Bacillus subtilis strains that have beneficial effects against neurodegenerative diseases such as Alzheimer's disease

Abstract

The present invention relates to novel Bacillus subtilis strains that have a beneficial effect against Alzheimer's disease.

Description

[Technical Field]

[0001] The present invention relates to novel Bacillus subtilis strains that have a beneficial effect on Alzheimer's disease, both alone and in combination with booster substances, and their use as probiotics. The invention further relates to the use of the strains of the invention as food supplements and as pharmaceuticals. [Background technology]

[0002] There are several risk factors identified for neurodegenerative diseases, such as obesity, cardiovascular disorders, and diabetes. However, with aging, the risk of cognitive decline or the risk of developing neurodegenerative diseases, such as Alzheimer's disease, increases exponentially. As a result, a significant number of people in the aging population will suffer from dementia. This increase poses a challenge to patients, their families, and the medical system. It is known that neurodegenerative diseases, such as Alzheimer's disease and dementia, can be detected as early as 20 years before the onset of symptoms. Therefore, there is growing interest in reducing the risk factors for developing neurodegenerative diseases, which can prevent and / or cure the diseases.

[0003] New research and development has uncovered a connection between the gut and the brain that can be influenced by the gut microbiota. The present invention relates to a novel Bacillus subtilis strain that has shown efficacy in reducing neurodegeneration and delaying Alzheimer's disease in different types of nematodes, Caenorhabditis elegans (C. elegans). Recent advances have reported that feeding Bacillus subtilis (NCIB3610 (DSM10) and JH642) to the nematode N2 strains CF1038 and PS3551 differentially extended their lifespan, due to biofilm formation and production of nitric oxide and the quorum-sensing pentapeptide CSF, compared to C. elegans fed with normal Escherichia coli (E. coli OP50) bacteria. When all of the genes involved (biofilm formation, NO, CSF), were inactivated, the nematode's lifespan decreased (Non-Patent Document 1). Another publication investigated the anti-Alzheimer's effect of Bacillus subtilis NCIB3610 (DSM10) on specific C. elegans strains through biofilm formation and production of the quorum-sensing pentapeptide CSF. C. elegans mutants CL2120 and GMC101, which express A-beta protein in muscle cells of worms fed Bacillus subtilis, showed delayed neuronal deterioration, delayed paralysis, and performed better in behavioral tests. Comparable effects were observed in the C. elegans mutant CL2355, which has pan-neuronally expressed A-beta protein (Non-Patent Document 2). Furthermore, B. subtilis strains NCIB3610 (DSM10), 168 (DSM23778), and JH642 showed a protective effect against α-synuclein aggregation in the C. elegans mutant NL5901, which expresses human α-synuclein (Non-Patent Document 3). The biofilm-producing ability of the described Bacillus subtilis NCIB3610 (DSM 10) was tested in biofilm-promoting MSgg medium as well as in the liquid standard medium NGM alone. To ensure biofilm formation and therefore bacterial colonization of the probiotic components, it was necessary to test under conditions similar to those in the colon. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Donato, V et al. (2017) “Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signaling pathway” Nat. Commun. 8 , 14332

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[0005] It was therefore an object of the present invention to provide probiotic strains that are able to reduce the risk of developing neurodegenerative diseases by microbiome modulation under colon-like conditions. [Means for solving the problem]

[0006] The use of Bacillus subtilis strains as probiotic ingredients in the feed industry has long been disclosed as prior art in the art. The function of probiotics (also known as "direct-fed microorganisms" or "DFM") is to positively influence the intestinal microflora by supporting the growth of beneficial bacteria and / or suppressing the growth of pathogenic bacteria. Many neurodegenerative diseases are multifactorial, with no single clear pathway in the body. The present invention targets multiple pathways and favorably influences the release of several metabolites and exhibits in situ production. Surprisingly, the novel B. subtilis strain DSM34350 also demonstrated efficient biofilm formation in a medium mimicking the human colon environment (SCEM), whereas prior art studies have shown that B. subtilis strains NCIB3610 (DSM10), 168 (DSM23778), and JH642, while showing similar growth rates, were unable to establish biofilms under human colonic conditions. Furthermore, the novel strain exhibited significantly higher fibrinolytic enzyme activity under human colonic conditions compared with the conventional strains. Fibrinolytic enzymes, such as nattokinase, can degrade protein plaques, such as amyloid fibrils, that accumulate in the brains and other organs of Alzheimer's patients. Therefore, higher fibrinolytic activity may be beneficial for preventing amyloid plaque formation.

[0007] Further tests in different C. elegans strains confirmed significant differences: it could be shown that paralysis in the C. elegans mutant GMC101 could be significantly delayed, meaning that lifespan could be extended compared to worms fed the E. coli OP50 strain or the B. subtilis NCIB3610 (DSM10) strain. Other experiments have provided new insights by feeding B. subtilis DSM34350 to C. elegans to investigate aging-related neurodegeneration, and again, significant lifespan extension effects were observed. Bacillus subtilis DSM 34350 was identified by targeted screening of naturally occurring isolates and has been deposited on August 16, 2022, by Evonik Operations GmbH under the above accession number at the Leibniz Institute DSMZ German Collection of Microbial Cell Cultures (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig, Germany, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0008] A first subject of the present invention is therefore the Bacillus subtilis strain deposited at the DSMZ under number DSM 34350 or a preparation thereof. The B. subtilis strain deposited at the DSMZ under the number DSM 34350 exhibits the following characteristic sequences: a) a 16S rDNA sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, in particular 100% to the polynucleotide sequence set forth in SEQ ID NO: 1; b) a yqfD sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, in particular 100% to the polynucleotide sequence set forth in SEQ ID NO: 2; c) a gyrB sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, especially 100% to the polynucleotide sequence set forth in SEQ ID NO: 3; d) an rpoB sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, especially 100% to the polynucleotide sequence set forth in SEQ ID NO: 4; e) A groEL sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, especially 100% to the polynucleotide sequence set forth in SEQ ID NO: 5.

