) comprising administering to the subject an effective amount of a physiologically acceptable composition according to the invention.

The present invention provides the use of a physiologically acceptable composition according to the invention for the preparation of a product, which may be a pharmaceutical or food product, such as a medical or clinical food, for 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 intestinal barrier function.

The present invention provides the use of a physiologically acceptable composition according to the invention for the preparation of a product, which may be a pharmaceutical or food product (e.g. a medical or clinical food product), for the treatment of disorders defined by pro-inflammatory markers, such as rheumatoid arthritis, osteoarthritis, local dermatitis, psoriasis, allergies and obesity.

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

The invention will now be illustrated by the following examples, which are provided by way of illustration, and it should be understood that many variations may be made in the methods described and in the amounts shown without departing from the spirit of the invention and the scope of the appended claims.

Examples

Example 1: anti-inflammatory effects on intestinal epithelial cells.

Materials and methods

All yeasts are produced by non-GMO yeast strains. The yeast included in the experiment was Saccharomyces boulardii DSM 33954 (deposited at DSMZ, german collection of microorganisms and cell cultures) available as ABB1 from ABBiotek; saccharomyces cerevisiae Y1529 (deposited at ATTC), available from ABBiotek as ABB6 in a zinc-rich form; and kluyveromyces marxianus V21/012335 (deposited at national institute of metrology, port Melbourne Vic 3207), available as ABB7 from ABBiotek. As used herein, a combination of these yeasts is also available as ABB22 from ABBiotek.

The fermentation process produces primary growing yeasts whose growth occurs under sterile, aerobic conditions. During fermentation, temperature, pH and growth rate are tightly controlled. For Saccharomyces cerevisiae, zinc sulfate was added to the yeast extract at the end of the fermentation process to a concentration of about 10% on a dry weight basis. The obtained product or yeast extract is stored in cold storage to maintain cell viability. The frozen yeast extract is treated with an autoclaving system to obtain a tyndallized version of yeast prior to spray drying. Maltodextrin or other fillers may be used as a support to have standardized concentrations.

Three heat-inactivated yeasts are premixed and then subjected to a homogenization step. For the in vitro studies described below, the mixtures were prepared from stock solutions of each yeast.

The yeast concentration of each yeast stock was measured by flow cytometry. Samples were prepared for each yeast, which were normalized to 1x10 ⁷ cells/mL in cell culture. To obtain a combination, samples of each yeast strain were mixed in a uniform ratio of each strain, i.e. each concentration of the three strains in the mixture was 0.333x10 ⁷ cells/ml.

The in vitro immunomodulatory activity of yeasts and combinations thereof was studied by producing chemokines by Caco-2 cells in the presence and absence of pro-inflammatory stimuli. Caco-2 cells were cultured to confluence in 96-well plates. At the beginning of the experiment, the cells were washed once with medium without antibiotics. The monolayers were incubated with the test components in antibiotic-free medium at 37 ℃ for 1 hour in triplicate. Thereafter, the cells were incubated with medium containing the test components and 50. Mu.g/ml gentamicin (Invitrogen) in duplicate. A mixture of recombinant TNFα (10 ng/ml) and recombinant IFNγ (5 ng/ml) (R & D systems) was further used as one of the pro-inflammatory stimulator stimulation replicates (FIGS. 4-6). As a blank, medium alone, no stimulation in the case of fig. 1-3, and fig. 4-6 are mixtures of medium (recombinant tnfα (10 ng/ml) and recombinant ifnγ (5 ng/ml) under pro-inflammatory stimuli were used.

Supernatants were collected 24 hours after stimulation and stored at 20 ℃. IL-8, IP-10 and MCP-1 levels were measured using the Bio Plex assay (BioRad) according to the manufacturer's protocol.

After collection of culture supernatants, the metabolic activity of the cells was analyzed by WST-1 assay (Roche) according to the manufacturer's protocol to confirm the non-cytotoxicity of the tested components. These cells were found to be not metabolically active, indicating that any observed effects were not due to metabolic changes or cytotoxicity.

