MEANS AND METHODS FOR STABILIZING INTESTINAL FLORA AND IMPROVING HYGIENE

Description

[0001] The invention relates to uses of a bactericidal agent for enhancing the performance or promoting the well-being of vertebrates and / or humans, as a disinfectant and / or as a preservative in the environment of vertebrates and / or humans or as a means of personal hygiene in vertebrates and / or humans. Hintergrund

[0002] The digestive tract of vertebrates and humans represents an organ system in which food or feed is not only mechanically broken down, transported, and digested by the body's own enzyme-containing fluids, but also plays a particularly important role in human health. This applies especially to the intestine as part of the digestive tract, where the term "intestine" refers to all intestinal sections such as the duodenum, jejunum, and ileum (these three intestinal sections collectively referred to as the small intestine), the large intestine, and the rectum, along with intestinal appendages such as the ceca. The structure of the intestine, the intestinal sections, and the intestinal appendages can vary between the individual classes of vertebrates (mammals and humans, birds, reptiles, amphibians, and fish) as well as within the classes, but this will not be discussed in detail here.The importance of the intestine for health arises, among other things, from the multitude of microorganisms that enter the intestine from the outside and colonize it (the intestine can therefore rightly be described as a microbial ecosystem). These microorganisms are not only involved in digestive processes but can also influence the immune system and trigger immune reactions. Collectively, they contribute significantly to the physiological balance of the intestine. Disturbances of this balance, such as those caused by imbalances within the "intestinal flora" (the totality of all microorganisms present in the intestine) or by microbial intestinal infections and, subsequently, by inflammatory bowel diseases, have significant effects on the overall condition, performance, and well-being of the organism.

[0003] The microorganism groups that make up the intestinal flora are bacteria, archaea, yeasts, fungi, and protozoa, with bacteria constituting the majority of the intestinal flora. Bacteria can be divided into two large groups based on the structure of their cell walls: "Gram-positive" bacteria have a multilayered peptidoglycan shell, a macromolecule composed of sugars and amino acids, superimposed on their lipid bilayer cell membrane. In "Gram-negative" bacteria, the peptidoglycan shell is only thin. In Gram-negative bacteria, a second lipid bilayer is superimposed on this.

[0004] In general, an overabundance of one or another bacterial group compared to the natural state can cause a disturbance in intestinal physiology. Among the gram-positive and gram-negative bacteria, there are also numerous pathogenic, i.e., disease-causing strains. Known gram-negative pathogenic strains are found, for example, within the genus Salmonella and the type Escherichia coli. Among gram-positive bacteria, pathogenic strains are found, for example, within the genera Bacillus, Clostridium, Enterococcus, Staphylococcus and Streptococcus.

[0005] Bacterial intestinal infections are traditionally treated with antibiotics, which can have bactericidal or bacteriostatic effects against certain bacteria. "Antibiotics" in the conventional sense, therefore, refers to agents that contain representatives of active ingredient groups against bacteria, such as penicillins, cephalosporins, tetracyclines, macrolides, and sulfonamides, and that are administered in the form of pharmaceuticals. "Bactericidal" means that the bacteria are killed by the administered antibiotic, whereas "bacteriostatic" means that the growth and reproduction capacity of the bacteria are inhibited. After separation from the bacteriostatic antibiotic, the bacteria can multiply again. Bactericidal and bacteriostatic effects are also referred to below as "antibacterial" effects.Antibiotics act specifically and specifically on certain cellular and molecular structures of the bacteria they target. These target structures result in selective toxicity to the bacteria, thus preventing damage to the host organism's cells. Antibiotics can be used for therapeutic purposes or for disease prevention. In livestock farming, antibiotics were long used to enhance performance, even without veterinary indication. This practice has been banned in the European Union since 2006.

[0006] Bacteria can possess intrinsic resistance to antibiotics due to the lack of the corresponding target structures. Bacteria can also become resistant to antibiotics (acquired resistance). Acquired resistance is based on a process of mutation and selection under the selective pressure of the presence of one or more antibiotics in the bacteria's habitat. Furthermore, such acquired resistance can be genetically transferred from one bacterial species to another.

[0007] The realization that decades of sometimes excessive use of antibiotics in both animal husbandry and human medicine have contributed significantly to the emergence and, above all, the steady increase in pathogenic bacteria resistant to antibiotics worldwide has led to great concern and international efforts to significantly reduce the use of conventionally used antibiotics. Antibiotics can also have adverse health effects, such as causing diarrhea or immunosuppressive effects, which can then lead to secondary infections with other pathogenic microorganisms (e.g., other bacteria, yeasts, or fungi). All of this has led to an intensified search for alternatives to the use of antibiotics in both animal husbandry and human medicine.

[0008] Two important approaches to achieving the goal of a sustainable reduction in antibiotic use are 1) stabilizing the intestinal flora through alternative agents or active ingredients and 2) improving hygiene.

[0009] According to the current state of the art, the following groups of alternative agents or active ingredients are typically used to stabilize the intestinal flora: probiotics, prebiotics, phytogens, organic acids and minerals.

[0010] "Probiotics" are products containing living microorganisms (so-called probiotic bacteria such as lactic acid bacteria or probiotic yeasts) that colonize the intestine and thus positively influence the intestinal flora. This can occur, for example, by displacing pathogenic germs (competitive effect), whereby the underlying principle of action is competition for living space and nutrients. Probiotic bacteria can also produce antibacterial peptides and excrete metabolic products, such as short-chain fatty acids, which can exert positive effects on the immune system, for example, by modulating the formation of immunologically active signaling substances such as cytokines, interleukins, and others. Probiotic products also include spores, i.e., persistent stages of bacterial cells that germinate in the intestine after application and become active cells that subsequently multiply.

[0011] "Prebiotics" are products containing active ingredients that promote the growth of probiotic bacteria in the intestine. Prebiotic agents can, for example, be certain sugars or oligosaccharides (molecular chains consisting of a few, typically 2-7, sugar building blocks) that cannot be utilized by the organism to which the prebiotic agents are administered, but which can be metabolized by the bacteria in the intestine, usually in the posterior intestinal sections or in the intestinal appendages of higher animals and humans.