[0009] A further subject of the present invention is therefore a Bacillus subtilis strain, in particular a Bacillus subtilis strain having the above-mentioned characteristics, or a preparation thereof, wherein the Bacillus subtilis strain exhibits at least one, preferably all, of the following characteristics: a) a 16S rDNA sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, in particular 100% to the polynucleotide sequence set forth in SEQ ID NO: 1; b) a yqfD sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, in particular 100% to the polynucleotide sequence set forth in SEQ ID NO: 2; c) a gyrB sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, especially 100% to the polynucleotide sequence set forth in SEQ ID NO: 3; d) an rpoB sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, especially 100% to the polynucleotide sequence set forth in SEQ ID NO: 4; e) A groEL sequence having a sequence identity of at least 99.5%, preferably at least 99.8%, especially 100% to the polynucleotide sequence set forth in SEQ ID NO: 5.

[0010] A particular subject of the present invention is therefore also a Bacillus subtilis strain that exhibits the following characteristics: a) the 16S rDNA sequence set forth in SEQ ID NO: 1; b) the yqfD sequence set forth in SEQ ID NO:2; c) the gyrB sequence set forth in SEQ ID NO: 3; d) the rpoB sequence set forth in SEQ ID NO: 4; e) The groEL sequence set forth in SEQ ID NO:5.

[0011] The strains of the present invention are preferably characterized by at least one, more preferably all, of the following additional characteristics: The strain is preferably capable of growing under anaerobic conditions and, more preferably, under human colonic conditions. The Bacillus subtilis strain is characterized by being able to establish a biofilm under colonic conditions after 24 hours at 37° C., with a biofilm strength of at least 0.1, preferably determined by measuring the absorbance at 565 nm. It is preferred if the biofilm is established in SCEM or TSB medium at pH 7. In particular, the Bacillus subtilis strain is characterized by having fibrinolytic activity, preferably nattokinase activity, under colonic conditions, preferably determined by an average halo diameter of at least 20 mm produced on fibrin agar plates after 24 hours. Preferably, the Bacillus subtilis strain is characterized by the ability to produce short chain fatty acids, preferably acetate, butyrate and lactate under colonic conditions, preferably at levels higher than 0.1 g / l or higher than 0.2 g / l after 26 hours. Without wishing to be bound by any theory, it is believed that the Bacillus subtilis strains of the present invention promote human health, particularly gut health or mental health, through a pleiotropic mode of action that includes fibrinolytic activity and production of short-chain fatty acids. As many neurodegenerative diseases do not have one clear pathway in the body but are multifactorial, multiple pathways are targeted in the present invention to favorably affect and demonstrate in situ production of several metabolites.

[0012] In a preferred embodiment of the invention, the strains and preparations of the invention are administered orally to animals or humans. Therefore, further subject matter of the present invention are compositions such as feed, food, drinking water and rearing water, as well as therapeutic compositions, which contain the Bacillus subtilis strain according to the invention and / or preparations thereof. A further subject of the present invention is also the use of the Bacillus subtilis strains and / or preparations according to the invention as probiotic ingredients (DFM) in feed or food products. Preferred food products according to the invention are dairy products, especially yogurt, cheese, milk, butter and quark. The cells of the strains according to the invention may be present, in particular in the compositions of the invention, as spores (dormant), as vegetative cells (growing), as transitional cells (transitioning from vegetative to spore or vice versa), or as a combination of at least two, in particular all, of these cells. In a preferred embodiment, the compositions of the invention comprise mainly or exclusively spores. Additionally or alternatively, cells of the strain may also be used in a non-viable, inactivated form, as even non-viable cells are expected to have a probiotic effect. Methods for inactivating cells are known to those skilled in the art.

[0013] The methods and uses of the strains and preparations of the present invention can be therapeutic or non-therapeutic. A further subject of the present invention is a food or feed composition comprising a Bacillus subtilis strain according to the invention or a preparation thereof and at least one further feed or food ingredient selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, anticoccidial agents, acid-based products, pharmaceuticals and combinations thereof, preferably manganese or thiamine. A further subject of the present invention is therefore also a pharmaceutical composition comprising the above-described strain of the invention or a preparation thereof and a pharmaceutically acceptable carrier. A particular subject of the present invention is 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 increasing the immune response of a human and / or establishing or maintaining a healthy intestinal microflora in a human, wherein the strains and / or preparations of the present invention are administered to a human. A further subject of the present invention is therefore also the use of the strains and / or preparations of the invention for promoting the health of a human and / or for improving the general physical condition of a human and / or for increasing the disease resistance of a human and / or for increasing the immune response of a human and / or for establishing or maintaining a healthy intestinal microflora in a human, wherein the strains and / or preparations of the invention are administered to a human.