As in the other examples, a one-way anova was performed and then statistical differences between control conditions and test conditions were calculated using Dunnett's post hoc test. The significance thresholds used in the figures are as follows: * p <0.05, < p <0.01 and p <0.001.

Results

FIG. 1 depicts the production of IP-10 chemokines in vitro after incubation of Caco-2 cells with 3 yeasts and combinations thereof in the absence of a pro-inflammatory stimulus.

In particular, for a yeast composition comprising Saccharomyces boulardii, saccharomyces cerevisiae and Kluyveromyces marxianus (from now on referred to as composition ABB C22), a synergistic decrease in pro-inflammatory cytokines in intestinal epithelial cells was observed compared to the individual yeasts (FIGS. 2-3). Synergistic reduction of pro-inflammatory cytokines in intestinal epithelial cells following pro-inflammatory challenges with tnfα/ifnγ was observed with yeast composition ABB C22 compared to single yeasts (fig. 4-5). Furthermore, the composition ABB C22 was observed to be superior to compositions comprising only the yeasts saccharomyces boulardii and saccharomyces cerevisiae (fig. 2-6), indicating that kluyveromyces marxianus plays a key role in achieving positive effects on multiple markers, even synergistic in at least some respects.

Example 2: anti-inflammatory effects on immune cells.

Materials and methods

Yeast and combinations thereof were prepared as in example 1.

The in vitro immunomodulatory activity of yeasts and combinations thereof was studied by following the cytokine production of the THP-1 cell line (macrophages). Human THP-1 cell lines were cultured at 1X10 ⁵ cells/well in 96-well plates in the presence of 100nM phorbol 12-myristate 13-acetate (PMA, sigma) and incubated for 48 hours to induce differentiation of THP-1 monocytes into macrophages. The cells were washed and incubated in medium for an additional 72 hours. Thereafter, the cells were incubated with the test component for 1 hour, after which the cells were incubated for a further 16 hours with or without LPS (100 ng/ml, sigma) in the presence of the test component. All conditions were tested in triplicate.

Supernatants were collected after stimulation and stored at-20 ℃. TNF- α and IL-10 levels were measured using ELISA assays (IL-10 human uncoated ELISA kit, TNF- α human uncoated ELISA kit, life Technologies) according to the manufacturer's protocol. The TNF-alpha/IL-10 ratio was calculated as a measure of the anti-inflammatory effect of the tested components.

After collection of culture supernatants, the metabolic activity of the cells was analyzed by WST-1 assay (Roche) according to the manufacturer's protocol to confirm the non-cytotoxicity of the tested components. These cells were found to be not metabolically active.

Results

A reduction in tnfα/IL-10 ratio in immune cells as a measure of anti-inflammatory effect was observed for the yeast composition ABB C22 compared to the individual yeasts and compared to the composition comprising only the yeasts saccharomyces boulardii and saccharomyces cerevisiae (fig. 7).

Example 3: intestinal barrier integrity after challenge.

Materials and methods

Yeast and combinations thereof were prepared as in example 1.

The effect of yeasts and combinations thereof on intestinal barrier function when challenged was investigated by tracking transepithelial electrical resistance (TEER) on the intestinal cell layer.

Caco-2 cells (2X 10 ⁴ cells/cm 2) were seeded and cultured on a Transwell polycarbonate cell culture insert (Greiner Bio one) having an average pore size of 0.4 μm and a diameter of 0.33cm ² until 1000. OMEGA.was fully differentiated. TEER was measured with EVOM epithelial ohm meter (World Precision Instruments) as an indicative measure of barrier integrity.

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

TEER values under individual conditions after exposure to pathogens correlate with TEER values themselves at t=0 and are expressed as Δteer (Ω. cm 2). Negative controls (ETEC H10407 only) and positive controls that were not exposed to pathogens and test components were included. All conditions were tested in triplicate.

Transepithelial flux using FITC dextran (Sigma) was measured at different time points after TEER measurement.

