Bacillus strains with improved growth

Abstract

The present invention relates to Bacillus strains with improved growth and / or germination properties and their use in feed, food and pharmaceutical compositions.

Description

[Technical field]

[0001] The present invention relates to endospore-forming Bacillus subtilis strains with improved growth and / or germination properties and their use in feed, food and pharmaceutical compositions. [Background technology]

[0002] Endospore-forming Bacillus strains are often used as probiotics in the feed and food industry. Their usefulness as probiotics is due in particular to their ability to inhibit the growth of pathogenic bacteria. However, probiotics of the phylum Bacillota often have the additional function of being multifunctional feed or food additives with beneficial effects on animal and human health. A specific characteristic of strains useful as probiotics is the ability to form endospores, which allows them to survive adverse conditions, such as those encountered during the manufacture of feed products, particularly the high temperatures during the feed pelleting process, or after ingestion of the feed by an animal, particularly the acidic environment of the stomach. When the environment becomes more favorable, such as that of the intestine, the Bacillus can be converted from the endospore into a vegetative state. Once in the vegetative state, it becomes metabolically active and can produce beneficial substances and also grow, thereby enhancing the desired probiotic activity at the target location.

[0003] Since proliferation of cells at a target location is one important factor for enhancing a desired activity of cells, such as the inhibition of pathogenic bacteria, it is important to identify factors and conditions that can enhance the proliferation function of cells and / or provide cells with improved proliferation and / or germination properties at a target location. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Chen et al.(2014);PLoS One 9(4):e95781 [Non-Patent Document 2] Zhang et al.(2013);J Bacteriol.195(8):1735-40 [Non-Patent Document 3] Setlow(2003);Curr Opin Microbiol.6(6):550-6 Summary of the Invention [Problem to be solved by the invention]

[0005] No mutants showing increased germination due to mutations in the gerKC gene have been described so far. According to the present invention, it has surprisingly been found that a point mutation in the GerKC gene encoding a spore germination protein significantly enhances the proliferation and / or germination functions of cells, apparently due to enhanced activity of the spore germination protein GerKC. Furthermore, it has been found that mutations in the gerKC gene, in particular those that enhance the germination ability of the cells, do not alter important properties of the parent strain from which the Bacillus cells are derived, and in particular no detrimental effects in terms of safety and risk aspects are observed, nor are any negative effects on the probiotic activity, so that mutant strains of the gerKC gene can be used as probiotics in the feed and food industries. The gene gerKC encodes a subunit of the GerK germination receptor (Non-Patent Document 1). Certain nutrients can bind to the germination receptor in the inner membrane of the spore, thereby inducing germination of Bacillus spores (Non-Patent Document 2). Both germination receptors GerB and GerK are required for spore germination, which is induced by the germinants L-asparagine, D-glucose, D-fructose and potassium ions (AGFK) (Non-Patent Document 3). The amino acid substitutions in the GerKC protein in the strain of the present invention change the structure of the protein to make it more responsive to external germinants and to more rapid spore growth and / or germination properties. The amino acid substitutions may make the protein more accessible to germinants or more rapidly induce activation of the signal transduction cascade. The GerKC subunit of the GerK germinant receptor is a lipoprotein bound to the inner membrane of the spore, while the GerKA and GerKB subunits are integral inner membrane proteins. The exact effect of the amino acid substitutions in the GerKC protein cannot be identified with certainty at present, since the mechanism of how germinants bind to the germinant receptor and how the signal transduction cascade directing germination is initiated is still unknown (Non-Patent Document 3). [Means for solving the problem]

[0006] Thus, a first subject of the present invention is an endospore-forming Bacillariophylum strain having improved growth and / or germination properties, in particular compared to the wild-type and / or parent strain, wherein the improved growth and / or germination properties are preferably due to increased activity of the germinant receptor GerK, in particular increased activity of the germinant protein KC (GerKC). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Thus, the term "improved growth and / or germination properties" according to the present invention preferably means that the respective strain exhibits improved growth and / or germination properties compared to the wild type and / or parent strain from which it originates. To determine the improved growth and / or germination properties, preferably a TSB (tryptic soy broth) medium is used, in particular as disclosed in Example 6. Preferably, the Bacillus strain has at least one mutation, in particular 1 to 10 mutations, more preferably 1 to 5 mutations, in particular 2 to 4 mutations, especially exactly 2 mutations, in at least one subunit of the germination receptor GerK, in particular compared to the receptor subunit sequence of the wild-type and / or parent strain from which it is derived. More preferably, the Bacillus strain has at least one mutation, in particular 1 to 10 mutations, more preferably 1 to 5 mutations, especially 2 to 4 mutations, in particular exactly 2 mutations, in the spore germination protein KC (GerKC), in particular compared to the protein sequence of the wild-type and / or parent strain from which it is derived. Thus, the mutant gerKC gene has preferably 1 to 10, more preferably 2 to 5, especially 2 to 4 point mutations, especially exactly 2 point mutations, compared to the gerKC gene of the wild-type and / or parent strain from which it is derived, especially compared to the gerKC sequence of Bacillus velezensis CECT 5940 strain disclosed as SEQ ID NO: 2. Thus, the mutant gerKC gene has preferably 1 to 10, more preferably 2 to 5, especially 2 to 4 point mutations, especially exactly 2 point mutations, compared to the gerKC gene of the wild-type and / or parent strain from which it is derived, especially compared to the gerKC sequence of Bacillus velezensis CECT 5940 disclosed as SEQ ID NO: 2.

[0008] In a preferred embodiment of the invention, the protein GerKC comprises in its protein sequence a glycine at position 11 and / or a serine at position 72. In a particularly preferred embodiment of the invention, this is achieved by the following mutations, starting from the protein encoded by the consensus sequence of GerKC, in particular as disclosed in SEQ ID NO: 5, and in particular starting from the GerKC sequence of strain CECT 5940, disclosed in SEQ ID NO: 2: substitution of the cysteine ​​at position 11 of GerKC by a glycine (C11G) and substitution of the proline at position 72 of GerKC by a serine (P72S), while the coding gene gerKC preferably comprises the following mutations, in particular compared to the consensus gerKC sequence disclosed in SEQ ID NO: 5, and in particular compared to the gerKC sequence of strain CECT 5940, disclosed in SEQ ID NO: 1: substitution of the thymine at position 31 of GerKC by a guanine (T31G) and substitution of the cytosine at position 214 by a thymine (C214T). In addition to said specific one or more mutations, further mutations may be contained in said sequence, preferably up to 8, more preferably up to 5 to 3, especially 1 or 2 further mutations may be present, but in a highly preferred embodiment of the invention, in addition to said specific one or two mutations, no further mutations are present in each sequence compared to the parent and / or wild-type sequence from which it is derived. In a particularly preferred embodiment, the only difference in the genomic sequence between the parent and / or wild-type strain and the strain according to the invention is said mutation in gene gerKC.

[0009] In a highly preferred embodiment of the invention, the gerKC gene has the sequence set forth in SEQ ID NO:3 and the GerKC protein has the sequence set forth in SEQ ID NO:4. Thus, a further subject of the present invention is also a protein having at least 95%, preferably at least 98 or 99%, sequence identity to the GerKC sequence of Bacillus veresensis CECT 5940 represented as SEQ ID NO: 2, wherein preferably said protein sequence comprises at least one mutation selected from T31G and C214T, preferably both mutations. Preferably, the protein GerKC is encoded by the consensus sequence represented as SEQ ID NO: 5, preferably comprising at least one mutation selected from T31G and C214T, preferably both mutations. Even more preferably, T31G and C214T are preferred, wherein the protein. Highly preferred is the protein GerKC having the sequence represented as SEQ ID NO: 4.