[0014] Another aspect of the present invention relates to a pharmaceutical or non-pharmaceutical composition further comprising a targeted release formulation for delayed release or enteric or colonic release. A targeted release formulation according to the present invention is a formulation that ensures delivery of the components of the preparation according to the present invention to a specific target in the body. Preferred formulations of the preparation promote intestinal or colonic delivery in the lower small intestine or large intestine. Targeted release formulations can be obtained by adding an enteric polymer to the matrix of the dosage form or by adding a coating, preferably an enteric coating, to the dosage form. [Brief explanation of the drawings]

[0015] [Figure 1]This figure compares the results of the paralysis test for C. elegans GMC101, which were maintained at 25°C for 8 days and fed E. coli OP50 starting at the L4 stage, when fed A) B. subtilis DSM10 or B) B. subtilis DSM34350. Statistical analysis was performed using a two-way analysis of variance (ANOVA-2) followed by Bonferroni's multiple comparison test. All p values ​​less than 0.05 were considered significant (*p<0.5; **p<0.01; ***p<0.001; ****p<0.0001 are symbols representing statistical values ​​according to Bonferroni's multiple comparison test; $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols representing statistical values ​​according to ANOVA-2, which explains the variation between conditions). [Figure 2] This figure compares the results of locomotor activity assays of C. elegans GMC101, which were maintained at 25°C for 8 days and fed E. coli OP50 starting at the L4 larval stage, when fed either B. subtilis DSM10 or B. subtilis DSM34350. Statistical analysis was performed using two-way analysis of variance (ANOVA-2) followed by Bonferroni's multiple comparison test. All p values ​​less than 0.05 were considered significant (*p<0.5; **p<0.01; ***p<0.001; ****p<0.0001 are symbols representing statistics according to Bonferroni's multiple comparison test; $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols representing statistics according to ANOVA-2, which explains the variation between conditions). [Figure 3] Figure 1 shows the results of a biofilm assay for the disclosed strain DSM34350 compared to other state-of-the-art strains described in the context of treating neurodegenerative diseases, with (A) the general ability to grow under simulated human colon conditions and (B) the ability to form biofilms under these conditions. Statistical analysis was performed using one-way ANOVA. Means with the same letter are not significantly different. [Figure 4]Figure 1 shows fibrinolytic activity results for the disclosed strain DSM34350 compared to other state-of-the-art strains described in the context of treating neurodegenerative diseases. A baseline halo diameter of 9 mm is equivalent to inactivity. Statistical analysis was performed using one-way ANOVA. Means with the same letter are not significantly different. DETAILED DESCRIPTION OF THE INVENTION

[0016] According to the present invention, a colon-specific delivery system is a delivery system that directly targets a substance or drug to the colon. The advantage of a colon-specific delivery system is its local effect in disorders such as ulcerative colitis, Crohn's disease, irritable bowel syndrome, and carcinoma. In these cases, targeted drug delivery to the colon ensures direct treatment at the site with lower dosage and fewer systemic side effects. In addition to local therapy, the colon can also be used as a portal vein entry point for drugs into the systemic circulation, and molecules that are poorly degraded / absorbed in the upper intestine, such as proteins and peptides, can be better absorbed from the more benign environment of the colon. Colon-specific drug delivery is believed to be beneficial in the treatment of colon-related diseases and the oral delivery of protein and peptide drugs. Generally, each colon-specific drug delivery system is designed based on one of the following mechanisms, with varying degrees of success: 1. pH-dependent polymer coating, 2. pH-independent biodegradable polymer coating, and 3. Delivery systems based on the metabolic activity of colonic bacteria.

[0017] Enteric coatings are barriers applied to oral medications to prevent their dissolution or disintegration in the gastric environment. Most enteric coatings function by providing a surface that is stable at the highly acidic pH found in the stomach but rapidly degrades at higher (alkaline) pHs. For example, they do not dissolve in the gastric acid of the stomach (pH approximately 3) but begin to dissolve in the environment present in the distal small intestine (the proximal pH range of the distal small intestine is approximately 5.6 to 7.4). Colon-targeted (drug) delivery systems are designed to selectively release drugs in response to the colonic environment without prematurely releasing the drug in the upper GI tract. Because the pH of the colon is relatively higher than that of the upper GI tract, intestine-specific delivery systems can include pH-dependent drug delivery systems. Thus, colon-targeted delivery systems are designed using pH-dependent polymers such as cellulose acetate phthalate (CAP), hydroxypropylmethylcellulose phthalate (HPMCP) 50 and 55, and copolymers of methacrylic acid and methyl methacrylate (e.g., Eudragit® S100, Eudragit® L, Eudragit® FS, and Eudragit® P4135F). Thus, in an advantageous configuration, the colon-specific delivery system preferably comprises a coating comprising at least one pH-dependent polymer or biodegradable polymer selected from methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, zein. It is preferable to use a polymer polymerized from 10 to 30% by mass of methyl methacrylate, 50 to 70% by mass of methyl acrylate, and 5 to 15% by mass of methacrylic acid as the coating. The disclosed polymer dispersion may preferably contain 15-50% by weight of a polymer polymerized from 20-30% by weight methyl methacrylate, 60-70% by weight methyl acrylate, and 8-12% by weight methacrylic acid, with the most preferred polymer being polymerized from 25% by weight methyl methacrylate, 65% by weight methyl acrylate, and 10% by weight methacrylic acid. A 30% by weight aqueous dispersion of a polymer polymerized from 25% by weight methyl methacrylate, 65% by weight methyl acrylate and 10% by weight methacrylic acid corresponds to the commercial product EUDRAGUARD® biologic. The ratio of monomers totals 100%. Functional polymers are 2-30mg / cm 2 , preferably 5-20 mg / cm 2is applied in an amount of