[0012] "Phytogenics" are products containing active ingredients derived from plants or plant parts that can exert an antibacterial effect in the intestines. Phytogenic active ingredients include certain plant oils (such as thymol, carvacrol, clove oil, and cinnamon oil) or saturated, medium-chain fatty acids with 6-12 carbon atoms. The latter are found, for example, in coconut oil.

[0013] "Organic acids" include short-chain, saturated fatty acids such as propionic acid and butyric acid, or other low-molecular-weight acids such as benzoic acid and citric acid. They are primarily known as preservatives, for example, for feed and food, and consequently as agents for ensuring the hygienic status of materials. Organic acids have various antibacterial effects. Propionic acid, for example, disrupts intracellular carbohydrate metabolism and DNA synthesis. Benzoic acid inhibits intracellular enzymes that degrade reactive oxygen species (e.g., certain radicals) and are thus involved in the cell's own detoxification process.

[0014] "Minerals" that serve as nutrients (macroelements and trace elements) can also have a direct effect on the intestinal flora. One example is zinc, which, in the form of zinc oxide in suitable formulations and even subpharmacological dosages, has a bactericidal effect on risk germs, such as Escherichia coli, owns.

[0015] There are several disadvantages of the above-mentioned means of stabilising the intestinal flora according to the state of the art.

[0016] Probiotics and prebiotics generally only have an indirect effect on the intestinal flora. Furthermore, it is not possible to differentiate between the effects on gram-positive and gram-negative bacteria, especially since typical probiotic bacteria (such as species of the genera Bacillus, Lactobacillus, Enterococcus and Bifidobacterium ) are themselves gram-positive. Prebiotics also only work indirectly by promoting the growth of probiotic bacteria. The lactic acid produced by the lactic acid bacteria typically used as probiotics can be absorbed in the small intestine in large quantities, i.e. if the probiotic bacteria multiply excessively, and can cause health problems. In principle, the multiplication of probiotic bacteria in the intestine is not controllable. In the case of overproduction, antibiotics must even be administered, which is then particularly contraindicated. It is now also known that excessive colonization of the intestine, particularly the small intestine, with bacteria can have negative health effects.

[0017] Phytogens often act against gram-negative bacteria, such as species of the genus Salmonella or Escherichia coli. In higher concentrations, phytogens added to feed or food can cause changes in taste or odor, thus impairing feed or food intake. Many phytogens are dedicated flavoring agents. Medium-chain fatty acids are effective against Gram-positive bacteria, but their effectiveness depends heavily on the specific composition of the acid mixture.

[0018] Organic acids can also negatively affect the taste and odor of feed and food. They can also have toxic effects, which makes handling these substances very difficult and necessitates special safety precautions during technical processing and application.

[0019] Zinc is a heavy metal. It is harmful to the environment if it enters the environment, for example, due to inadequate absorption via feces. Therefore, current efforts are aimed at limiting the use of zinc in livestock farming.

[0020] Due to the disadvantages described, there is a fundamental need for additional means to stabilize the intestinal flora, particularly by modulating the proportion of gram-positive bacteria in the intestine.

[0021] Improving hygiene was mentioned as a second important approach to sustainably reducing the use of antibiotics. "Hygiene" is generally understood here to mean measures that reduce the contamination of materials (solids and their surfaces, as well as liquids) in the environment of animals and humans, as well as on the external and internal surfaces of the body, with risky or disease-causing microorganisms by exerting an "antimicrobial" effect, i.e., killing the microorganisms ("biocidal" effect) or inhibiting their growth and reproduction ("biostatic" effect). These primarily include measures for "disinfection" and "preservation," as well as for body cleansing. According to these functions, we speak of "disinfectants," "preservatives," and "hygiene products."

[0022] Disinfectants have a biocidal effect against various types of microorganisms and, for practical reasons, are usually available in liquid or gaseous form. They are often a component of "cleaning agents" and usually contain inorganic or organic substances, less frequently biochemical substances, as active ingredients. Examples of inorganic and organic active ingredients include alcohols (such as ethanol and 1-propanol), aldehydes (such as formaldehyde), oxidizing agents (such as peracetic acid, chlorine, chlorine dioxide, hydrogen peroxide, sodium hypochlorite, ozone, or iodine), phenols (such as chloroxylenol), and surfactants (such as cetyltrimethylammonium bromide).

[0023] An example of an inorganic substance used for disinfection is silver. Silver can also be used to disinfect the surfaces of materials. Surfaces that have a biocidal effect thanks to a suitable coating are used, among other things, for surgical instruments. Upon contact with the surface, the microorganisms are killed. In addition to silver, copper, quaternary ammonium compounds, and organosilanes are also suitable for biocidal surface coating. Organosilanes have a bactericidal effect by mechanically damaging the outer membrane of Gram-negative bacteria. In this way, they prevent the bacteria from adhering to the surface and simultaneously kill them. Other special types of nanostructuring of surfaces can also impart a biocidal effect to surfaces.

[0024] Preservatives increase the shelf life of feed, food, cosmetics, pharmaceuticals, wood, paints, and coatings by suppressing the growth of microorganisms. Organic acids such as benzoic acid and citric acid and their salts, sulfur compounds such as sulfur dioxide and sulfites, nitrogen compounds such as nitrates and nitrites, parabens, etc. are commonly used as preservatives with biocidal or biostatic effects.

[0025] Antimicrobial agents are also a component of personal hygiene products for humans and animals. This affects external body surfaces and structures such as the skin and skin appendages (e.g., hair, bristles, feathers, nails, claws, or beaks), as well as internal surfaces and structures such as the oral cavity (the mouth) with the teeth and throat. Antimicrobial agents contained in hygiene products include, for example, sulfur compounds (e.g., methyl(chloro)isothiazolinone), acids (e.g., benzoic acid, sorbic acid, formic acid, and salicylic acid), organic alcohols (e.g., farnesol), phenols (e.g., triclosan), chlorine compounds (e.g., chlorhexidine), metal compounds (e.g., copper sulfate), and aldehydes (e.g., formaldehyde).

[0026] Antimicrobial substances, such as those used in hygiene measures or health applications, can also be formulated in the form of nanoparticles or immobilized on the surface of suitable carrier materials as so-called microparticles (microbeads, microspheres). Nanoparticles typically have diameters of up to 100 nm. Microparticles can have diameters of up to several micrometers. Both nanoparticles and microparticles are produced using special, sometimes complex chemical-biochemical-physical processes.