[0010] The microorganisms according to the invention can in principle be obtained by any kind of method, i.e. GMO or non-GMO methods. However, preferably, the microorganisms are not genetically modified, i.e. non-GMO. This means that the microorganisms are preferably either naturally occurring microorganisms or natural mutants of naturally occurring microorganisms or microorganisms obtained by another method classified as non-GMO. The term "natural mutants" refers to mutants that arise from naturally occurring microorganisms and / or parent strains, but not by genetic modification of the microorganisms, by applying classical genetic engineering and / or biotechnological methods, such as site-directed mutagenesis. Said natural mutants can be obtained by classical methods of natural selection, such as growing the microorganisms in the presence of UV light and / or by applying high temperature or protoplast formation and / or in the presence of a specific antibiotic to which the parent strain is sensitive. Natural mutants can furthermore, but less preferably, be obtained using mutagens, i.e. chemicals that induce the formation of mutants. The formation of natural mutants using mutagens is less preferred, therefore in a preferred embodiment of the invention, the natural mutants and / or non-GMO mutants are obtained without using said mutagens. If the variants are obtained by applying genetic and / or biotechnological methods, such as site-directed mutagenesis, preferably methods are applied which are classified as non-GMO. Non-GMO methods according to the invention are preferably characterized in that no heterologous genetic information is introduced into the microorganism. In a particularly preferred embodiment of the invention, the microorganisms of the invention are non-naturally occurring mutants, in particular non-GMO and / or natural mutants as defined above, which preferably have the same characteristics as the parent strains from which they are derived, except for the function of improved growth and / or germination properties.

[0011] The microorganisms according to the invention are preferably furthermore non-pathogenic, in particular non-pathogenic for humans and animals, and preferably also non-pathogenic for plants. The microorganisms according to the present invention are preferably selected from the list of microorganisms listed as direct-fed microorganisms ("DFM") or probiotics, as listed on the list of Generally Recognized as Safe ("GRAS") or relevant lists, such as, but not limited to, the EFSA QPS list, the AAFCO list, or the list by the Chinese Ministry of Agriculture (MOA).

[0012] The microorganism according to the invention is preferably susceptible to at least 5 antibiotics, more preferably at least 6, 8 or 10 antibiotics, especially at least 11 or 12 antibiotics, wherein the antibiotics are preferably selected from tylosin, nalidixic acid, trimethoprim, apramycin, sulfonamides, ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol, nourseothricin, daptomycin and virginiamycin. In a highly preferred embodiment, the microorganism according to the invention is susceptible to all of the above antibiotics. "Susceptible to an antibiotic" means that the growth of the microorganism is inhibited by the antibiotic under conditions in which the microorganism would otherwise grow. Susceptibility to antibiotics is preferably tested according to CLSI guidelines (M07-A8 and M45-A2). Bacillus strains are preferably considered susceptible for vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol if growth is detected only at or below the breakpoint concentrations specified in EFSA Journal 2012;10(6):2740. For tylosin, nalidixic acid, trimethoprim, apramycin, sulfonamides, ampicillin, nourseothricin, daptomycin and virginiamycin, for which no breakpoints have been given by EFSA for the genus Bacillus, the breakpoint is preferably 4 mg / L.

[0013] In a preferred embodiment of the present invention, the microorganism is an endospore-forming bacterium. The endospore-forming bacterium cell of the present invention may be present, particularly in the composition of the present invention, as a spore (dormant), as a vegetative cell (growing), as a transitional cell (in transition from growth phase to sporulation phase), or as a combination of at least two, particularly all, of these types of cells. In a preferred embodiment, the composition of the present invention mainly comprises spores or only comprises spores. The microorganisms of the present invention are selected from the phylum Bacillota (formerly known as Firmicutes), preferably from the genus Bacillus, more preferably from the following species: Bacillus subtilis, B. amyloliquefaciens, B. velezensis, Bacillus licheniformis, Bacillus maltophilus ... licheniformis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus megaterium, Bacillus lentus, Bacillus laterosporus, Bacillus alevi, Bacillus cereus, Bacillus badius, Bacillus thuringiensis, Bacillus coagulans, Bacillus siamensis, Bacillus glycinifermentans, Bacillus methylotrophicus, Bacillus thurii The bacterial strain is selected from Bacillus subtilis, Bacillus thuringensis, Bacillus polyfermenticus, Bacillus vallismortis, Bacillus tequilensis, Bacillus atrophaeus, Bacillus mojavensis, Bacillus sonorensis, Bacillus inaquosus and Bacillus safensis, and most preferably Bacillus subtilis, Bacillus beruerensis, Bacillus amyloliquefaciens, Bacillus pumilus or Bacillus megaterium, and among the above, most preferably Bacillus amyloliquefaciens or Bacillus beruerensis. In a further preferred embodiment of the invention, the microorganism of the phylum Bacillario is of the genus Clostridium, preferably C. butyricum, C. tyrobutyricum or C. acetobutylicum. In a highly preferred embodiment of the invention, the microorganism according to the invention is of the species Bacillus veresensis, more preferably the genomic sequence of said microorganism is at least 99%, in particular at least 99.5% or 99.8%, especially at least 99.9% or 99.99% identical to the genomic sequence of the Bacillus veresensis strain CECT 5940. In a particularly preferred embodiment, the only difference in the genomic sequence between the strain CECT 5940 and the strain according to the invention is said mutation in the gerKC sequence, and the strain according to the invention preferably has improved growth and / or germination properties compared to the strain CECT 5940. The strain CECT 5940 is sold under the trade name Ecobiol® by the company Evonik Operations.

[0014] In a highly preferred embodiment of the invention, the microorganisms according to the invention furthermore have probiotic activity and / or can be used as direct-feeding microorganisms. The microorganism according to the invention particularly preferably further comprises: (a) the ability to grow rapidly in large-scale bioreactors; (b) the ability to grow in the presence of bile, preferably in the presence of 2 to 4 mM bile; (c) the ability to grow in the presence of formate, acetate, propionate, and / or lactate; (d) the ability to produce organic acids, especially lactic acid; (e) the ability to grow under high salt conditions; (f) the ability to form endospores; (g) the ability to grow anaerobically; (h) the ability to inhibit pathogenic bacteria, particularly Clostridium perfringens, Salmonella Enteritidis, Salmonella typhimurium, Escherichia coli, Clostridioides difficile, and Vibrio parahaemolyticus; (i) the ability to produce at least one enzyme, preferably an enzyme selected from amylase, lipase, catalase, cellulase, xylanase and protease; (j) susceptibility to selected antibiotics; (k) the ability to produce biosurfactants, in particular surfactin; (l) Function to regulate the microbiome; The microorganisms of the present invention are preferably capable of rapid growth in large-scale bioreactors, which means in particular heterotrophic growth in the absence of light with a doubling time of less than 60 minutes in a stirred tank reactor.

[0015] The microorganism of the present invention is further preferably characterized in that it is capable of growing in the presence of 2 mM bile, preferably in the presence of 4 mM bile, in particular characterized in that in the presence of 2 mM bile, it has an AUC5 performance value of at least 0.5, preferably at least 0.65, in particular at least 0.8, and an AUC10 performance value of at least 1.2, preferably at least 1.4, in particular at least 1.6; and / or it is further characterized in that it is capable of growing in the presence of 0.3% by weight bile, in particular in the presence of 0.3% by weight chicken bile and / or in the presence of 0.3% by weight porcine bile, and preferably characterized in that it is capable of surviving exposure to 0.3% by weight bile, in particular 0.3% by weight chicken bile and / or 0.3% by weight porcine bile, for at least 3 hours, preferably at least 5-8 hours.

[0016] The microorganism of the present invention is further preferably characterized in that it is capable of growing anaerobically, in particular in that it is capable of degrading water-insoluble cellulose and / or protein under anaerobic conditions. The microorganisms of the invention are further preferably characterized in that at least 50%, preferably at least 70 or 90% of the spores survive exposure to 99° C. for 20 minutes. The microorganism of the present invention is preferably used to prepare a feed or food additive composition by mixing the microorganism with a suitable carrier.