[0018] Another preferred subject in this context is the use of the pharmaceutical composition as defined above as a medicament.The present invention provides a therapeutic composition for use in the prevention or treatment of Alzheimer's disease. The compositions of the invention, in particular feed, food and pharmaceutical compositions as well as drinking or rearing water, preferably contain the strains of the invention and contain about 1 x 10 3 ~Approx. 2×10 12 CFU / g feed or ml water, specifically about 1 x 10 3 or approximately 1 x 10 4 or approximately 1 x 10 5 or approximately 1 x 10 6 or approximately 1 x 10 7 or approximately 1 x 10 8 or approximately 1 x 10 9 or approximately 1 x 10 10 or approximately 1 x 10 11 or approximately 1 x 10 12 CFU / g feed or ml water, preferably about 1 x 10 4 ~Approx. 1×10 10 CFU / g feed or ml water, more preferably 1 x 10 4 ~1×10 7 CFU / g feed or ml water is administered to animals. In this regard, preferred amounts of the strains and / or preparations of the invention in the feed, food and water compositions of the invention are preferably in the range of 0.1% to 10% by weight, more preferably 0.2% to 5% by weight, and especially 0.3% to 3% by weight. Food or feed compositions according to the present invention also include dietary supplements in the form of pills, capsules, tablets or liquids.

[0019] Furthermore, the present invention also relates to a method for improving the health, in particular the intestinal health, of a subject in need thereof, such as a human or an animal, comprising administering to said human or animal a Bacillus subtilis strain or formulation according to the present invention. The strains and preparations of the present invention can be obtained by culturing the strains of the present invention according to methods known in the art, including by using media and other methods such as those described in U.S. Pat. No. 6,060,051, European Patent No. 0287699, or U.S. Patent Application Publication No. 2014 / 0010792. Conventional large-scale microbial cultivation processes include submerged fermentation, solid-state fermentation, or liquid-surface culture. As nutrients become depleted toward the end of the fermentation, the cells of the strain begin to transition from the growth phase to the sporulation phase, resulting in the end product of fermentation consisting primarily of spores, metabolic products, and residual fermentation medium. Sporulation is part of the natural life cycle of these strains and is generally initiated by cells in response to nutrient limitation. Fermentation is configured to obtain a high level of colony-forming units of Bacillus subtilis cells and promote sporulation. The bacterial cells, spores, and metabolic products in the culture medium obtained from the fermentation can be used directly or can be concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, depth filtration, and evaporation. The concentrated fermentation broth can be washed, for example, via a dialysis process, to remove residual fermentation broth and metabolites. The fermentation broth or broth concentrate may be dried using conventional drying processes or methods, such as spray drying, freeze drying, tray drying, fluidized bed drying, drum drying, or evaporation, with or without the addition of a carrier. The resulting dried product may be further processed, such as by milling or granulation, to achieve a particular particle size or physical form. Carriers, such as those mentioned above, may also be added after drying.

[0020] Preparations of the strains of the invention may be cell-free preparations, or preparations containing cell debris or preparations containing a mixture of intact cells and cell debris. Cell-free preparations of the strains of the invention can be obtained, for example, by centrifugation and / or filtration of the fermentation broth. Depending on the technique used, these cell-free preparations may not be completely free of cells, but may contain a smaller number of cells. Since the cells secrete compounds such as metabolites, enzymes and / or peptides into the surrounding medium, the cell supernatant contains a mixture of such compounds, in particular metabolites, enzymes and / or peptides, secreted by the cells. Thus, in a preferred embodiment of the invention, the strain preparation is the supernatant of the fermentation broth.

[0021] Compositions containing cell debris of strains can be obtained by applying techniques known to those skilled in the art, for example, by applying mechanical means or high pressure to disrupt the cells. Depending on the level of force applied, compositions containing only disrupted cells or a mixture of cell debris and intact cells can be obtained. Cell homogenization can be achieved, for example, using a French cell press, ultrasonicator, homogenizer, microfluidizer, ball mill, rod mill, pebble mill, bead mill, high-pressure grinding roll, vertical shaft impactor, industrial blender, high-shear mixer, paddle mixer, and / or Polytron homogenizer. A suitable alternative is enzymatic and / or chemical treatment of the cells. The cell-free preparations of the present invention also include preparations obtained by first applying the above techniques to disrupt cells, followed by removal of cell debris and remaining intact cells, which may be achieved in particular by centrifugation and / or filtration.

[0022] Preparations of the strains of the invention may contain as active compounds at least one metabolite, preferably a mixture of metabolites, and / or at least one enzyme selected from proteases, in particular nattokinase, subtilisin, xylanase and / or cellulase, and / or at least one peptide, and / or combinations thereof.