[0027] The state-of-the-art disinfection and preservation agents and methods, as well as personal hygiene products described above, also have various disadvantages, regardless of their formulation. Many disinfectant substances are toxic and harmful to health or highly aggressive towards materials, requiring special safety precautions during handling and application. Disinfectants and preservatives, or their active ingredients, that enter wastewater can severely pollute the environment. In principle, all chemical and biochemical active ingredients are of concern, as they can potentially be absorbed into the body through the skin or mucous membranes, particularly in the context of personal hygiene. Long-term health risks from the use of nanoparticles as a special type of formulation cannot yet be foreseen, as nanoparticles, like particulate matter, can penetrate cells and cause physiological changes.

[0028] There is therefore also a great need for new and alternative means to improve hygiene. Beschreibung

[0029] Having said that, the object of the present invention is to provide a novel use of an agent which 1) as a feed additive or food additive stabilizes the intestinal flora in vertebrates and humans by having a bactericidal effect on gram-positive bacteria.This makes it possible to modulate the balance between gram-positive and gram-negative bacteria within the bacterial intestinal flora and in particular also the number of risky or pathogenic gram-positive bacteria in the intestine, whereby the disadvantages of the above-mentioned alternative means for stabilising the intestinal flora according to the state of the art are avoided and 2) can be used as a disinfectant to improve the hygienic condition of solid materials or liquids and as a preservative to preserve solid and liquid feed and foodstuffs as well as other products such as cosmetics, and furthermore for personal hygiene, i.e. overall as part of measures to improve hygiene, whereby the disadvantages of the above-mentioned means for disinfection, preservation and personal hygiene according to the state of the art are also avoided here.

[0030] The invention relates to various uses, which are described in claims 1, 2, 3 and 4. Advantageous embodiments of the invention are specified in the subclaims.

[0031] The agent according to the invention contains spores of the fungus Myceliophthora thermophila. The agent is a bactericidal substance and is also referred to in this application as "bactericidal agent" or "bactericide".

[0032] Myceliophthora thermophila is a thermophilic, filamentous fungus from the division of Ascomyceten, the class of Sordariomycetes, the order of the Sordariales and the family of Chaetomiaceae. Myceliophtora thermophila was described in particular by Apinis (Nova Hedwigia 5:57-78, 1963) and by van Oortschot (Persoonia 9:401-408, 1977). Myceliophthora thermophila occurs in compost, hay, and warm soils, from where it can be isolated. The vegetative spores develop singly or in short rows from ampulla-shaped thickenings of conidial hyphae and have a diameter of 3-5 µm. In the more mature stage, they have a rough surface. Previously used synonyms for Myceliophthora thermophila are Chrysosporium thermophilum and Sporotrichum thermophile. The teleomorph (the sexual stage) is called Thielavia heterothallica (earlier Corynascus heterothallicus ). Industrially, Myceliophthora thermophila primarily used for the production of technical enzymes for the degradation of plant biomass. The fungus is accessible to the public, particularly through deposits of strains at recognized depositories, e.g., at the American Type Culture Collection (ATCC) under the depository number ATCC 42464, at the Westerdijk Fungal Biodiversity Institute under the depository number CBS 117.65, and at the German Collection of Microorganisms and Cell Cultures (DSMZ) under the depository number DSM1807. The strain of the fungus Myceliophtora thermophila, The specimen used for the investigations detailed in the working examples was deposited with the Westerdijk Fungal Biodiversity Institute, CBS Collection, Uppsalaan 8, 3508 AD Utrecht, under the identification number Myceliphtora thermophila #154. The depository assigned the accession number CBS 146310 to the deposit.

[0033] It was surprisingly found that spores of the fungus Myceliophthora thermophila, derived from a culture of this fungus have a bactericidal effect on Gram-positive bacteria without the need for any special treatment of the spores. This has been shown by comparative studies in which the effect of a spore suspension of Myceliophthora thermophila on a selection of gram-positive and gram-negative bacteria, and also tested for bacteriostatic or bactericidal activity. The antibacterial effect was demonstrated independently in three different test procedures: 1) In the "PREMI test," the potential antibacterial effect within a nutrient agar is tested by monitoring the germination of spores and the growth of vegetative cells of the test bacterium. Bacillus stearothermophilus after the addition of a liquid and the test substance, is measured by the color change of an acid formed during these processes. 2) In the "4-plate test," the antibacterial effect is tested by the decrease in turbidity of a nutrient agar into which the respective test bacteria have been poured, whereby the test substance is placed into a punched hole in the agar. 3) In the bacterial growth test in liquid nutrient medium, the optical density of the bacterial suspension, which increases with the growth of the respective test bacteria, is measured photometrically after the addition of the test substance. In all three test procedures, a spore suspension of Myceliophthora thermophila antibacterial effects against gram-positive bacteria. In contrast, the spore suspension showed antibacterial effects against gram-negative bacteria Escherichia coli No antibacterial effect in the 4-plate test and in the bacterial growth test with liquid nutrient medium.

[0034] That it is the spores of Myceliophthora thermophila The active principle underlying the bactericidal effect against Gram-positive bacteria was demonstrated in a further study in which the spores were removed from the spore suspension by filtration. The remaining solution without spores showed no bactericidal effect. Therefore, it can be assumed that the underlying active principle is not a water-soluble substance that is either formed constitutively in the cytoplasm of the living spores and released through the spore wall into the surrounding solution, or that is present on the surface of the spores as their constitutive component and from there readily enters the solution.

[0035] Further studies have shown that the bactericidal effect against Gram-positive bacteria also applies to non-living, inactivated spores. For this purpose, the spores were killed with an acid mixture (Schaumacid®< from H. Wilhelm Schaumann GmbH & Co KG) so that they were no longer able to germinate. This also clearly speaks against a bactericidal substance formed and then secreted by the spores during active, cytoplasmic metabolism as the active principle. Furthermore, the bactericidal effect remained undiminished when the spores were dried and then resuspended. The bactericidal effect was also maintained when the spores were induced to germinate and spores subsequently formed by the developing fungal mycelium were harvested and tested for their antibacterial effect. The bactericidal effect of the spores is therefore stable across generations.