[0017] In a particularly preferred embodiment of the invention, the microorganism of the invention is preferably used in the form of a dry fermentation medium, where "dried" means that the residual water content is preferably 0.1 to 10% by weight, more preferably 0.2 to 5% by weight, especially 0.3 to 4% by weight. A further subject of the invention is therefore also a feed or food additive containing the microorganism according to the invention, which may in particular be provided in the form of a dry fermentation medium. The feed or food additive according to the invention preferably comprises the microorganism or the dry fermentation medium of the microorganism in an amount of 0.1 to 10% by weight, more preferably in an amount of 0.2 to 5% by weight, in particular in an amount of 0.3 to 3% by weight. The feed or food additive further preferably comprises a carrier, in particular in an amount of at least 80% by weight, preferably in an amount of at least 90 or at least 95% by weight, in particular in an amount of 90 to 99.9% by weight or 95 to 99.8% by weight or 97 to 99.7% by weight. In a preferred embodiment of the invention, the feed or food additive consists exclusively of a mixture of the microorganism or the dry fermentation medium of the microorganism and said carrier. The feed or food additive preferably contains 1×10 microorganisms. 9 ~1×10 11 cfu (colony forming units), more preferably 5 x 10 per gram of feed or food additive 9 ~5×10 10 The amount of cfu.

[0018] Carriers included in the feed or food additive are preferably selected from inert formulation ingredients added to improve recovery, efficacy or physical properties and / or to aid packaging and administration. The carriers may be added individually or in combination. The carriers may be selected from anti-caking agents, antioxidants, bulking agents, binders, structuring agents, coating agents and / or protective agents. Examples of useful carriers are polysaccharides (particularly starch, maltodextrin, methylcellulose, gums, chitosan and / or inulin), protein sources (particularly skim milk powder and / or sweet whey powder), protein hydrolysates (particularly peptones such as soy peptone and / or yeast extract), peptides, sugars (particularly lactose, trehalose, sucrose and / or dextrose), lipids (particularly lecithin, vegetable oils and / or mineral oils), salts (particularly sodium chloride, sodium carbonate, cellulose, cellulose acetate ... Examples of suitable carriers for animal feed additives include calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate and / or sodium citrate), and silicates (particularly clays, especially beolite clay, amorphous silica, fumed / precipitated silica, zeolites, Fuller's earth, Bayliss®, Clintporite, montmorillonite, diatomaceous earth, talc, bentonite, and / or silicates such as aluminum silicate, magnesium silicate and / or calcium silicate). Suitable carriers for animal feed additives are described in the official publications of American Feed Control Officials, Inc., published annually. See, for example, Official Publication of American Feed Control Officials, Sharon Krebs, editor, 2006 edition, ISBN 1-878341-18-9. The carriers can be added after concentrating the fermentation medium containing the microorganism of the invention and / or during and / or after drying the fermentation medium.

[0019] The residual moisture content of the feed or food additive according to the present invention is preferably 0.1 to 6 mass%, preferably 0.3 to 5 mass%, more preferably 0.5 to 4 mass%, and further preferably has a water activity of 0.05 to 0.6, preferably 0.1 to 0.5 (aw value).

[0020] A further subject of the present invention is also the preparation of a microorganism according to the invention or a dried fermentation medium of said microorganism, preferably at a concentration of 1×10 9 ~1×10 11 cfu (colony forming units), more preferably 5 x 10 per gram of feed or food additive. 9 ~5×10 10 A method for preparing the feed or food additive according to the invention, in particular by mixing with the above-mentioned carriers, in an amount to adjust the amount of cfu.

[0021] The above feed additives, like the microorganisms themselves, can be used in the preparation of feed and pharmaceutical compositions and can be added to drinking water and breeding water. In a preferred embodiment, 0.1 kg to 100 kg of feed additive, in particular 1 kg to 10 kg of feed additive, are used per tonne of feed, drinking water or breeding water to provide a composition that can be used to feed animals. Feed and food compositions can be prepared by mixing the feed or food additive with conventional feed or food ingredients, respectively. A further subject of the present invention is therefore also a feed or food composition comprising a microorganism and / or a feed or food additive according to the invention and preferably at least one further feed or food ingredient. A further subject of the present invention is therefore also the use of a microorganism according to the invention and / or a feed or food additive according to the invention for preparing a feed or food composition. A further subject of the present invention is therefore also a method for preparing a feed or food composition according to the invention, in which the microorganism according to the invention and / or the feed or food additive according to the invention are mixed with further feed or food ingredients. A further subject of the present invention is also a pharmaceutical composition comprising a microorganism according to the invention and at least one pharma- ceutically acceptable carrier. A further subject of the invention is therefore also the use of a microorganism according to the invention and / or a feed or food additive according to the invention for preparing a pharmaceutical composition, in particular a pharmaceutical composition for the treatment, prevention or alleviation of the course of intestinal diseases. A further subject of the invention is therefore also a process for preparing a pharmaceutical composition according to the invention, comprising mixing the microorganism according to the invention and / or the feed or food additive according to the invention with a pharma- ceutically acceptable carrier. The phylum Bacillus, in particular the species Bacillus beruezensis, and especially the Bacillus beruezensis CECT 5940 strain, are generally known to have beneficial effects on the intestine of animals and humans.

[0022] Thus, when administered to an animal, the microorganisms of the invention preferably serve to promote the health of the animal, and / or improve the general physical condition of the animal, and / or improve the feed conversion of the animal, and / or reduce the mortality rate of the animal, and / or increase the survival rate of the animal, and / or improve the weight gain of the animal, and / or increase the disease resistance of the animal, and / or increase the productivity of the animal, and / or modulate the immune response of the animal, and / or establish or maintain a healthy gut microbiota in the animal, and / or improve the meat quality of the animal, in particular the meat elasticity and / or meat toughness, and / or reduce the shedding of bacterial and / or viral pathogens through the faeces of the animal. In particular, the microorganisms and compositions of the invention may be used to help re-establish a healthy balance of the gut microbiota after the administration of antibiotics for therapeutic purposes.

[0023] A further subject of the present invention is therefore a method for promoting the health of an animal, and / or improving the general body condition of said animal, and / or improving the feed conversion rate of said animal, and / or reducing the mortality rate of said animal, and / or increasing the survival rate of said animal, and / or improving the weight gain of said animal, and / or increasing the productivity of said animal, and / or increasing the disease resistance of said animal, and / or modulating the immune response of said animal, and / or establishing or maintaining a healthy intestinal microflora in said animal, and / or improving the meat quality of said animal, and / or reducing the shedding of bacterial and / or viral pathogens via the faeces of said animal, wherein at least one microorganism and / or at least one feed additive and / or at least one composition of the present invention is administered to the animal. A further subject of the present invention is therefore also a method for promoting human health and / or improving the general physical condition of a human and / or increasing the disease resistance of a human and / or modulating the immune response of a human and / or for establishing or maintaining a healthy intestinal microflora in a human, in which at least one microorganism and / or at least one composition according to the invention is administered to the human.

[0024] A further subject of the present invention is therefore also the use of at least one microorganism and / or at least one feed or food additive and / or at least one composition of the invention (for preparing a composition) for promoting the health of an animal and / or improving the general physical condition of an animal and / or improving the feed conversion rate of an animal and / or reducing the mortality rate of an animal and / or increasing the survival rate of an animal and / or improving the weight gain of an animal and / or increasing the productivity of an animal and / or increasing the disease resistance of an animal and / or modulating the immune response of an animal and / or establishing or maintaining a healthy intestinal microflora in an animal and / or improving the meat quality of an animal and / or reducing the shedding of bacterial and / or viral pathogens via the faeces of an animal, wherein the at least one microorganism and / or the at least one feed additive and / or the at least one composition of the invention is administered to an animal. A further subject of the present invention is therefore the use of at least one microorganism and / or at least one composition of the invention (for the preparation of a composition) for promoting human health and / or improving the general physical condition of a human and / or for increasing the disease resistance of a human and / or for modulating the immune response of a human and / or for establishing or maintaining a healthy intestinal microflora in a human, wherein at least one microorganism and / or at least one composition of the invention is administered to a human.