[0023] Preparations containing effective mixtures of metabolites such as those contained in the strains of the present invention and / or the cell preparations described above can be obtained, for example, by the method described in U.S. Patent No. 6,060,051. In particular, the preparations can be obtained by precipitating the metabolites contained in the preparations using an organic solvent such as ethyl acetate, followed by redissolving the precipitated metabolites in a suitable solvent. The metabolites can then be purified by size exclusion filtration, which separates the metabolites into different fractions based on molecular weight cutoffs. Preferably, according to the present invention, an effective amount of the strains and / or preparations of the present invention is always used in embodiments of the present invention. The term "effective amount" refers to an amount that provides at least one beneficial effect to the animal and / or the environment, in particular with regard to the above-mentioned characteristics, compared to an animal receiving the same diet (including feed and other compounds) except for the strains and / or preparations and / or compositions of the present invention. For therapeutic applications, preferably, a therapeutic amount of the strains and / or preparations of the present invention is used. The term "therapeutic amount" refers to an amount sufficient to improve, reverse, or prevent a disease state in a human or 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 bacteria in the intestine at various doses, (ii) increase or maintain the level of beneficial bacteria, and / or (iii) promote human or animal health, particularly intestinal health, at various doses. [Example]

[0024] method 1. Biofilm Assay Substances produced by Bacillus subtilis DSM10 under biofilm formation have been shown to be associated with anti-aging effects, prolonged survival of C. elegans wild-type strains, and delayed onset of disease in C. elegans Alzheimer's mutants (Non-patent documents 1 and 4). Test strain B. subtilis DSM34350 and reference strain B. subtilis DSM10 were reactivated by spreading 50 μl of glycerol frozen stock onto tryptic soy agar (TSA) and incubating at 37° C. for 24 hours. Cell material from the plate was used to inoculate a preculture of 10 ml tryptic soy broth (TSB) in a 100 ml Erlenmeyer flask. The flask was incubated at 200 rpm and 37° C. for 16 hours. Biofilm assays were performed in 6.25 g / l Bacto tryptone (BD), 2.6 g / l D-glucose, 0.88 g / l NaCl, 2.7 g / l KHCO 3 , 0.43g / l KH2PO4, 1.7g / l NaHCO 3 The experiments were carried out under colonic conditions in a human simulated colon environment medium (SCEM) containing 4 g / L bile salt no. 3 and 4 g / L sucrose, as well as in TSB as a control. Precultures of the test strains were used to inoculate 200 μl of SCEM medium in 96-well plates in six replicates to an optical density (OD) of 0.2. The plates were incubated at 37°C without shaking to allow biofilm formation. After 24 h, absorbance at 600 nm was measured from each well using a TecanSpark instrument to determine general features of growth in each medium. The cultures were then removed from the wells by inverting the plate and shaking the contents into a waste container. The wells were washed twice by completely filling each well with sterile water, inverting the plate, and shaking the contents out. Biofilms that remained attached to the wells were stained by adding 250 μl of 0.1% crystal violet solution and incubating at room temperature (RT) for 30 min. All wells were then washed three or four times with sterile water until the staining stopped flowing. After drying the plates overnight at room temperature, 225 μl of 99.9% ethanol was added and incubated at room temperature for 15 minutes to degrade the stained biofilms. The absorbance of the stained biofilms was measured at 595 nm using a TecanSpark instrument (100 flashes, 100 ms rest time). If the absorbance exceeded the linear range of the instrument, the samples were diluted with ethanol and measured again.

[0025] 2. Fibrinolysis Assay A fibrinolysis assay was applied to evaluate the fibrinolytic activity of test strains under human colonic conditions using enzymes such as nattokinase, which is required to degrade protein plugs such as insoluble misfolded amyloid fibrils that accumulate in the brain and other organs of Alzheimer's patients (Non-Patent Documents 7 and 8). The solutions used were fibrinogen stock (0.6 g / 100 ml in 50 mM pH 7.4 sodium phosphate buffer), thrombin stock (B) (10 μg / ml in 50 mM pH 7.4 sodium phosphate buffer), agarose solution (2% agarose in 50 mM pH 7.4 sodium phosphate buffer), and plasmin stock (10 μg / ml in 100 mM sodium phosphate, 25% glycerol, pH 7.3). Fibrin agar plates were freshly prepared before each test by mixing 5 ml of fibrinogen stock, 5 ml of agarose solution, and 0.1 ml of thrombin stock in a Petri dish and allowing to polymerize at room temperature for 1 hour before use. The test strain Bacillus subtilis DSM34350 and the reference strain Bacillus subtilis DSM10 were reactivated by spreading 50 μl of glycerol frozen stock onto TSA and incubating at 37°C for 24 hours. Cell material from the plates was used to inoculate 10 ml SCEM in 100 ml Erlenmeyer flasks and 10 ml preculture in TSB as a control. The flasks were incubated at 200 rpm and 37°C for 16 hours. Cultures were diluted to an OD600 of 2 in SCEM, and 50 μl of the diluted culture was applied to 9 mm-diameter holes cut in fibrin agar. Pure SCEM served as a negative control, and 50 μl of plasmin stock served as a positive control. Plates were incubated for 24 hours, and the diameter of the clear halo was measured, which is proportional to the fibrinolytic activity of the test strain.