[0036] Heat-treated spores (by autoclaving) of Myceliophthora thermophila showed a greatly reduced bactericidal effect compared to non-heat-treated spores.

[0037] With the onset of germination, the bactericidal effect of the spores of Myceliophthora thermophila lost.

[0038] The bactericidal effect of the spores of Myceliophthora thermophila against gram-positive bacteria was particularly evident when the spores were produced by culturing the fungus on potato dextrose agar or on vermiculite together with potato infusion and glucose.

[0039] Spores of the closely related species Myceliophthora lutea showed no antibacterial effect against Gram-positive bacteria under otherwise identical conditions. Spores of other species of filamentous fungi, for example strains of the species Aspergillus oryzae, Neurospora intermedia and Chaetomium thermophilum (to the same family as Myceliophthora thermophila belonging to the genus) showed no antibacterial activity against Gram-positive bacteria. It can therefore be assumed that the antibacterial activity against Gram-positive bacteria is not a general property of spores of filamentous fungi or of fungal spores in general.

[0040] Details of the described investigations are given in the working examples.

[0041] The mechanism underlying the bactericidal effect of the spores of Myceliophthora thermophila The underlying mechanism has not yet been clarified in detail. However, the results of previous investigations suggest the following characteristic properties of the active principle, which can be considered features of the invention: 1) The active principle underlying the observed bactericidal effect is a property of the spore surface, i.e., the spore surface is "bioactive" in this sense. The development of the bactericidal effect requires either direct contact between the bacterial cells and the spore surface or a substance mediating the effect, which is either a signaling substance that must come into contact with the spore surface or a precursor of a bactericidal substance that is converted into the bactericidal substance by an enzyme (or several enzymes) bound to the spore surface.In both cases, the substance mediating the effect cannot originate from the spore suspension used in the studies described above. 2) At the same time, the bactericidal effect is clearly not limited to the immediate environment of the spores; that is, once initiated, it spreads spatially, and the bactericidal effect also develops at a certain distance from the location of the spores. This is demonstrated by the studies of the bactericidal effect of spores of . Myceliophthora thermophila in test procedures in which solid culture media were used in which neither the test bacteria nor the fungal spores are mobile due to their respective sizes. 3) It is conceivable, but unlikely and implausible to assume that the postulated signal substance or precursor - unless the signal for initiating the bactericidal effect is the physical contact of the bacterial cells with the spore surface - are substances that are readily present in the culture media used in the test procedures described above under the conditions that vary in the individual investigations. It is more likely, and this is postulated here, that these are substances that are associated with the presence of Gram-positive bacteria in the respective culture media.are generated by the bacteria themselves, so that one can assume that the mechanism underlying the bactericidal effect "recognizes" the presence of the gram-positive bacteria.

[0042] The agent according to the invention has the advantages that accrue to active ingredients immobilized on microparticles through this special type of formulation. These generally include improved stability. Compared to nanoparticles, the fungal spores have the advantage of not being able to penetrate cells of the intestinal epithelium due to their size. One advantage over microparticles coated with antimicrobial active ingredients, as available according to the state of the art and for which the bactericidal effect requires direct contact of the bacterial cells with the surface of the particles, is the range of the bactericidal effect of the agent according to the invention beyond the immediate region on the spore surface, so that not every bacterial cell needs to have come into contact with the surface of at least one microparticle at least once in order to be killed.In the case where microparticles according to the prior art are equipped with a bactericidal mechanism acting at a distance, the particular advantage of the agent according to the invention described here is that it only exerts its bactericidal effect when the fungal spores are within range of Gram-positive bacteria. This means that the bactericidal effect does not develop automatically, but rather when needed. A further practical advantage of the agent according to the invention compared to the use of antimicrobial microparticles according to the prior art is that the spores of... Myceliophthora thermophila The bactericidal effect after cultivation of the fungus is evident. Furthermore, no further necessary and costly coating of the spores or other complex chemical-biochemical-physical treatments are required. Furthermore, the spores do not harm the environment, as spores from Myceliophthora thermophila occur ubiquitously under natural conditions.

[0043] In summary, it is therefore assumed that the bactericidal effect of spores of Myceliophthora thermophila with their special, mechanistic properties is a completely new discovery. A comparable agent, i.e., an agent with identical active properties based on microparticles with a suitable coating or other treatment, would require extremely complex processing steps and molecular configurations of the microparticles. The inventive properties of the spores of Myceliophthora thermophila are also not obvious, since a bactericidal effect against Gram-positive bacteria is not among the known and expected properties of fungal spores in general. However, with the newly acquired knowledge described here, it cannot be fundamentally ruled out in the future that antimicrobial effects with the special inventive features described here may also occur under production conditions other than those mentioned above and also with spores of other fungal species.

[0044] US 2002 / 0102246 A1 describes an enzymatic composition that can be used for disinfection in laundries, on solid surfaces, in water systems, on the skin, on teeth, or on mucous membranes, as well as for the preservation of food products, cosmetics, paints, and coatings. This comprises a phenol-oxidizing enzyme system and an enhancer. The phenol-oxidizing enzyme system can, for example, be a laccase or a laccase-related enzyme together with oxygen or another oxidizing agent. The patent focuses particularly on laccases produced by fungi. Laccase-producing fungi include, among others, Myceliophthora thermophila. A laccase as a component of the mechanism of the bactericidal action of the spores of Myceliophthora thermophila However, this could not be demonstrated experimentally. Furthermore, an external study (see the appendix) showed that the spores of Myceliophthora thermophila They also have a bactericidal effect against anaerobic, Gram-positive bacteria under anaerobic (oxygen-free) and reducing conditions. This excludes laccase and other oxidases as involved in the mechanism of action.

[0045] US 7,288,264 B1 describes various antimicrobial articles or agents that kill microorganisms on contact, including microparticles. However, the invention does not describe articles or agents in which the antimicrobial effect is not limited to the immediate surface of the articles.

[0046] WO 2011 / 033275 A1 describes bacterial spores coated with a therapeutically active agent on their surface. This invention also does not describe microbial systems in which the antimicrobial effect is not limited to the immediate surface of the spores.

[0047] US 2006 / 0247 150 A1 describes compositions for disinfecting and cleaning processes containing fungal spores. However, according to this invention, the spores do not have any bactericidal activity.