[0025] Further subject matter of the present invention is therefore also at least one microorganism and / or at least one feed or food additive and / or at least one composition according to the invention for promoting the health of animals and / or humans and / or for improving the general physical condition of animals and / or humans and / or for improving the feed conversion rate of animals and / or for reducing the mortality rate of animals and / or for increasing the survival rate of animals and / or for improving the weight gain of animals and / or for increasing the productivity of animals and / or humans and / or for increasing the disease resistance of animals and / or humans and / or for modulating the immune response of animals and / or humans and / or for establishing or maintaining a healthy intestinal microflora in animals and / or humans and / or for improving the meat quality of animals and / or for reducing the shedding of bacterial and / or viral pathogens via the faeces of animals. "Enhancing animal productivity" refers specifically to any of the following: production of more or better quality eggs, milk or meat, or increasing the number of offspring, or improving survival of the offspring. By using the microorganisms, feed or food additives and compositions of the invention, preferably an improvement in at least one of the above characteristics is achieved, preferably meaning an improvement of at least 1%, more preferably at least 3 or at least 5%, compared to a suitable negative control. As a negative control, an average known in the animal husbandry field may be used, but preferably an animal that is subjected to the same treatment as the animals to be tested, but that is not administered the microbial preparation and / or composition of the invention, is used.

[0026] In particular, the microorganisms, preparations and compositions of the invention may be administered or fed to an animal in an amount effective to inhibit and / or reduce the growth of pathogenic bacteria in the intestine of the animal, including, in particular, Listeria, Enterococci, Staphylococci, Aeromonas, Streptococci, Campylobacter, Shigella, Haemophilus and Brachyspira, in addition to Clostridium, Salmonella, Vibrio and Escherichia coli. Relatedly, the method of the invention may be used to reduce the amount of pathogenic bacteria, viruses and protozoa excreted in animal feces. The method of the invention may also be used to maintain or enhance the growth of beneficial bacteria, such as lactic acid bacteria, in the intestine of the animal. The compositions of the present invention can maintain an overall healthy gut microbiome by reducing pathogenic bacteria and / or enhancing or maintaining beneficial bacteria.

[0027] A further subject of the present invention is therefore a method for inhibiting and / or reducing the growth of pathogenic bacteria and / or for maintaining and / or enhancing the growth of beneficial bacteria, in particular in the gastrointestinal tract of an animal or human, in which a microorganism, preparation and / or composition according to the invention is administered to an animal or human and in which the pathogenic bacteria are preferably of the genus Clostridium, in particular Clostridium perfringens, Clostridium difficile, C. novyi, C. septica, C. erythropoeias ... um and Clostridium colinum; the genus Listeria, in particular L. monocytogenes, L. seeligeri and L. welshimeri; the genus Salmonella, in particular the subspecies Salmonella enterica, arizonae, bongori and in particular the serovars Salmonella gallinarum, Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, Salmonella cholerasuis, Salmonella heidelberg, Salmonella dublin, Salmonella hadar, Salmonella typhi, Salmonella paratyphi and Salmonella infantis; Enterococcus , especially Enterococcus faecalis, Enterococcus faecium and Enterococcus cecorum; Staphylococci, especially Staphylococcus aureus; Aeromonas, Streptococci, especially Salmonella suis and Salmonella gallinaceus; Campylobacter, especially Campylobacter jejuni and Campylobacter coli.coli, E. coli, Haemophilus, in particular Haemophilus parasuis, Brachyspira, in particular Brachyspira hyodysenteriae, and Vibrio, in particular Vibrio parahaemolyticus and V. harveyi, and the beneficial bacteria are preferably selected from Lactobacillus, in particular Lactobacillus and Bifidobacterium. The pathogenic microorganisms are in a preferred embodiment selected from Clostridium, in particular Clostridium perfringens, Salmonella, in particular S. enterica, and Vibrio, in particular Vibrio parahaemolyticus. In a preferred embodiment of the invention, the amount of at least one pathogenic bacterium, in particular the amount of Clostridium perfringens, Salmonella enteritidis and / or Vibrio parahaemolyticus, is reduced by at least 0.5 log, more preferably by at least 1 log, 2 log or 3 log. Thus, further subject of the present invention are microorganisms, preparations and compositions according to the invention for inhibiting and / or reducing the growth of pathogenic bacteria and / or for maintaining and / or enhancing the growth of beneficial bacteria, the pathogenic bacteria being preferably those of the genus Clostridium, in particular Clostridium perfringens, Clostridium difficile, Clostridium novyi, Clostridium septicum and Clostridium colinum, the genus Listeria, in particular Listeria monocytogenes, Listeria seeligeri and Listeria welshimeri, the genus Salmonella, in particular Salmonella arizonae, bongori and in particular serotypes, Salmonella gallinarum, Salmonella prolum, Salmonella typhimurium, Salmonella enterica serovar Ty ... Salmonella cholera, Salmonella heidelberg, Salmonella dublin, Salmonella harder, Salmonella typhi, Salmonella paratyphi and Salmonella infantis; Enterococci, in particular Enterococcus faecalis, Enterococcus faecium and Enterococcus secorrum; Staphylococci, in particular Staphylococcus aureus; Aeromonas, Streptococci, in particular Salmonella suis and Salmonella gallina In a preferred embodiment, the pathogenic microorganism is selected from the genus Clostridium, in particular Clostridium difficile and Clostridium perfringens, the genus Salmonella, in particular Salmonella typhimurium and Salmonella enteritidis, the genus Vibrio, in particular Vibrio parahaemolyticus and Vibrio harveyi, and the beneficial bacteria is preferably selected from the genus Lactobacillus and Bifidobacterium. In a preferred embodiment, the pathogenic microorganism is selected from the genus Clostridium, in particular Clostridium difficile and Clostridium perfringens, the genus Salmonella, in particular Salmonella typhimurium and Salmonella enteritidis, the genus Vibrio, in particular Vibrio parahaemolyticus and Escherichia coli.

[0028] The occurrence and / or increased proliferation of pathogenic bacteria may result in or cause the occurrence of certain diseases. For example, the occurrence and / or increased proliferation of Clostridium perfringens may result in the occurrence of intestinal diseases, especially the occurrence of necrotic enteritis in pigs and poultry. The occurrence and / or increased proliferation of Clostridium perfringens may also result in the occurrence of further diseases, such as bacterial enteritis, necrotic dermatitis and cholangiohepatitis. Even the mildest forms of infection with Clostridium perfringens may already be accompanied by diarrhea, which may cause the litter to become wet, which may lead to secondary diseases, such as footpad dermatitis. Clostridium perfringens type C is generally considered to be the main cause of necrotic enteritis and necrotic hemorrhagic enteritis in piglets, while type A is associated with intestinal diseases in suckling and feeding pigs, with mild necrotic enteritis and villous atrophy. C. difficile is an important emerging pathogen causing diarrhea primarily in neonatal pigs. Affected piglets can suffer from respiratory distress, abdominal bloating, and scrotal edema.

[0029] S. aureus subsp. aureus can cause digital knot disease in chickens and streptococcal mastitis in sows, and can produce toxins that cause food poisoning in humans.

[0030] Salmonella is an important cause of human food poisoning, often associated with the consumption of meat, such as poultry, pork or products derived from them. Therefore, controlling Salmonella is an important challenge for the meat production industry. In Europe, a baseline study carried out in 2005 on the prevalence of Salmonella in egg-laying flocks showed that at a global level in the EU, 20.3% of large-scale egg-laying hens were bacteriologically positive for Salmonella Enteritidis. In some countries, the prevalence was even higher than 80% [European Food Safety Authority (2006), "Preliminary report: analysis of a baseline study on the prevalence of Salmonella in egg-laying flocks of chickens (Gallus gallus)"].

[0031] Vibrio parahaemolyticus is responsible for diseases in shellfish, particularly early mortality syndrome (EMS), also known as acute hepatopancreatic necrosis (AHPND), which affects both giant black tiger prawn (Penaeus monodon) and white shrimp (Penaeus vannamei). Pathogens can cause further diseases such as polyarthritis, fibrous polyserositis, postweaning intestinal disorders such as postweaning diarrhea and edema disease, and swine dysentery. A preferred subject of the present invention is therefore in particular produced by Clostridium , in particular Clostridium difficile or Clostridium perfringens, and / or Salmonella , in particular Salmonella Enteritidis, preferably Salmonella enteritidis subsp. or Salmonella typhimurium, and / or Vibrio , in particular Vibrio parahaemolyticus, and / or Escherichia coli, for treating diseases.