Results

Protection of intestinal epithelial integrity was measured after incubation with an infectious agent known to disrupt the epithelial monolayer (here E.coli ETEC). The TEER of the yeast combination ABB C22 had a higher increase relative to the negative control after 1 and 2 hours of incubation than either the single yeast or the combination of saccharomyces cerevisiae and saccharomyces boulardii (fig. 8).

Example 4: intestinal barrier integrity is established.

Materials and methods

Yeast and combinations thereof were prepared as in example 1.

The effect of yeasts and combinations thereof on intestinal epithelial cell growth and differentiation was tracked during intestinal cell monolayer formation.

Caco-2 cells (2X 10 ⁴ cells/cm 2) were seeded on Transwell polycarbonate cell culture insert (Greiner Bio one) with an average pore size of 0.4 μm and a diameter of 0.33cm ². After cell attachment overnight, the test component was added to the apical side of the cells.

Test components were prepared and stored in aliquots at 20 ℃. A new aliquot was taken every two days to update the composition. To avoid the yeasts tested (when capable of growing under aerobic conditions) causing excessive growth of epithelial cells, 10% yeast conditioned medium and combinations thereof, as well as heat killed yeasts, were used.

TEER was measured every 2 days using EVOM epithelial voltmeter (World Precision Instruments) as an indicative measure of cell growth and barrier integrity formation.

Results

The increase in TEER was used to measure spontaneous formation of intestinal epithelium over time. After 16 days, an increase in TEER was observed for the three yeasts compared to the negative control, and only saccharomyces cerevisiae was maintained until day 20 (fig. 9a, upper panel). In contrast, relative to the control, from day 16 onwards, an increase in TEER of the combination ABB C22 was observed to remain and increase until day 22 (fig. 9b, lower panel).

A higher slope of TEER increase was observed for the combination ABB C22 compared to the individual yeast strains and the combination of saccharomyces cerevisiae and saccharomyces boulardii, indicating a faster TEER increase (trend line slope in fig. 10).

PCT/RO/134 table

Claims (26)

1. A physiologically acceptable composition comprising (i) at least one component selected from the group consisting of inactivated saccharomyces cerevisiae (s.boulardii) yeast, saccharomyces boulardii lysate, saccharomyces boulardii cell wall component and saccharomyces boulardii extract, further comprising (ii) at least one component selected from the group consisting of inactivated saccharomyces cerevisiae (s.cerevisiae) yeast, saccharomyces cerevisiae lysate, saccharomyces cerevisiae cell wall component and saccharomyces cerevisiae extract, and further comprising (iii) at least one component selected from the group consisting of inactivated kluyveromyces marxianus (k.marxianus) yeast, kluyveromyces marxianus lysate, kluyveromyces marxianus cell wall component and kluyveromyces marxianus extract.

2. The physiologically acceptable composition of claim 1, comprising an inactivated saccharomyces boulardii, an inactivated saccharomyces cerevisiae, and an inactivated kluyveromyces marxianus.

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

4. A physiologically acceptable composition according to any of claims 1-3, wherein (inactivated) saccharomyces cerevisiae is present at a concentration of at least 10 ⁶ cells/gram (based on the total weight of the yeast component), preferably 10 ⁹ cells/gram (based on the total weight of the yeast component) to 4x10 ¹⁰ cells/gram (based on the total weight of the yeast component), wherein (inactivated) saccharomyces cerevisiae is present at a concentration of at least 10 ⁶ cells/gram (based on the total weight of the yeast component), preferably 10 ⁸ cells/gram (based on the total weight of the yeast component) to 5x10 ⁹ cells/gram (based on the total weight of the yeast component), and wherein (inactivated) kluyveromyces marxianus is present at a concentration of at least 10 ⁶ cells/gram, preferably 10 ⁶ cells/gram (based on the total weight of the yeast component) to 5x10 ⁹ cells/gram (based on the total weight of the yeast component).

5. The physiologically acceptable composition according to any of claims 1 to 4, wherein at least one of the yeasts is a mineral-enriched yeast, preferably a zinc-enriched yeast, in particular a zinc-enriched saccharomyces cerevisiae.

6. The physiologically acceptable composition according to any of claims 1 to 5, wherein the 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 CCTCCM2012116, 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, 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 V21/012335 and Kluyveromyces marxianus Z17.