[0026] 3. Short-chain fatty acid (SCFA) production The production of SCFAs by the gut microbiota has been reported to be associated with diverse beneficial effects on the host and to regulate brain metabolic fitness (Non-Patent Document 6). A decrease in the amount of SCFAs correlates with the induction of inflammatory processes in the body and can affect the permeability of the blood-brain barrier, leading to neuroinflammation (Non-Patent Document 5). While butyrate and acetate have primarily been reported to exert beneficial effects on brain health, lactic acid produced by Bacillus subtilis strains can serve as a substrate for cross-feeding commensal bacteria to produce butyrate in the human intestine. Test strain Bacillus subtilis DSM34350 was reactivated by spreading 50 μl of a glycerol frozen stock onto TSA and incubating at 37°C for 24 hours. Cell material from the plate was used to inoculate a 10 ml preculture of TSB in a 100 ml Erlenmeyer flask. The flask was incubated at 200 rpm and 37°C for 16 hours. Short-chain fatty acid (SCFA) production of strain DSM34350 was tested in SCEM under human colonic conditions. Precultures of the test strains were used to inoculate 1000 μl of SCEM medium in 48-well plates in triplicate to an optical density (OD) of 0.2. Plates were incubated at 37°C, 400 rpm, and under anaerobic conditions for 26 hours. SCFA acetate and lactate were quantified by high-performance liquid chromatography.

[0027] 4. Analysis of anti-Alzheimer's effects in C. elegans The in vivo anti-Alzheimer's effects of B. subtilis DSM10 and DSM34350 were tested in the transgenic C. elegans strain GMC101. The mutant expresses the full-length human A-beta-1-42 peptide, which is responsible for the formation of extracellular insoluble amyloid plaques found in the brains of human Alzheimer's disease. The peptide is produced in the body wall muscle cells of the worm under the control of a temperature-sensitive promoter. Shifting larval stage 4 or young adult animals from 20°C to 25°C triggers peptide expression in the muscle cells, rapidly paralyzing (immobilizing) the worms. Strains were tested by culturing both B. subtilis strains and E. coli OP50, a standard non-probiotic C. elegans feeding strain, in TSB medium at 37°C for 24 hours (200 rpm) and harvesting by centrifugation at 3000 rpm for 15 minutes. For each probiotic, the supernatant was carefully removed and the resulting pellet was resuspended in S medium buffer (5.85 g / l NaCl, 1 g / l KHPO, 6 g KHPO, HO, 1 ml / L cholesterol (5 mg / ml in ethanol), 3 ml / l 1 M CaCl, 3 ml / l 1 M MgSO, 10 ml / l 1 M potassium citrate, 10 ml / l trace metals solution [1.86 g disodium EDTA, 0.69 g FeSO x 7 HO, 0.2 g MnCl x 4 HO, 0.29 g ZnSO x 7 HO, 0.025 g CuSO x 5 HO, make up to 1 L with HO], and 0.6 ml / l TWEEN® 20) to reach an OD600. 50 μl of the resuspended culture was plated onto nematode growth medium (NMG) agar containing 3 g / l NaCl, 2.5 g / l peptone, 20 g / l agar, 1 ml / l cholesterol (5 mg / mL in ethanol), 1 ml / l 1 M CaCl, 1 ml / l 1 M MgSO, and 25 ml / l 1 M (pH 6.0) KPO and allowed to dry overnight at room temperature. Worms were maintained at 20°C on NGM agar plates containing the bacterial strain of interest from the L1 to L4 larval stages. Worms were then transferred to probiotic or feeding strain plates and incubated at 25°C to induce amyloid expression. Every two days, nematode populations were transferred to freshly seeded plates containing the strain of interest. Five plates (n=5) with 10 worms per plate were tested per strain. Worm populations were observed once daily under a stereomicroscope. Before each motility monitoring, the plate containing the worms was tapped three times on the bench to ensure uniform stimulation among the populations. Following this initial stimulation, the following three parameters were monitored: - Spontaneous movement without further stimulation (the insect's motor function is observed for 30 seconds). If not observed; - Head or tail movement after stimulation (single light touch of the insect's claws on the head and tail). If not observed; - Paralysis (no response after 3 additional stimuli) was recorded continuously. Motility endpoints obtained daily for each plate containing 10 worms were used to calculate the percentage of worms exhibiting (1) spontaneous movement, (2) head / tail movement, and (3) paralysis. The percentages obtained for each condition were compared with the control (worms fed OP50 bacteria). Time point D0 corresponds to the time point immediately preceding population migration at 25°C; the entire population was considered 100% mobile at this point. In all experiments, statistical analysis was performed using a two-way analysis of variance (ANOVA-2) followed by Bonferroni's multiple comparison test. All P values ​​less than 0.05 were considered significant (*p<0.5; **p<0.01; ***p<0.001; ****p<0.0001 are symbols representing statistics according to Bonferroni's multiple test / $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols representing statistics according to ANOVA-2, which accounts for variation between conditions). GraphPad Prism5 was used for all statistical analyses.