[0032] A further preferred subject in this context is therefore a therapeutic composition comprising the strain and / or preparation and / or composition of the invention for treating and / or preventing necrotic enteritis and / or necrotic hemorrhagic enteritis, in particular subclinical necrotic enteritis and / or necrotic hemorrhagic enteritis, in animals, preferably pigs or poultry.A further preferred subject in this context is therefore a therapeutic composition comprising the microorganism and / or preparation and / or composition of the invention for treating and / or preventing premature mortality syndrome in animals, preferably animals kept in aquaculture, more preferably crustaceans, in particular shrimp and prawns. A further preferred subject in this context is therefore a therapeutic composition for treating and / or preventing diseases caused by food poisoning comprising the microorganism and / or preparation and / or composition of the invention. Another preferred subject in this context is therefore a therapeutic composition for the treatment and / or prevention of bacterial enteritis, necrotic dermatitis, cholangiohepatitis, clostridiosis, diarrhoea, dyspnoea, abdominal distension, scrotal oedema, digital bloating, padding of the feet, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrous polyserositis, post-weaning intestinal disorders such as post-weaning diarrhoea and oedema diseases, dysentery, osteomyelitis, inflammation of the joints and / or bone tissue, pericarditis, splenomegaly, hepatomegaly, nephromegaly, congestion, necrosis, infarction in the liver or spleen, valvular endocarditis, sepsis and / or meningitis in animals, preferably pigs or poultry, comprising the microorganism and / or preparation and / or composition of the invention.

[0033] A further subject of the present invention is therefore also the treatment and / or prevention of diseases, in particular intestinal diseases, associated with bacterial infections by Clostridium, in particular Clostridium perfringens, and / or Salmonella, in particular Salmonella, preferably Salmonella subsp. Enteritidis and / or Vibrio, in particular Vibrio parahaemolyticus, by administering a microorganism and / or a preparation and / or composition according to the invention to an animal in need thereof, the animal being preferably a pig or a poultry. A further subject of the present invention is therefore also the treatment and / or prevention of diseases, in particular intestinal diseases, preferably necrotizing enteritis or necrotic hemorrhagic enteritis, in particular asymptomatic necrotizing enteritis or asymptomatic necrotizing hemorrhagic enteritis, by administering a microorganism and / or a preparation and / or a composition according to the invention to an animal in need thereof, the animal being preferably a pig or a poultry. A further subject of the present invention is therefore also the treatment and / or prevention of diseases in aquatic animals, in particular early mortality syndrome, by administering a microorganism and / or a preparation and / or a composition according to the invention to an animal in need thereof, the animal being preferably an animal reared in aquaculture, more preferably a crustacean, in particular shrimp and prawn. A further subject of the present invention is therefore also the treatment and / or prevention of diseases caused by food poisoning, comprising the administration of a microorganism and / or a preparation and / or a composition according to the invention to an animal or human in need thereof. A further subject of the present invention is therefore the treatment and / or prevention of diseases caused by food poisoning, by applying the microorganisms and / or preparations and / or compositions of the invention to animal products, in particular meat or eggs, to avoid poisoning in humans. A further subject of the present invention is therefore the treatment and / or prevention of diseases, preferably of pigs or poultry, selected from bacterial enteritis, dermatitis gangrenosum, cholangiohepatitis, clostridiosis, diarrhoea, dyspnoea, abdominal distension, scrotal oedema, digital bloating, padding of the feet, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrous polyserositis, postweaning intestinal disorders such as postweaning diarrhoea and oedema diseases, dysentery, osteomyelitis, inflammation of the joints and / or bone tissue, pericarditis, splenomegaly, hepatomegaly, nephromegaly, congestion, necrosis, infarction in the liver or spleen, valvular endocarditis, septicaemia and / or meningitis.

[0034] The methods and uses of the microorganisms, feed or food additives and compositions of the invention may be pharmaceutical or non-pharmaceutical, in particular therapeutic or non-therapeutic. In particularly preferred embodiments of the invention, the methods and uses are non-pharmaceutical, in particular non-therapeutic, preferably feed and food applications. Preferred food compositions according to the invention are fermented foods and / or dairy products, in particular yoghurt, cheese, milk, butter, curd and natto. The microorganisms according to the invention may be used in particular in beverages and milk substitutes, such as infant formulas. Pharmaceutical compositions according to the invention preferably contain at least one pharma- ceutically acceptable carrier. They may further contain other ingredients, such as conventional food ingredients, as further listed below.

[0035] The pharmaceutical compositions according to the invention are preferably liquid, paste, aerosol, suppository or dry compositions, in particular for administration to the pharynx / respiratory tract, the gastrointestinal tract, the urogenital tract or the skin. Administration can be carried out by using medical devices such as sprays, pumps or inhalers. A further subject of the present invention is therefore also a method for preparing a pharmaceutical composition, in which the microorganism according to the invention is mixed with a pharma- ceutically acceptable carrier and, optionally, with further compounds, and the pharmaceutical composition can be provided in the form of a medical device. A further subject of the invention is in particular also a food supplement containing a microorganism according to the invention and at least one pharma- ceutically acceptable carrier.

[0036] In a preferred embodiment of the present invention, the microorganisms and compositions of the present invention are orally administered to animals or humans. Administration is preferably carried out in the form of feed and food compositions, but can also be carried out by applying the microbial preparation or feed or food additive of the present invention to drinking water or rearing water. Thus, a further subject of the present invention is water, in particular an aqueous solution or aqueous suspension, containing at least one microorganism or feed or food additive according to the present invention. In particular, when administered to drinking water or rearing water, the microorganisms and compositions of the present invention may be applied in the form of compositions, particularly tablets, that dissolve easily after exposure to drinking water or rearing water and release the microorganisms into the aqueous environment. Such compositions, particularly tablets, are further preferred embodiments of the present invention. These compositions preferably include a carrier, such as a betaine salt, a citrate salt, a carbonate salt and / or a bicarbonate salt, as disclosed, for example, in WO 2020 / 225020. The feed, food and pharmaceutical compositions, as well as drinking water, rearing water, effluent or wastewater of the present invention preferably have a concentration of about 1 × 10 to about 1 × 10 10 CFU / g feed or ml water, preferably about 1 x 10 4 ~Approx. 1×10 9 CFU / g feed or ml water, more preferably 1×10 6 ~1×10 8 Contains microorganisms in CFU / g feed or ml water. Similarly, a preferred amount of the feed or food additive of the present invention in the feed, food and water composition of the present invention is in the range of 0.01% to 10% by mass, more preferably 0.05% to 5% by mass, and particularly 0.1% to 2% by mass.

[0037] The feed, food and pharmaceutical compositions of the present invention may comprise further carriers or further conventional feed ingredients or combinations thereof. Suitable carriers for the preparation of feed, food and pharmaceutical compositions are the same as those mentioned above for the preparation of feed or food additives starting from the microorganism of the invention or the fermentation medium of said microorganism. Suitable conventional animal feed ingredients which may be contained in a composition according to the invention and / or which may be used in the preparation of a feed or food composition starting from the microorganism and / or microbial fermentation medium and / or feed or food additive according to the invention include one or more of the following: proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, carriers, coccidiostats, acid-based products and / or medicines, such as antibiotics. Carbohydrate-containing ingredients that may be used in the present invention are, for example, forage, roughage, wheat meal, corn meal, sunflower meal or soybean meal, and mixtures thereof.

[0038] Protein-containing ingredients that may be used in the present invention are, for example, soy protein, pea protein, wheat gluten, corn gluten, rice, canola meal, marine animal meals, particularly fish meal, land animal meals, and mixtures thereof. "Marine animal meals" include meat meal, meat and bone meal, blood meal, liver meal, poultry meal, silkworm meal, silkworm pupa meal, and combinations thereof.