[0048] The agent according to the invention can be used to stabilize the intestinal flora or the bacterial flora of skin surfaces, body orifices, mouth (or mouth), throat or other external or internal body surfaces in vertebrates and humans, provided that it contains inactivated spores of Myceliophthora thermophila It is expected that the product will positively influence the composition of the intestinal flora or other bacterial flora by modulating the number of gram-positive bacteria and increasing resistance to gram-positive, disease-causing bacteria. The latter is particularly important for the intestinal flora during sensitive stages of rearing and growth, reducing the risk of diarrhea and other bacterial diseases. Overall, the well-being of animals and humans is expected to improve significantly through the use of the product. Furthermore, the product can be used in therapeutic doses for the prevention and treatment of diseases in humans and vertebrates.

[0049] Furthermore, the agent can be used to improve hygiene, i.e. as a disinfectant and as a preservative for the treatment of solid and liquid materials as well as for the personal hygiene of humans and animals with the aim of killing gram-positive bacteria or preventing the growth of gram-positive bacteria. Ausführungsformen

[0050] According to one embodiment, the agent according to the invention contains a culture of the fungus Myceliophtora thermophila spores of this fungus. According to a further embodiment, the agent contains a culture of the fungus Myceliphthora thermophila isolated spores of this fungus. According to a further embodiment, the spores are isolated from the culture of the fungus by harvesting them from the culture. According to a further embodiment, the spores are repeatedly harvested from the culture. According to a further embodiment, spores together with the culture or parts of the culture are used as a bactericidal agent. According to a further embodiment, the agent according to the invention contains inactivated, i.e., killed, spores of the fungus. Myceliophthora thermophila.

[0051] According to a variant of these embodiments, the agent according to the invention contains spores killed by one or more acids approved under feed law, food law, or the EU Biocide Regulation, as well as other relevant regulations and laws. In the case of use as a feed additive, for example, by the product Schaumacid® from H. Wilhelm Schaumann GmbH & Co. KG, Ovelgünner Straße 27, 39365 Eilsleben. Treatment with acids can be used for the production of the agent according to the invention.

[0052] According to a further embodiment, the agent according to the invention is a solid which contains spores of Myceliophthora thermophila A formulation as a solid is particularly advantageous if the agent is to be mixed into other solids without introducing additional moisture into the preparation.

[0053] According to a variant of this embodiment, the spores are harvested dry from the fungal culture, for example, by suctioning them off the fungal culture and then collecting them, or by adsorbing them from the fungal culture with the aid of adhesive substances, such as sucrose or lactose, and separating the adhesive substance, together with the adhered spores, from the residual fungal culture material by sieving. This creates a solid spore formulation on a carrier material.

[0054] According to a further variant of this embodiment, the spores are taken up from the fungal culture using a saline solution (a saline-containing aqueous solution, where the salt is, for example, table salt), and the spore suspension is then dried. The saline solution may contain additional substances that promote the suspension of the spores in the saline solution, for example, surface-active substances such as Tween 20 or Tween 80 or other surfactants.

[0055] According to a variant of this embodiment, the spore suspension is dried by freeze-drying, vacuum drying, or spray drying. Freeze-drying, vacuum drying, or spray drying are particularly gentle drying methods and suitable for increasing the shelf life of the spores.

[0056] According to a further variant of this embodiment, additional substances are added to the solid spore formulation to improve the physical properties and further processing, for example, mixing into solid or liquid preparations, or to increase the shelf life. These can be carriers or binders, such as salts of acetic acid or bentonite, flow agents such as diatomaceous earth, preservatives such as organic acids, and surfactants such as Tween 20 or Tween 80, as well as other substances.

[0057] According to a further embodiment, the agent according to the invention is provided in the form of a paste or a cream which kills the spores of Myceliophthora thermophila Formulations of the agent in the form of a paste or cream are particularly suitable for disinfecting the surfaces of solid materials or for personal hygiene.

[0058] According to a further embodiment, the agent according to the invention is in the form of a liquid preparation which contains the spores of Myceliophthora thermophila Liquid formulations of the product are also very suitable for disinfecting surfaces of solid materials or external and internal body surfaces, among other things.

[0059] According to a variant of this embodiment, the liquid formulation is based on water or alcohol.

[0060] According to a further variant of this embodiment, the liquid formulation based on water or alcohol contains, in addition to the spores of Myceliophthora thermophila Other substances or materials that improve the physical properties and further processing or that increase the shelf life of the liquid formulation. These may be substances that maintain the suspension of the spores in the liquid.

[0061] According to a further embodiment, the agent according to the invention is used as a feed additive or food additive for stabilizing the intestinal flora of vertebrates, in particular mammals and birds, in particular farm animals (such as cattle, horses, sheep, goats, pigs, and poultry) and domestic animals (such as dogs and cats), and humans, by modulating the number of gram-positive bacteria. When the agent according to the invention is used in farm animals, an increase in performance parameters during animal growth, such as mean daily live weight gain and feed efficiency (the amount of feed consumed per unit of live weight gain), is to be expected. Furthermore, increased resistance to the negative effects of gram-positive bacteria is to be expected across all life stages.This is particularly important during critical stages of rearing, for example, in piglets during the weaning phase. Overall, the use of the agent according to the invention, particularly in intensive livestock farming, contributes to improved animal welfare. A study (results not shown) demonstrated that the bactericidal effect of spores from... Myceliophthora thermophila is stable towards treatment with a protease, so that it can be assumed that the bactericidal effect of the spores against Gram-positive bacteria is retained after passage through the stomach and in the intestine. The fungal spores did not cause clinical signs of illness or mortality in a study conducted according to the officially established method OECD Test Guideline No. 423 of December 17, 2001. The LD 50 is above 5000 mg / kg body weight when ingested by rats. The fungal spores are excreted in the feces. Studies have shown that the fungal spores do not inhibit methane-producing bacteria, so the feces can be used for biogas production.

[0062] According to a variant of this embodiment, the agent according to the invention is mixed in solid form into feed or food.

[0063] According to a further variant of this embodiment, the agent according to the invention is added in solid or liquid form to drinking water and drinking water or, in the case of humans, to other drinks (such as fruit or vegetable juices).