[0028] 5. Identification of compounds with biofilm-enhancing effects Since the biofilm-forming function of B. subtilis was associated with anti-aging effects, supplementary ingredients with potential biofilm-enhancing effects were tested in strain DSM34350. Test strain Bacillus subtilis DSM34350 was reactivated by spreading 50 μl of a glycerol frozen stock onto tryptic soy agar (TSA) and incubating at 37°C for 24 hours. Cell material from the plate was used to inoculate a preculture of 10 ml tryptic soy broth (TSB) in a 100 ml Erlenmeyer flask. The flask was incubated at 200 rpm and 37°C for 16 hours. Biofilm assays were performed under colonic conditions in SCEM with and without the test substances manganese, thiamine, and phenylalanine in combination with tryptophan. To test the substances, 0.015–0.04 mg / ml manganese in the form of manganese sulfate tetrahydrate, 0.68 μg / ml thiamine in the form of thiamine × HCl, and a combination of 0.05 mg / ml each of phenylalanine and tryptophan were individually added to SCEM medium, and the biofilm-forming ability of each additive was examined compared to the SCEM control. Precultures of the test strains were used to inoculate 200 μl of each medium variation in 96-well plates in six replicates to an optical density (OD) of 0.2. The plates were incubated at 37°C without shaking to allow biofilm formation. After 24 hours, absorbance at 600 nm was measured from each well using a Tecan Spark instrument to determine the general ability of the strains to grow in each medium. The cultures were then removed from the wells by inverting the plate and shaking the contents into a waste container. The wells were washed twice by completely filling each well with sterile water, inverting the plate, and shaking the contents out. Any biofilms that remained attached to the wells were stained by adding 250 μl of 0.1% crystal violet solution and incubating at room temperature (RT) for 30 minutes. All wells were then washed three or four times with sterile water until the stain no longer washed away. After drying the plate overnight at room temperature, 225 μl of 99.9% ethanol was added and incubated at room temperature for 15 minutes to disintegrate the stained biofilms. The absorbance of the stained biofilms was measured at 595 nm in a Tecan Spark instrument (100 flashes, 100 ms rest time). If the absorbance exceeded the linear range of the instrument, the samples were diluted with ethanol and measured again.

[0029] 6. Comparison with other strains in the treatment of neurodegenerative diseases The following strains were tested under colonic conditions (SCEM medium) in the biofilm formation and fibrinolysis assays described above (1. and 2.) to compare the described strain DSM34350 with other state-of-the-art strains described in the context of the treatment of neurodegenerative diseases.

[0030] Table 1: Current strains of Bacillus subtilis (DSM10 and JH642) selected for comparison in fibrinolysis and biofilm formation assays with DSM34350, complemented by further benchmark strains with high genome-wide average nucleotide identity (gANI) (DSM1090, SMY, PY79, and DSM23778).

[0031] [Table 1] Biofilm assays were performed with six technical replicates, and fibrinolysis assays with three technical replicates. Statistical analysis was performed using one-way ANOVA.

[0032] result 1. Biofilm formation under colonic conditions A crystal violet-based biofilm assay was applied to test biofilm formation under colon-like conditions in both B. subtilis DSM10 and DSM34350. Both general growth in terms of absorbance at 600 nm and biofilm intensity after crystal violet staining were measured in TSB and SCEM media (Table 2). Both strains showed growth in both media, but only DSM34350 was able to establish a biofilm in SCEM medium. In complete medium (TSB), both strains built biofilms, but the biofilm intensity of DSM34350 was more than six-fold higher.

[0033] Table 2: Growth and biofilm formation of B. subtilis DSM34350 and B. subtilis DSM10 in TSB and SCEM media

[0034] [Table 2] Values ​​are the average of six replicates.

[0035] 2. Nattokinase activity under colonic conditions The fibrinolytic activity of enzymes such as nattokinase was tested under colon-like conditions using a plate-based fibrinolysis assay for both B. subtilis DSM10 and DSM34350. The mean clearance halo after 24 hours was measured for both strains grown in TSB as a control and in SCEM and is proportional to the fibrinolytic activity (Table 3). B. subtilis DSM34350 showed significantly higher fibrinolytic activity in both media than DSM10. The activity of B. subtilis DSM34350 in SCEM was increased compared to TSB, and the halo size was approximately 10 mm longer compared to B. subtilis DSM10.

[0036] Table 3: Nattokinase activity of B. subtilis DSM34350 and B. subtilis DSM10 in TSB and SCEM media as the average halo diameter produced on fibrin agar plates.

[0037] [Table 3] 3. Short-chain fatty acid (SCFA) production Bacillus subtilis DSM34350 was cultured in SCEM under anaerobic conditions, and production of the short-chain fatty acids acetate and DL-lactate was quantified after 26 hours. This strain was able to produce both SCFAs under conditions similar to the human colon, providing a potential substrate for cross-feeding to other commensal microorganisms in the human gut to produce butyrate (Table 4).

[0038] Table 4: Production of short-chain fatty acids acetate and DL-lactate by Bacillus subtilis DSM34350 under anaerobic conditions in SCEM.

[0039] [Table 4] 4. Analysis of anti-Alzheimer's effects in C. elegans The anti-Alzheimer's effects of B. subtilis DSM10 and B. subtilis DSM34350 were evaluated in the transgenic C. elegans strain GMC101, which expresses human amyloid-β in muscle cells and therefore develops increased paralysis and reduced spontaneous movement over time. Feeding GMC101 with B. subtilis DSM10 significantly (p<0.05) delayed overall paralysis compared to the non-probiotic strain E. coli OP50 (Figure 1A). B. subtilis DSM34350 delayed the progression of paralysis even more significantly (p<0.0001) (Figure 1B). At a single time point, the proportion of paralyzed worms was significantly reduced on day 6 with B. subtilis DSM10 and on days 4, 6, and 8 with B. subtilis DSM34350. After 8 days, fewer than 65% of worms were paralyzed when fed B. subtilis DSM34350, whereas 84% ​​of worms fed E. coli OP50 were paralyzed at the same age. Figure 1 shows the results of a paralysis test on C. elegans GMC101, which were maintained at 25°C for 8 days and fed E. coli OP50 starting at the L4 stage. The results were obtained by feeding A) B. subtilis DSM10 or B) B. subtilis DSM34350. Statistical analysis was performed using a two-way analysis of variance (ANOVA-2) followed by Bonferroni's multiple comparison test. All p values ​​less than 0.05 were considered significant (*p<0.5; **p<0.01; ***p<0.001; ****p<0.0001 are symbols representing statistical values ​​according to Bonferroni's multiple comparison test; $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols representing statistical values ​​according to ANOVA-2, which explains the variation between conditions). After two days, a complete cessation of spontaneous locomotion, recorded without stimulation of the worms, was found for the E. coli OP50-fed group (Figures 2A and 2B). B. subtilis DSM10 and B. subtilis DSM34350 showed a significant effect of delaying the inability to spontaneously move for 2 to 3 days. For both strains, a complete cessation of spontaneous locomotion was found only after 6 days. Significant differences for single time points were found up to day 2 for B. subtilis DSM10 and day 3 for B. subtilis DSM34350.