[0039] The fats are usually marine animal oils, vegetable oils or microbial oils, in particular microalgae oils, or combinations thereof. Examples of vegetable oils include soybean oil, rapeseed oil, sunflower seed oil, canola oil, cottonseed oil, linseed oil and palm oil. Marine animal oils include fish oils, as well as oils from krill, clams, squid or shrimp, and also fatty oils from fish of the families Engraulidae, Carangidae, Clupeidae, Osmeridae, Scombridae and / or Ammodytidae. Examples of microalgae oils include, in particular, Labyrinthulea oils, preferably Schizochytria or Thraustochytria oils. In addition to isolated oils, defatted biomass itself can also be used as fat source, i.e., in particular marine animal meals, preferably fishmeal, or vegetable meals, in particular soybean meal, rapeseed meal, sunflower meal, canola meal, cottonseed meal and / or linseed meal.

[0040] Protein-containing ingredients further containing fat that can be used in the present invention are, for example, fish meal, krill meal, bivalve meal, squid meal or shrimp shells, as well as combinations thereof. The feed according to the present invention preferably has a total protein content of 20-50% by mass and / or a total fat content of 1-15% by mass and / or a total carbohydrate content of 20-60% by mass. Further probiotics (DFM) and / or microorganisms which may be used in the present invention in combination with the microorganisms and feed additives of the present invention are preferably bacteria selected from the species Bacillus subtilis, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus lentus, Bacillus pumilus, Bacillus laterosporus, Bacillus coagulans, aerobic spore-forming bacteria, Bacillus cereus, Bacillus badius, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus beruerensis, Enterococcus faecium, and Pediococcus acidilactici.Preferred examples are Bacillus pumilus DSM 32539 and Bacillus subtilis DSM 32540 (both deposited at the DSMZ on June 14, 2017 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure) and their derivatives, Bacillus licheniformis DSM 32314 and Bacillus subtilis DSM 32315 (both deposited at DSMZ on May 12, 2016 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure) and its derivatives, Bacillus subtilis PB6 (described in U.S. Pat. No. 7,247,299 and deposited under ATCC Accession No. PTA-6737) (sold by Kemin under the trademark CLOSTAT(Canadian)®), Bacillus subtilis C-3102 (described in U.S. Pat. No. 4,919,936 and deposited in Japan at the National Institute of Advanced Industrial Science and Technology under the trademark FERM BP1096) (sold by Calpis under the trademark CALSPORIN®), Bacillus subtilis DSM 17299 (sold by Chr. Hansen under the trademark GalliPro®), Bacillus licheniformis DSM 17236 (sold under the trademark GalliProTect® by Chr. Hansen), a mixture of Bacillus licheniformis DSMZ 5749 and Bacillus subtilis DSMZ 5750 spores (sold under the trademark BioPlus® YC by Hansen), Bacillus subtilis DSM 29784 (sold under the trademark Alterion® by Adisseo / Novozymes), Bacillus subtilis sold under the trade name PORCBOOST® by Chr. Hansen, Bacillus licheniformis DSM 28710 sold under the trademark B-Act® by Hevepharma, a three-strain Bacillus probiotic sold under the trademark Enviva® PRO by DuPont, the probiotic PureGro™ sold by DSM, or the Bacillus coagulans microorganism described in U.S. Pat. No. 6,849,256.In particular, in food compositions, further probiotics known to be useful for human health may be used, such as bacteria producing lactic acid bacteria of the order Lactobacillales, more preferably the family Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae or the family Streptococci, or of the order Bifidobacteriales, preferably the order Bifidobacteriales, in particular the genus Bifidobacterium. If said further probiotics are not formulated as part of the composition of the invention, they may be administered together with the composition of the invention (at the same time or at different times).

[0041] The prebiotics that may be used in the present invention are preferably oligosaccharides, in particular oligosaccharides selected from galactooligosaccharides, sialyloligosaccharides, lactulose, lactosucrose, fructooligosaccharides, palatinose or isomaltose oligosaccharides, glycosylsucrose, maltooligosaccharides, isomaltooligosaccharides, cyclodextrins, gentiooligosaccharides, soybean oligosaccharides, xylooligosaccharides, dextran, pectin, polygalacturonan, rhamnogalacturonan, mannan, hemicellulose, arabinogalactan, arabinan, arabinoxylan, resistant starch, mebiose, chitosan, agarose, inulin, tagatose, polydextrose, and alginates.

[0042] Enzymes which may be used in the feed composition according to the invention and which may aid in the digestion of the feed are preferably phytase (EC 3.1.3.8 or 3.1.3.26), xylanase (EC 3.2.1.8), galactanase (EC 3.2.1.89), galactosidase, in particular α-galactosidase (EC 3.2.1.22), protease (EC 3.4), phospholipase, in particular phospholipase A1 (EC 3.1.1.32), A2 (EC 3.1.1.4), C (EC 3.1.4.3) and D (EC 3.1.4.4), lysophospholipase (EC 3.1.1.5), amylase, in particular α-amylase (EC 3.2.1.1), lysozyme (EC 3.2.1.17), glucanases, in particular β-glucanases (EC 3.2.1.4 or EC 3.2.1.6), glucoamylases, cellulases, pectinases, or any mixture thereof. Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P and HiPhos™ (DSM Nutritional Products), Natuphos™ (BASF), Finase® and Quantum® Blue (AB Enzymes), Phyzyme® XP (Verenium / DuPont) and Axtra® PHY (DuPont). Other suitable phytases include those described, for example, in WO 98 / 28408, WO 00 / 43503, and WO 03 / 066847. Examples of commercially available xylanases include Ronozyme® WX and G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium) and Axtra® XB (Xylanase / beta-glucanase, DuPont). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).

[0043] Vitamins that may be used in the present invention are, for example, vitamin A, vitamin D3, vitamin E, vitamin K, e.g., vitamin K3, vitamin B12, biotin, choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, e.g., Ca-D-pantothenate, or combinations thereof.

[0044] Immunomodulatory agents that may be used in the present invention are, for example, antibodies, cytokines, spray-dried plasma, interleukins, or interferons, or combinations thereof. Minerals that may be used in the present invention are, for example, boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, calcium, phosphorus, magnesium, potassium, or sodium, or combinations thereof. Amino acids that can be used in the present invention are, for example, lysine, alanine, threonine, methionine, or tryptophan, or combinations thereof. The composition of the invention may further comprise betaine and / or choline and / or other physiologically available methyl group donors. The feed may further comprise polyunsaturated fatty acids, in particular DHA and / or EPA.

[0045] Therefore, a further embodiment of the present invention is also a method for preparing an animal feed composition, comprising mixing at least one microorganism and / or microbial preparation and / or feed additive of the present invention, in particular in an amount effective for promoting animal health, in particular intestinal health, with feed ingredients such as proteins, lipids and / or carbohydrates, and optionally further beneficial substances, preferably as mentioned above, to provide a feed product, which may also include, for example, a pelleting process. Standard pelleting processes known to those skilled in the art may be used, including extrusion of dry or semi-moist feed. The preferred pelleting temperature is from about 65°C to about 120°C. The microorganisms, microbial preparations, feed additives and compositions of the invention can be administered to the animal in the feed and / or drinking water and / or rearing water over multiple days throughout the life of the animal or during a particular stage or portion of the life of the animal. For example, the microorganisms and / or microbial preparations and / or compositions may be administered only in the starter feed or only in the finishing feed of the livestock.