[0064] According to a further variant of these embodiments, the dosage recommendation is made on the basis of the results of a microbiome analysis, i.e. an analysis of the composition of the intestinal flora, in particular on the basis of a determination of the ratio of the numbers of gram-positive to gram-negative bacteria, in particular by determining the numbers of risky gram-positive bacteria.

[0065] According to a further embodiment, the spores of Myceliophthora thermophila used in therapeutic doses for the treatment and prevention of bacterial diseases, especially intestinal diseases, as well as other diseases such as inflammation of the mucous membranes in humans and vertebrates.

[0066] According to a further embodiment, the spores of Myceliophthora thermophila in solid or liquid formulation for the disinfection of solid materials or surfaces of solid materials or liquids in contact areas of animals and humans with the aim of killing gram-positive bacteria there.

[0067] According to a variant of this embodiment, the spores of Myceliophthora thermophila used as a component of bedding in stables for the keeping of farm animals or pets.

[0068] According to a variant of this embodiment, an aqueous formulation of the spores or a formulation in alcohol is used as a disinfectant.

[0069] According to a further variant of this embodiment, the aqueous formulation or the formulation in alcohol contains further disinfectant substances, for example organic acids or surfactants.

[0070] According to a further variant of this embodiment, a pasty or gel-like formulation of the spores is used, which is spread onto the surfaces of solid materials and can be washed off after a suitable exposure time.

[0071] According to a further embodiment, the agent according to the invention is used for the preservation of solid and liquid materials. This is intended to suppress the growth of Gram-positive bacteria in these materials.

[0072] According to a variant of this embodiment, the agent according to the invention is used together with other preservatives, for example organic acids.

[0073] According to a further embodiment, the agent according to the invention is used for body hygiene in humans and animals.

[0074] According to a variant of this embodiment, the agent according to the invention is used, for example, in solid soaps, washing lotions, shampoos, toothpastes, or deodorants for the care of external and internal body surfaces in humans and vertebrates. In particular, the abrasive properties, which are determined by the size and surface texture of the spores, can also be utilized for cleaning purposes in hygiene products.

[0075] According to a further variant of this embodiment, the agent according to the invention is used in claw cleaning or as a component of udder dips in farm animals.

[0076] According to a further embodiment, the agent according to the invention is used to kill the following gram-positive bacteria: Bacillus subtilis, Bacillus stearothermophilus, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Clostridium perfringens and Clostridium tyrobutyricum.

[0077] The invention is explained below with reference to the description of embodiments with reference to the attached figures. Ausführungsbeispiele

[0078] In the following, exemplary embodiments are presented with reference to the accompanying figures, without the invention being limited to these examples. The figures show: Abbildung 1 : Proof of the antibacterial effect of spore suspensions of Chaetomium thermophilum and Myceliophthora thermophila with the PREMI test. Abbildung 2 : Effect of different spore suspensions on Bacillus subtilis. Abbildung 3 : Proof of the antibacterial effect of spores of Myceliophthora thermophila, The fungus was cultivated on different growth substrates using the PREMI test. Abbildung 4 : Demonstration of the antibacterial effect of autoclaved and non-autoclaved spore suspension of Myceliophthora thermophila for gram-positive bacteria using the 4-plate test. Abbildung 5 : Determination of the minimum inhibitory concentration of the spore suspension of Myceliophthora thermophila against Bacillus subtilis. Abbildung 6 : Proof of the antibacterial effect of the spore suspension of Myceliaphthora thermophila on Escherichia coli. Abbildung 7 : Effect of spore suspension of Myceliophthora thermophila on Escherichia coli. Abbildung 8 : Effect of cycloheximide on Bacillus subtilis and spores of Myceliophthora thermophila. Abbildung 9 : Investigation of the bactericidal or bacteriostatic effect of the spore suspension of Myceliophthora thermophila. Abbildung 10 : Effect of inactivated spore suspension of Myceliophthora thermophila on Bacillus subtilis. Abbildung 11 : Effect of spore suspension of Myceliophthora thermophila with 2% foam acid and a saline control with 2% foam acid on Bacillus subtilis. Abbildung 12 : Effect of freeze-dried spores of Myceliophthora thermophila on Bacillus subtilis. Abbildung 13 : Effect of spores from the third generation of Myceliophthora thermophila on Bacillus subtilis and Kocuria rhizophila. Abbildung 14 : Storage stability of the antibacterial effect of the spores of Myceliophthora thermophila. Proof of the antibacterial effect of spore suspensions of Chaetomium thermophilum and Myceliophthora thermophila with the PREMI test

[0079] The PREMI test is a simple, broad-spectrum microbial screening test for the detection of antimicrobial substances, such as antibiotics. The test principle is based on the growth inhibition of Bacillus stearothermophilus. For this purpose, a standardized number of spores of Bacillus stearothermophilus embedded in a nutrient agar. After adding a liquid, the spores germinate and produce an acid, which causes the nutrient agar to change color from violet to yellow. When growth is inhibited by an antibacterial agent, which is also added, no color change occurs. Ampicillin is an effective antibiotic against Bacillus stearothermophilus and serves as a positive control in the test. Water serves as a negative control, as this should not inhibit growth.

[0080] The results (see Abbildung 1 ) show that a spore suspension of Myceliophthora thermophila, harvested from a culture on potato dextrose agar, the growth of Bacillus stearothermophilus The spore suspension of another thermophilic, filamentous fungus, Chaetomium thermophilum, showed no antibacterial effect against Bacillus stearothermophilus. Effect of different spore suspensions on Bacillus subtilis

[0081] In the bacterial growth test in liquid nutrient medium to detect the antibacterial effects of active ingredients, a preculture of the respective target bacterium is first established. A spore suspension containing a defined number of fungal spores is inoculated with the bacterial suspension and incubated. The growth rate of the target bacterium can be measured based on the turbidity (measured as OD 600 ). The concentration of spores in the spore suspension at which no bacterial growth occurs is the minimum inhibitory concentration (MIC).