[0040] Figure 2 shows the results of spontaneous locomotor activity tests for C. elegans GMC101, maintained at 25°C for 8 days on a diet of E. coli OP50 starting at the L4 stage, when fed A) B. subtilis DSM10 or B) B. subtilis DSM34350. Statistical analysis was performed using a two-way analysis of variance (ANOVA-2) followed by Bonferroni's multiple comparison test. All p values ​​less than 0.05 were considered significant (*p<0.5; **p<0.01; ***p<0.001; ****p<0.0001 are symbols representing statistical values ​​according to Bonferroni's multiple comparison test; $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols representing statistical values ​​according to ANOVA-2, which explains the variation between conditions). A second ANOVA-2 (DSM34350 vs. DSM10) was performed to assess the significant differences between B. subtilis DSM10 and B. subtilis DSM34350 (Table 5). B. subtilis DSM34350 significantly improves worm motility when compared to B. subtilis DSM10 treatment.

[0041] Table 5: ANOVA-2 results comparing B. subtilis DSM34350 with B. subtilis DSM10

[0042] [Table 5] 5. Identification of compounds with biofilm-enhancing effects Because the biofilm-forming function of B. subtilis was associated with anti-aging effects, supplementary ingredients with potential biofilm-enhancing effects were tested in strain DSM 34350. Manganese, thiamine, and a combination of phenylalanine and tryptophan were investigated for their effects on DSM 34350 biofilm formation under colonic conditions during SCEM. When comparing the strength of biofilms established by DSM34350, increased values ​​were observed for all test substances and concentrations compared to the control without added substance (Table 6). For different concentrations of manganese, an inverse dose effect was observed: as less manganese was added, higher biofilm formation was induced. Thiamine at 0.05 mg / ml enhanced biofilm formation comparable to the lowest manganese concentration, while the combination of phenylalanine and tryptophan only slightly enhanced biofilm formation compared to the control.

[0043] Table 6: Growth and biofilm formation of Bacillus subtilis DSM34350 in SCEM medium with and without potential enhancing compounds.

[0044] [Table 6] Values ​​are the average of six replicates. In summary, both manganese and thiamine have been identified as suitable additives for inducing biofilm formation associated with anti-aging and anti-Alzheimer's effects in DSM34350 (Non-Patent Documents 1 and 4).

[0045] 6. Comparison with other strains in the treatment of neurodegenerative diseases To compare the described strain DSM34350 with other state-of-the-art strains described in the context of treating neurodegenerative diseases, the strains were tested in biofilm formation and fibrinolysis assays under colonic conditions (SCEM medium). Biofilm assays were performed with six technical replicates, and fibrinolysis assays with three technical replicates. The results are shown in Figures 3 and 4. Statistical analysis was performed using one-way ANOVA. Means with the same letter are not significantly different. Although other strains than the described strain DSM34350 were also able to grow under simulated colonic conditions (Fig. 3A), biofilm formation of DSM34350 was significantly higher compared to all other strains (Fig. 3B). The fibrinolytic activity of the disclosed strain DSM34350 was significantly higher under simulated colonic conditions compared to all other strains tested (Figure 4). References

[0046] [Table 7]

Claims

1. A Bacillus subtilis strain deposited at the Leibniz Institute DSMZ, German Collection of Microbial and Cell Cultures (DSMZ) under DSM 34350 or a preparation thereof.

2. 10. Use of the Bacillus subtilis strain or a preparation thereof according to claim 1 as a probiotic ingredient (DFM) in feed or food.

3. 10. A food or feed composition comprising the Bacillus subtilis strain or a preparation thereof according to claim 1 and at least one further feed or food ingredient selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, anticoccidial agents, acid-based products, pharmaceuticals and combinations thereof, or manganese or thiamine.

4. A pharmaceutical composition comprising the Bacillus subtilis strain of claim 1 or a preparation thereof and a pharmaceutically acceptable carrier.

5. 5. The composition of claim 3 or 4, further comprising a delayed release or a targeted release formulation for enteric or colonic release.

6. 6. The composition of claim 4 or 5 for use as a medicine.

7. 6. The composition of claim 4 or 5 for use in treating or preventing Alzheimer's disease.

8. A non-therapeutic method for improving the health or intestinal health of a subject in need thereof, comprising administering the Bacillus subtilis strain or a preparation thereof described in claim 1.

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