[0046] The microorganisms, microbial preparations and compositions of the present invention can further be used in a wide range of doses, for example, in a daily dose range of about 1 mg to about 500 mg per kilogram of live weight, in particular in a range of about 5 mg to about 200 mg, in a range of about 10 mg to about 100 mg, or in a range of about 20 to about 60 mg. According to the invention, in an embodiment of the invention, it is preferred that an effective amount of the microorganism and / or microbial preparation and / or feed or food additive and / or composition of the invention is always used. The term "effective amount" refers to an amount that brings about at least one beneficial effect on an animal and / or environment, in particular with regard to the above-mentioned characteristics, when compared to an animal or environment that is not administered the microorganism and / or microbial preparation and / or composition of the invention, but is administered the same diet (including feed and other compounds) or the same composition, respectively. For therapeutic applications, preferably therapeutic amounts of the microorganisms and / or microbial preparations and / or feed or food additives and / or compositions of the present invention are used. The term "therapeutic amount" refers to an amount sufficient to improve, reverse or prevent a disease state in an animal. Optimal dosage levels for different animals can be easily determined by those skilled in the art by evaluating, inter alia, the ability of the composition to (i) inhibit or reduce pathogenic viruses and / or bacteria in the gastrointestinal tract, particularly in the intestine, or in the oral or nasopharyngeal cavity, or on the skin or gills, at different doses, (ii) increase or maintain the level of beneficial bacteria, at different doses, and / or (iii) promote the health of the animal, particularly the intestinal health, at different doses.

[0047] The method of the present invention may be used with all kinds of animals, particularly all kinds of vertebrates, such as mammals, aquatic animals and birds. Animals that may benefit from the present invention include, but are not limited to, farm animals, pets, exotic animals, zoo animals, and animals used for sport, recreation or work. In a highly preferred embodiment of the invention, the animals are livestock raised for consumption or food production, such as poultry, pigs and ruminants. The poultry may be selected from production or livestock poultry, but also from luxury poultry or wild birds. Preferred productive poultry in this context are chickens, turkeys, ducks and geese. Production livestock in this context are preferably poultry optimized for juvenile production or poultry optimized for meatiness. Preferred luxury poultry or wild birds are peacocks, pheasants, quails, chukkars, guinea fowl, quails, egyptians, ptarmigans, pigeons and swans, with quails being particularly preferred. Further preferred poultry are ratites, especially ostriches and emus, as well as parrots. The ruminant according to the present invention is preferably selected from cows, goats and sheep. In one embodiment, the composition of the present invention can be fed to pre-ruminant animals to improve their health, in particular to reduce the incidence of diarrhea in these animals. Pre-ruminant animals are ruminant animals ranging from birth to about 12 weeks of age, including calves. Pets are preferably selected from dogs, cats, domestic birds and domestic exotic animals.Sport animals may in particular be horses.

[0048] Aquatic animals according to the invention are preferably fishes, in particular Actinopterygii or crustaceans, such as, in particular, tilapia and other cichlids, carps and other cyprinids, salmon and other salmonids, catfish, in particular African catfish and Pangasius, tuna, sea bass, cod, smelt, milkfish, gourami, sea bass, in particular barramundi, sea fish of the Sparidae family, grouper and snakehead fish. Salmon and other salmonid fish in this context include Atlantic salmon, sockeye salmon, cherry salmon, Chinook salmon, Chum salmon, Coho salmon, Danube salmon, Pacific salmon, pink salmon and trout. The aquatic animal according to the invention is particularly preferably a tilapia. Crustaceans include, inter alia, shrimp, lobster, crab, prawns and crayfish. Preferred shrimps in this context include the genera Litopenaeus, Farfantepenaeus and Penaeus, in particular Penaeus stylirostris, Penaeus vannamei, Penaeus monodon, Penaeus chinensis, Penaeus occidentalis, Penaeus californiensis, Penaeus semisulcatus, Penaeus esculentu, Penaeus setiferus, Penaeus japonicus, Penaeus azuesco, Penaeus serrata ... Penaeus aztecus, Penaeus duorarum, Penaeus indicus, and Penaeus merguiensis. Penaeus bannameji, also called white legged shrimp, is highly preferred in the present invention. The aquatic animals, in particular shrimp, treated or fed with the microorganisms and / or preparations and / or compositions according to the invention may in particular be larval, post-larval or juvenile shrimp. The aquatic animal may in particular be a fish which is subsequently processed into fishmeal or fish oil. In this context, the fish is preferably herring, pollock, cod or anchovy, blue whiting, capelin, driftfish, jack, mackerel, menhaden, sardine or scad fish. The fishmeal or fish oil thus obtained can be used in aquaculture for cultivating edible fish or crustaceans. The aquatic animals may further be oysters, clams, cockles, arkshells, bivalves, mussels or scallops. However, the aquatic animals may also be small organisms used as feed in aquaculture. These small organisms may take the form of, for example, nematodes, small crustaceans or rotifers.

[0049] The farming of aquatic animals may take place in ponds, tanks, basins, or isolated areas of the sea or lakes or rivers, in particular in this case in cages or net racks. Aquaculture can be used to farm finished food fish, but also for the farming of juvenile fish that are released to replenish wild fish stocks. In salmon farming, the fish are preferably first grown to smolts in freshwater tanks or artificial waterways and then grown in floating cages or net racks in the sea, ponds or rivers, preferably fixed in bays or fjords. Therefore, the feed composition according to the present invention is preferably a feed for use in raising the above-mentioned animals.

[0050] The preferred use of the animal feed according to the present invention is for cattle, pigs, poultry, fish, crustaceans or pets, with pigs, especially weaned piglets, and poultry being particularly preferred.Spore-forming microorganisms, especially those of the Bacillus genus, are particularly suitable for this use, since they produce heat-resistant spores that can be mixed into the animal feed before the pelleting process.The microorganisms of the present invention are particularly preferably used in milk replacers for cattle, goats, sheep and pigs, or as post-pelleting spray-on coatings for pelleted feed materials.

[0051] The microorganisms and fermentation media that can be used in the present invention can be obtained by culturing the microorganisms of the present invention by methods known in the art, including using media and other methods described, for example, in Elisashvili et al. (Probiotics and Antimicrobial Proteins 11, 731-747 (2019)), Ren et al. (AMB Express 8:21 (2018)) or the Food and Agriculture Organization of the United Nations (FAO) report 179 entitled "Probiotics in Animal Nutrition" (Bajagai et al., FAO (2016)). Conventional large-scale microbial cultivation processes include submerged fermentation, solid-state fermentation, or liquid surface culture. The fermentation is configured to obtain a high level of colony forming units of microbial cells. Optionally, the fermentation medium thus obtained may be washed, for example via a diafiltration process, to remove residual fermentation medium and metabolites. The fermentation medium used for the preparation of the feed or food additive according to the present invention is preferably prepared by culturing the microorganisms of the present invention at a concentration of 5×10 0.01% after the fermentation is completed. 10 ~5×10 12 cfu / g, more preferably 1×10 11 ~4×10 12 Contains cells in amounts of cfu / g. EXAMPLES

[0052] Comparison of genomic DNA between mutant and parent strain CECT 5940 The parent strain CECT 5940 was deposited in the Spanish Culture Collection CECT in April 2001 and has been marketed as a probiotic product for many years. It was initially classified as Bacillus amyloliquefaciens but has recently been reclassified as Bacillus verruezensis. Sequence comparison of the mutants by mapping showed that the genomic DNA of the mutants is otherwise identical, with only two point mutations in the gene sequence encoding the germination protein KC (GerKC). The two point mutations in the mutant gene result in an exchange of a cysteine ​​at position 11 of GerKC to a glycine (C11G) and a proline at position 72 of GerKC to a serine (P72S). EXAMPLES

[0053] Comparison of morphological characteristics of mutant and parent strains Bacillus CECT 5940 is reported as an aerobic, spore-forming Gram-positive bacillus. The vegetative cells are rod-shaped (0.7-0.9 μm long × 1.8-3.0 μm wide) and are most often linked in chains. They have a peripheral flagellum and are slightly mobile. The spores are ellipsoidal and there is no deformation of the sporangium. Two point mutations in the germination receptor subunit gerKC have no effect on the phenotypic appearance of the strain and its cultivation conditions. EXAMPLES

[0054] Comparison of antimicrobial susceptibility between mutant and parent strains Bacillus CECT 5940 strain was found to be sensitive to chloramphenicol, clindamycin, daptomycin, erythromycin, gentamicin, kanamycin, streptomycin, nourseothricin, tetracycline, vancomycin and virginiamycin M1. Phenotypic antibacterial resistance is not altered by genetic modification in the gerKC gene. Both strains are sensitive to the antibiotics tested. EXAMPLES

