Bacillus subtilis strains having positive impact on neurodegenerative diseases such as alzheimer's disease

Abstract

The present invention relates to a novel Bacillus subtilis strain which has a positive effect on Alzheimer's disease.

Description

Field of the Invention

[0001] The present invention relates to a novel Bacillus subtilis strain and its use as a probiotic, which has a positive effect on Alzheimer's disease alone and in combination with enhancing substances. The present invention also relates to the use of the strain of the present invention as a food supplement and a pharmaceutical product. Background Art

[0002] Several risk factors for neurodegenerative diseases have been identified, such as obesity, cardiovascular damage, and diabetes. However, the risk of cognitive decline or developing neurodegenerative diseases such as Alzheimer's disease increases exponentially with age. As the population ages, this leads to a large number of people being affected by cognitive diseases. This increase is a challenge for patients, their families, and the healthcare system. It is well known that neurodegenerative diseases such as Alzheimer's disease and dementia can be detected in people 20 years before symptoms begin. Therefore, the focus is on reducing the risk factors for developing neurodegenerative diseases, thereby potentially preventing and / or curing such diseases.

[0003] New research finds a connection between the gut and the brain that may be influenced by the gut microbiota. The present invention relates to a new strain of Bacillus subtilis that has been shown to delay neurodegenerative decline and Alzheimer's disease state in different types of Caenorhabditis elegans.

[0004] It is documented in the prior art that Bacillus subtilis (NCIB3610 (DSM 10) and JH642) fed to N2, CF1038 and PS3551 types of C. elegans showed different life extension effects due to biofilm formation and production of nitric oxide and quorum sensing pentapeptide CSF compared to C. elegans fed with typical E. coli OP50 bacteria. When all of these genes (biofilm formation, NO, CSF) are inactivated, the life span of C. elegans is shortened (Donato, V et al., Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway. Nat. Commun. 8, 14332 doi: 10.1038 / ncomms14332 (2017)). Another publication studied the anti-Alzheimer's effect of Bacillus subtilis NCIB3610 (DSM 10) on certain types of Caenorhabditis elegans due to biofilm formation and the production of quorum sensing pentapeptide CSF. C. elegans mutants CL2120 and GMC101 expressing A-β protein in muscle cells of larvae fed with Bacillus subtilis showed that neuronal degeneration can be delayed, paralysis is slowed, and they perform better in behavioral tests. The same effect can be observed in C. elegans mutant CL2355 with pan-neuronally expressed A-β-protein (Cogliati, S et al. Bacillus Subtilis Delays Neurodegeneration and Behavioral Impairment in the Alzheimer's Disease Model Caenorhabditis Elegans. Journal of Alzheimer's disease: JAD 73 (3): 1-18 doi: 10.3233 / JAD-190837 (2019)). In addition, Bacillus subtilis strains NCIB3610 (DSM 10), 168 (DSM23778) and JH642 showed a protective effect against α-synuclein aggregation in the Caenorhabditis elegans mutant NL5901 expressing human α-synuclein (Goya, M et al. Probiotic Bacillus subtilis Protects against α-Synuclein Aggregation in C.elegans. Cell Rep, 30 (2), 367-380. e367.https: / / doi.org / 10.1016 / j.celrep.2019.12.078 (2020)).

[0005] The biofilm-producing ability of the Bacillus subtilis NCIB3610 (DSM 10) was tested only in MSgg medium, which promotes biofilm formation, and in the liquid standard medium NGM. In order to ensure biofilm formation and thus bacterial colonization of the probiotic ingredients, it is necessary to perform the tests under conditions similar to the colon. Summary of the invention

[0006] Therefore, the object of the present invention is to provide a probiotic strain capable of reducing the risk of developing neurodegenerative diseases through microbiome modulation under colon-like conditions.

[0007] The use of Bacillus subtilis strains as probiotic ingredients in the feed industry has previously been disclosed in the prior art. Probiotics (also known as "direct fed microorganisms" or "DFMs") function to influence the intestinal flora in a positive way by supporting the growth of beneficial bacteria and / or inhibiting the growth of pathogenic bacteria.

[0008] Many neurodegenerative diseases do not have a well-defined pathway in the body, but are multifactorial diseases. The present invention targets multiple pathways and shows positive impact and in situ production of several metabolites.

[0009] Surprisingly, the new Bacillus subtilis strain DSM 34350 also shows effective biofilm formation in human simulated colon environment medium (SCEM), while the Bacillus subtilis NCIB3610 (DSM10), 168 (DSM 23778) and JH642 recorded in the prior art have similar growth rates, but cannot build biofilm under human colon conditions. In addition, compared with the strain, the new strain shows significantly higher fibrinolytic activity under human colon conditions. Fibrinolytic enzymes (such as nattokinase) can decompose protein plaques accumulated in the brain and other organs of Alzheimer's patients, such as amyloid fibrils. Therefore, higher fibrinolytic activity is considered to be beneficial to prevent the formation of amyloid plaques.