[0082] In a first study, spore suspensions of Myceliophthora thermophila, Aspergillus oryzae and Neurospora intermedia and a bacterial suspension with Bacillus subtilis used as a target bacterium (see Abbildung 2 ). As a control, a saline solution was added to the bacterial suspension. In the presence of the saline solution, a significant growth of Bacillus subtilis The spore suspensions of Aspergillus oryzae and Neurospora intermedia also showed no anti-bacterial, growth-inhibiting effect on Bacillus subtilis. In contrast, the spore suspension of Myceliophtora thermophila a significant inhibitory effect on the growth of Bacillus subtilis. As a control, the turbidity of the spore suspension was also measured by Myceliophthora thermophila measured photometrically without the addition of the bacterial suspension. It can be seen that the fungal spores germinate after about 19 hours. Consequently, the increase in turbidity after about 19 hours when using the spore suspension of Myceliophthora thermophila together with the suspension of Bacillus subtilis on the germination of spores of Myceliophthora thermophila, not on a growth of Bacillus subtilis, due to.

[0083] The growth-inhibiting effect of the spore suspension of Myceliophthora thermophila on Bacillus subtilis was confirmed in a further study (see Abbildung 8 ). In this study, the spore solution of Myceliophthora thermophila Cycloheximide was added. Cycloheximide is a translation inhibitor that causes the death of eukaryotic cells. This approach was used to demonstrate that the antibacterial effect of the spore suspension of Myceliophthora thermophila on Bacillus subtilis also persists beyond the 19 h period. As a control, saline solution with the same concentration of cycloheximide was added to the bacterial suspension. As the control showed, the cycloheximide itself had no effect on the growth of Bacillus subtilis.

[0084] In a further study, the minimum inhibitory concentration (MIC) of the spore suspension of Myceliophthora thermophila on the growth of Bacillus subtilis determined approximately. Abbildung 5 shows that the MIC for Bacillus subtilis at a spore concentration between 1·10 4< CFU / mL and 1·10 6< CFU / mL (CFU = Colony Forming Units). A spore concentration in the spore suspension of 1·10 6< CFU / mL clearly led to a complete inhibition of the growth of Bacillus subtilis. This spore concentration corresponds to a ratio of 100 fungal spores per bacterial cell. Results from another study (not shown) show that the MIC can be narrowed to the range between 1 10 4 < CFU / mL and 5 10 5 < CFU / mL. In the study, 5 10 5 < CFU / mL corresponds to a ratio of 50 fungal spores per bacterial cell. Proof of the antibacterial effect of spores of Myceliophthora thermophila, The fungus was cultivated on different growth substrates, with the PREMI test

[0085] Another study, the results of which are published in Abbildung 3 shown, shows that the spores of Myceliophthora thermophila have an antibacterial effect when cultivated on potato dextrose agar as a special nutrient medium. Furthermore, spores could be produced using a growth substrate consisting of vermiculite, potato infusion, and glucose, which also exhibited the antibacterial effect. A solution of potato dextrose agar and saline, as used for harvesting the spores, showed no antibacterial effect. Demonstration of the antibacterial effect of autoclaved and non-autoclaved spore suspension of Myceliophthora thermophila for gram-positive bacteria using the 4-plate test

[0086] The 4-plate test is another microbiological detection method that enables the detection of antibacterial agents. This method is an agar diffusion test. The test substances are placed into punched holes in various culture media containing the test bacteria (target bacteria). Kocuria rhizophila, Micrococcus luteus, Bacillus megaterium and Bacillus subtilis subsp. spizizenii These bacteria are gram-positive. The presence of antibacterial substances is indicated after appropriate incubation by the formation of zones of inhibition. These are spatial areas within the nutrient agar in which the target bacteria cannot grow and thus cloud the agar. The diameter of the zone of inhibition correlates with the strength of the antibacterial effect. Abbildung 4 shows the results of the 4-plate test. The top right shows which type of sample was added to each well in the nutrient agar. "Spore extract" refers to the solution from which the spores of Myceliiophthora thermophila were removed by filtration with a 0.2 µm syringe filter. The antibiotic discs served as a positive control. The spore suspension with spores of Myceliophthora thermophila showed a growth-inhibiting effect on all four test bacteria. After autoclaving, the spore suspension of Myceliophthora thermophila only at Bacillus subtilis an inhibitory effect. The solution without fungal spores showed no effect. The presence of the spores of Myceliophthora thermophila is therefore a prerequisite for the antibacterial effect. Proof of the antibacterial effect of the spore suspension of Myceliaphthora thermophila on Escherichia coli

[0087] A similar test procedure to the 4-plate test was used to determine the antibacterial effect of the spores of Myceliophthora thermophila for gram-negative bacteria. This test differs only in the bacterial suspension used from Escherichia coli, which was poured into the nutrient agar. Escherichia coli It is a gram-negative bacterium. The result is a Abbildung 6 The spore suspensions of Myceliophthora thermophila and Aspergillus oryzae showed no growth-inhibiting effect on Escherichia coli.

[0088] Also in gram-negative bacteria of the genus Salmonella No antimicrobial effects were observed with the 4-plate test (results not shown).

[0089] Abbildung 7 shows the result of the determination of the MIC of the spore suspension of Myceliophthora thermophila opposite Escherichia coli, measured with the bacterial growth test using liquid nutrient medium. Even a spore suspension concentration of 1 10 8 < CFU / mL, applied to a bacterial concentration of 1 10 4 < CFU / mL, did not inhibit the growth of Escherichia coli. Investigation of the bactericidal or bacteriostatic effect of the spore suspension of Myceliophthora thermophila

[0090] To test whether the antibacterial effect of the spore suspension of Myceliophthora thermophila bactericidal or bacteriostatic, a small piece of agar from the inhibition zone of the agar plate was inoculated with the test strain Micrococcus luteus from the 4-plate test (see Abbildung 4 for orientation) and transferred to an uninoculated CASO plate (see Abbildung 9 ). As a negative control, a piece of the overgrown agar outside the inhibition zone was punched out and transferred to a CASO plate. The agar piece punched out of the inhibition zone according to the 4-plate test showed no growth in CASO medium. The agar piece punched out from the area outside the inhibition zone showed growth of Micrococcus luteus This result shows that the effect of the spore suspension of Myceliophthora thermophila is bactericidal. Effect of inactivated spore suspension of Myceliophthora thermophila on Bacillus subtilis

[0091] For this study, the bacterial growth test with liquid nutrient medium was used. For this purpose, the spores of Myceliophthora thermophila inactivated using different methods. The UV-treated spore suspension was irradiated with UV light for 2 hours. The acid-treated spore suspension was mixed with 2% Schaumacid (H. Wilhelm Schaumann GmbH, An der Mühlenau 4, 25421 Pinneberg), incubated for 48 hours at 4°C, and then neutralized. The frozen spore suspension was kept frozen at -20°C for 5 days. All samples were plated on potato dextrose agar to check whether the spores were actually killed and thus no longer germinate. Of the various treatments, only the treatment of the spores with the acid preparation (Schaumacid) achieved the antibacterial effect against Bacillus subtilis (see Abbildung 10 ). It made no difference whether the sample was stored at 4°C or 30°C during treatment (results not shown).