7. The physiologically acceptable composition according to claim 6, wherein the saccharomyces boulardii is saccharomyces boulardii DSM 33954; wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae S288C or Saccharomyces cerevisiae Y1529; and wherein the Kluyveromyces marxianus is Kluyveromyces marxianus V21/012335.

8. Physiologically acceptable composition according to any of claims 1 to 7, wherein the saccharomyces boulardii-based fraction of the composition is 0.05-99.95wt.%, preferably 5-95wt.%, in particular 15-50wt.%, based on total yeast components; wherein the saccharomyces cerevisiae based fraction of the composition is 0.05-90wt.%, preferably 10-90wt.%, more preferably 10-80wt.%, in particular 15-50wt.%, based on total yeast components; and wherein the kluyveromyces marxianus-based fraction of the composition is 0.05-99.95wt.%, preferably 5-95wt.%, in particular 15-50wt.%, based on total yeast components, provided that: the total amount of the three fractions is 100wt.% or less.

9. The physiologically acceptable composition according to any one of claims 1 to 8, comprising: 15-50wt.% of inactivated saccharomyces boulardii cells based on total microbiological material of the composition; 15-50wt.% of inactivated saccharomyces cerevisiae cells based on total microbiological material of the composition; and 15-50wt.% of inactivated kluyveromyces marxianus cells based on the total microbial material of the composition, provided that: the total amount of the three fractions is 100wt.% or less.

10. The physiologically acceptable composition according to any of claims 1 to 9, wherein the composition is a pharmaceutical product, a nutraceutical product or a cosmeceutical product.

11. The physiologically acceptable composition according to any of claims 1 to 10, wherein the composition is a nutritional product, preferably a product selected from the group consisting of: dairy products, infant formulas, fruit-based products, cereal products, snack foods, vegetable beverages, smoothies, and isotonic beverages.

12. The physiologically acceptable composition according to any of claims 1 to 11, wherein the composition is an oral rehydration salt.

13. The physiologically acceptable composition according to any of the preceding claims for use in the treatment of humans or animals by therapy.

14. The physiologically acceptable composition according to any one of claims 1 to 13 for use in maintaining or improving intestinal health or gastrointestinal function in an individual.

15. The physiologically acceptable composition according to any one of claims 1 to 14 for use in the treatment of gastrointestinal disorders.

16. The physiologically acceptable composition for use according to claim 15, wherein the gastrointestinal disorder involves an impairment of intestinal barrier function.

17. The physiologically acceptable composition for use according to claim 14, 15 or 16, wherein the gastrointestinal disorder is selected from the group consisting of diarrhea, inflammatory bowel disease and irritable bowel syndrome.

18. The physiologically acceptable composition for use according to claim 17, wherein the gastrointestinal disorder is bacterial-induced diarrhea.

19. The physiologically acceptable composition according to any of claims 1 to 18 for use in the treatment of a disorder defined by a pro-inflammatory marker, such as rheumatoid arthritis, osteoarthritis, local dermatitis, psoriasis, allergy or obesity.

20. Physiologically acceptable composition for use according to any of claims 13 to 19, wherein the composition is to be administered into the gastrointestinal tract, preferably orally, or wherein the composition is to be administered topically.

21. The physiologically acceptable composition for use according to any of claims 13 to 20, wherein the composition is for use in treating humans.

22. Use of a physiologically acceptable composition according to any of claims 1 to 12 as a food additive, feed additive, functional food in human nutrition, functional food in animal nutrition, food additive for nutraceutical or functional ingredient.

23. Use of a physiologically acceptable composition according to any of claims 1 to 12 as a probiotic, a metazoan, a paraprobiotic, a prebiotic, a symbiotic or a probiotic substitute.

24. A medical device comprising the physiologically acceptable composition according to any one of claims 1-12.

25. The medical device of claim 24, wherein the device is selected from the group of patches and plasters.

26. Saccharomyces boulardii yeast with accession number DSM 33954 (deposited in DSMZ, german collection of microorganisms and cell cultures).