[0055] Comparison of production of unwanted antibacterial substances To assess the production of antibacterial substances relevant for therapeutic use in humans and animals according to EFSA guidance (EFSA, 2018), the inhibitory activity of culture supernatants of Bacillus CECT 5940 against the reference strains Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 was measured. No inhibition was observed for both Bacillus CECT 5940 wild type, indicating a lack of production of antibacterial substances, including aminoglycosides. The lack of production of antibacterial substances relevant for therapeutic use in humans and animals is not altered by the genetic modification in the gerKC gene of Bacillus CECT 5940. EXAMPLES

[0056] Comparison of toxin production between mutant and parent strains Cytotoxicity studies on Vero C1008 epithelial cells confirmed the absence of toxigenic potential for Bacillus CECT 5940 wild type, which was also true for the mutant strain. No cytotoxicity was observed for the negative controls, Bacillus CECT 5940 and the mutant strain. In contrast, the positive control, i.e., the supernatant of Bacillus cereus strain DSM-31, showed significant cytotoxicity even when applied at the lowest concentration to Vero cells. Thus, the genetic modification does not change the "non-cytotoxic" status. EXAMPLES

[0057] Evaluation of the growth behavior of engineered strains The growth behavior of the mutant strain was evaluated by measuring the onset of metabolic activity of spore cultures. A resazurin-containing redox dye was used as an indicator of metabolic activity, since upon reaction with reduced electron metabolites generated after germination, the dye changes color at the beginning of the elongation phase. The increase in metabolic activity can be measured at 550 nm (Nagler et al. (2014); Appl Environ Microbiol 80(4):1314-1321; O'Brien et al. (2000); Eur J Biochem. 5421-6)). The increase in metabolic activity of the mutant strain was compared with that of the parent strain CECT 5940. Spore cultures of both strains were prepared and cultivated under the same conditions in germination-inducing TSB medium containing nutrient germination triggers. The parent strain reached a certain metabolic activity after a specific incubation time, while the similar activity of the mutant strain was reached 26,5% earlier. This confirmed the expected improvement in growth of the mutant strain.

Claims

1. An endospore-forming Bacillota strain having improved growth and / or germination characteristics.

2. 2. The Bacillus strain of claim 1, comprising probiotic activity, the ability to inhibit pathogenic bacteria selected from Clostridium perfringens, Salmonella Enteritidis, Salmonella typhimurium, Escherichia coli, Clostridioides difficile, Vibrio parahaemolyticus, and combinations thereof, the ability to grow in the presence of bile, the ability to produce organic acids, and / or the ability to produce lactic acid.

3. The Bacillus strain of claim 1 , wherein the improved growth and / or germination properties are due to enhanced activity of the germination receptor GerK.

4. 4. The Bacillus strain of claim 3, wherein the improved growth and / or germination properties are due to at least one mutation, 1 to 10 mutations, 1 to 5 mutations, or 2 to 4 mutations in at least one subunit of the germination receptor GerK, said subunit preferably being the spore germination protein KC (GerKC).

5. 5. The Bacillus strain of claim 4, wherein the spore germination protein KC contains glycine at position 11 and / or serine at position 72 of its amino acid sequence.

6. The Bacillus strain of claim 4, wherein the sequence of the gerKC gene is represented by SEQ ID NO:3 and / or the sequence of the GerKC protein is represented by SEQ ID NO:

4.

7. The Bacillus phylum strain is a strain of the genus Bacillus, and may be any of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus licheniformis, Bacillus spp. licheniformis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus megaterium, Bacillus lentus, Bacillus laterosporus, Bacillus alevii, Bacillus cereus, Bacillus badius, Bacillus thuringiensis, Bacillus polyfermenticus, Bacillus valismortis, Bacillus 2. The Bacillus strain according to claim 1, which is a strain belonging to the phylum Bacillus selected from the group consisting of Bacillus tequilensis, Bacillus atrophaeus, Bacillus mojavensis, Bacillus sonorensis, Bacillus inaquosus and B. safensis, or selected from the group consisting of Bacillus subtilis, Bacillus beruerensis, Bacillus amyloliquefaciens, Bacillus pumilus or Bacillus megaterium, or which is Bacillus amyloliquefaciens or Bacillus beruerensis.

8. 2. The Bacillus strain of claim 1, which is a Bacillus beruerensis strain and the genomic DNA of said strain is at least 99%, at least 99.5%, at least 99.9%, or at least 99.99% identical to the genomic DNA of strain CECT 5940, and said strain has enhanced growth and / or germination characteristics compared to strain CECT 5940.

9. A feed or food additive comprising the Bacillus strain of claim 1.

10. A feed or food composition comprising a Bacillus strain according to claim 1 and at least one further feed or food ingredient, preferably selected from carriers, proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, carriers, anticoccidial agents, acid-based products and / or medicines, e.g. antibodies.

11. 10. A pharmaceutical composition comprising the Bacillus strain of claim 1 and a pharma- ceutically acceptable carrier for treating, preventing or ameliorating the course of intestinal disease.

12. 2. Use of the Bacillus strain according to claim 1 in the preparation of a feed or food composition.

13. 2. Use of the Bacillus strain according to claim 1 in the preparation of a pharmaceutical composition for treating, preventing or ameliorating the course of intestinal diseases.

14. 2. Use of a Bacillus strain according to claim 1 for the preparation of a composition for promoting the health of an animal, and / or improving the general body condition of the animal, and / or improving the feed conversion rate of the animal, and / or reducing the mortality rate of the animal, and / or increasing the survival rate of the animal, and / or improving the weight gain of the animal, and / or increasing the productivity of the animal, and / or increasing the disease resistance of the animal, and / or modulating the immune response of the animal, and / or establishing or maintaining a healthy intestinal microflora in the animal, and / or improving the meat quality of the animal, and / or reducing the shedding of bacterial and / or viral pathogens via the faeces of the animal.

15. 13. A method of feeding an animal, comprising feeding said animal the Bacillus strain of claim 1.

16. A GerKC protein represented by a sequence having at least 95%, preferably at least 98 or 99%, sequence identity to the GerKC sequence of Bacillus verruezensis CECT 5940 strain represented by SEQ ID NO: 2, said sequence containing at least one or both mutations selected from T31G and C214T, and having high growth and / or germination properties.

17. 10. A feed or food composition comprising a feed or food additive according to claim 9 and preferably at least one further feed or food ingredient selected from carriers, proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, carriers, anticoccidial agents, acid-based products and / or medicines, e.g. antibodies.

18. 10. Use of the feed or food additive according to claim 9 in the preparation of a feed or food composition.

19. 10. Use of a feed or food additive according to claim 9 for preparing a composition for promoting the health of an animal, and / or improving the general body condition of the animal, and / or improving the feed conversion rate of the animal, and / or reducing the mortality rate of the animal, and / or increasing the survival rate of the animal, and / or improving the weight gain of the animal, and / or increasing the productivity of the animal, and / or increasing the disease resistance of the animal, and / or modulating the immune response of the animal, and / or establishing or maintaining a healthy intestinal microflora in the animal, and / or improving the meat quality of the animal, and / or reducing the shedding of bacterial and / or viral pathogens via the faeces of the animal.

20. 11. Use of a feed or food composition according to claim 10 for preparing a composition for promoting the health of an animal, and / or improving the general body condition of the animal, and / or improving the feed conversion rate of the animal, and / or reducing the mortality rate of the animal, and / or increasing the survival rate of the animal, and / or improving the weight gain of the animal, and / or increasing the productivity of the animal, and / or increasing the disease resistance of the animal, and / or modulating the immune response of the animal, and / or establishing or maintaining a healthy gut microflora in the animal, and / or improving the meat quality of the animal, and / or reducing the shedding of bacterial and / or viral pathogens via the faeces of the animal.

21. 10. A method of feeding an animal, comprising feeding said animal a feed or food additive according to claim 9.

22. 11. A method of feeding an animal, comprising feeding said animal a feed or food composition according to claim 10.

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