[0092] For the purpose of control, it was investigated whether the antibacterial effect was caused by the acid preparation itself. For this purpose, saline solution was mixed with foam acid (2%), then neutralized and converted into a bacterial suspension of Bacillus subtilis given. How Abbildung 11 shows, Schaumacid alone had no antibacterial effect against Bacillus substilis. Effect of freeze-dried spores of Myceliophthora thermophila on Bacillus subtilis

[0093] To preserve the spores of Myceliophthora thermophila Freeze-drying proved to be suitable. A study with freeze-dried spores using the bacterial growth test with liquid nutrient medium showed that - even after prior treatment with 2% foam acid - the antibacterial effect of the spores towards Bacillus subtilis continued to exist (see Abbildung 12 ). The MIC is not changed by freeze-drying. Effect of spores from the third generation of Myceliophthora thermophila on Bacillus subtilis and Kocuria rhizophila

[0094] For the investigations described so far, a second generation spore suspension of Myceliophthora thermophila To investigate whether the antibacterial effect persists for a longer period, third-generation spores were tested using the bacterial growth assay with liquid nutrient medium. Bacillus substilils and Kocuria rhizophila The results in Abbildung 13 show that the fungal spores of the third generation also have the antibacterial effect against Bacillus substilis and Kocuria rhizophila showed. Storage stability of the antibacterial effect of the spores of Myceliophthora thermophila

[0095] The Abbildung 14 The results of a further study using the bacterial growth test with liquid nutrient medium show that the antibacterial effect of freeze-dried spores of Myceliophthora thermophila opposite Bacillus subtilis and Kocuria rhizophila remained stable when stored at room temperature for 12 weeks. At 40°C, overgrown PDA flasks could be stored stably for over 20 weeks. Storing the spore suspension at 4°C for over 20 weeks also did not result in any decrease in the antibacterial effect. External study on the antibacterial effect of spores of Myceliophthora thermophila

[0096] The antibacterial effect of the spores of Myceliophthora thermophila against Gram-positive bacteria was confirmed by a standardized study in an external laboratory. This study is attached in the appendix. Anlage: External study on the antibacterial effect of spores of Myceliophthora thermophila

[0097]

Claims

1. A use of a bactericidal agent for gram-positive bacteria containing spores of the fungus Myceliophthora thermophila for non-therapeutically increasing the performance of vertebrates and / or humans by stabilizing the intestinal flora.

2. A use of a bactericidal agent for gram-positive bacteria containing spores of the fungus Myceliophthora thermophila for non-therapeutically promoting the well-being of vertebrates and / or humans by stabilizing the bacterial flora of skin surfaces, body orifices, mouth (or else muzzle), throat, or other external or internal body surfaces.

3. A use of a bactericidal agent for gram-positive bacteria containing spores of the fungus Myceliophthora thermophila as a disinfectant and / or as a preservative for treating solid or liquid materials in order to improve hygiene in the environment of vertebrates and / or humans.

4. A use of a bactericidal agent for gram-positive bacteria containing spores of the fungus Myceliophthora thermophila as a non-therapeutic body hygiene agent for caring for external or internal body surfaces in vertebrates and / or humans.

5. The use according to any one of claims 1 to 4, wherein the bactericidal agent contains inactivated spores of the fungus M. thermophila.

6. The use according to claim 5, wherein the bactericidal agent contains an acid and / or spores of the fungus M. thermophila that have been inactivated by means of UV light.

7. The use according to any one of claims 1 to 6, wherein the bactericidal agent contains freeze-dried, vacuum-dried, or spray-dried spores of the fungus M. thermophila.

8. The use according to any one of claims 1 to 7, wherein the bactericidal agent contains a carrier and spores of the fungus M. thermophila.

9. The use according to any one of claims 1 to 8, with the bactericidal agent in the form of a solid.

10. The use according to any one of claims 1 to 8, with the bactericidal agent in a formulation as a paste or cream.

11. The use according to any one of claims 1 to 8, with the bactericidal agent in a liquid formulation.

12. The use according to claim 11, with the bactericidal agent in a liquid formulation based on water or alcohol.

13. The use according to any one of claims 1 to 12, with the bactericidal agent as a feed additive or food additive.

14. The use according to claim 13, with the bactericidal agent as a feed additive contained in a feed or as a food additive contained in a food.

15. The use according to any one of claims 1 to 14, wherein the agent is added to drinking water and the drinking water is administered to vertebrates and / or humans.

16. The use according to any one of claims 1 to 15, wherein the agent is added to a feed or food and the feed is administered to vertebrates or the food is administered to humans.

17. The use according to claim 3, wherein the agent is added to drinking water and surfaces in barns, cages, enclosures, or in other regions used for keeping livestock or pets are treated with the drinking water.

18. The use according to claim 17, wherein the surfaces of drinking troughs and / or feed troughs and / or stable bedding are treated with the agent.

19. The use according to claim 3, wherein the agent is used to preserve a feed or food.

20. The use according to any one of claims 1 to 19, wherein the agent is used to kill off or inhibit at least one of the following gram-positive bacteria: Bacillus subtilis, Bacillus stearothermophilus, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis, Clostridium perfringens, Clostridium tyrobutyricum.

21. A bactericidal agent for gram-positive bacteria containing spores of the fungus Myceliophthora thermophila for use in a method for treating or preventing bacterial diseases caused by gram-positive bacteria, in particular intestinal diseases and / or inflammation of the mucous membranes in humans and / or animals.

22. The agent according to claim 21, which is a bactericidal agent from any one of claims 5 to 13.