[0010] Further tests in different types of C. elegans confirmed significant differences. It could be shown that paralysis of the C. elegans mutant GMC101 could be significantly delayed, meaning that lifespan could be extended compared to larvae fed either Escherichia coli OP50 or Bacillus subtilis NCIB3610 (DSM 10).

[0011] New insights were gained in another experiment in which Bacillus subtilis DSM 34350 was fed to C. elegans to study ageing-related neurodegeneration. Here, too, a significant lifespan-extending effect was observed.

[0012] Bacillus subtilis DSM 34350 was identified by targeted screening of naturally occurring isolates. It was deposited by Evonik Operations GmbH under the accession number mentioned above at the Leibniz-Institut DSMZ Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, 38124 Braunschweig, Germany on August 16, 2022 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. DETAILED DESCRIPTION

[0013] Therefore, a first subject of the present invention is a Bacillus subtilis strain deposited at the DSMZ as DSM 34350; or a preparation thereof.

[0014] The Bacillus subtilis strain deposited at the DSMZ as DSM 34350 exhibits the following characteristic sequences:

[0015] a) a 16S rDNA sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 1;

[0016] b) a yqfD sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 2;

[0017] c) a gyrB sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 3;

[0018] d) an rpoB sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 4;

[0019] e) a groEL sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO:5.

[0020] Therefore, another subject of the present invention is a Bacillus subtilis strain, in particular a Bacillus subtilis strain having the characteristics as mentioned above, or a preparation thereof, wherein the Bacillus subtilis strain exhibits at least one, preferably all, of the following characteristics:

[0021] a) a 16S rDNA sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 1;

[0022] b) a yqfD sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 2;

[0023] c) a gyrB sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 3;

[0024] d) an rpoB sequence having at least 99.5%, preferably at least 99.8%, and in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO: 4;

[0025] e) a groEL sequence having at least 99.5%, preferably at least 99.8%, in particular 100% sequence identity with the polynucleotide sequence shown in SEQ ID NO:5.

[0026] Therefore, a particular subject of the present invention is also a Bacillus subtilis strain which exhibits the following characteristics:

[0027] a) 16S rDNA sequence shown in SEQ ID NO: 1;

[0028] b) the yqfD sequence shown in SEQ ID NO: 2;

[0029] c) the gyrB sequence shown in SEQ ID NO: 3;

[0030] d) the rpoB sequence shown in SEQ ID NO:4;

[0031] e) The groEL sequence shown in SEQ ID NO:5.

[0032] The strains of the present invention are preferably characterized by at least one, more preferably all, of the following additional characteristics:

[0033] The strain is preferably capable of growing under anaerobic conditions. In addition, it is preferably capable of growing under human colon conditions.

[0034] The Bacillus subtilis strain is characterized by being able to establish a biofilm after 24 hours at 37°C under colonic conditions, the biofilm preferably having a biofilm strength of at least 0.1, as determined by measuring the absorbance at 565 nm.

[0035] Preferably, the biofilm is constructed in SCEM medium or TSB medium at pH 7.

[0036] In particular, the Bacillus subtilis strain is characterized by having fibrinolytic activity, preferably nattokinase activity, under colonic conditions, preferably determined by a mean empty circle diameter of at least 20 mm produced after 24 hours on fibrin agar plates.

[0037] Preferably, the Bacillus subtilis strain is characterized by being able to produce short chain fatty acids, preferably acetate, butyrate and lactate, under colonic conditions, preferably more than 0.1 g / l or more than 0.2 g / l after 26 hours.

[0038] Without wishing to be bound by any theory, it is believed that the Bacillus subtilis strain according to the present invention enhances human health, particularly intestinal health or mental health, through a multifaceted mode of action, including fibrinolytic activity and production of short chain fatty acids. Since many neurodegenerative diseases do not have a clear pathway in the body, but are multifactorial diseases, multiple pathways are targeted using the present invention, showing a positive impact and producing several metabolites in situ.

[0039] In a preferred embodiment of the present invention, the strains and preparations of the present invention are administered orally to animals or humans.

[0040] Therefore, another subject of the present invention is a composition, such as feed, food, drinking and feeding water and therapeutic composition, comprising the Bacillus subtilis strain according to the invention and / or its preparations.

[0041] Another subject of the present invention is also the use of the Bacillus subtilis strain and / or the preparation according to the invention as a probiotic ingredient (DFM) in feed or food products.

[0042] Preferred foods according to the invention are dairy products, in particular yoghurt, cheese, milk, butter and quark.

[0043] In particular, in the composition of the present invention, the cells of the strain of the present invention may be present as spores (dormant), vegetative cells (growing), transitional cells (transitioning from the growth phase to the sporulation phase) or a combination of at least two of these cell types, in particular as a combination of all these types of cells. In a preferred embodiment, the composition of the present invention mainly comprises spores or only comprises spores.

[0044] Additionally, or as an alternative, the cells of the strain may also be used in a non-viable, killed form, since it is expected that non-viable cells also still have a probiotic effect. Methods for killing cells are known to those skilled in the art.

[0045] The methods and uses of the strains and preparations of the invention may be therapeutic or non-therapeutic.

[0046] Another subject of the present invention is a food or feed composition containing the Bacillus subtilis strain according to the invention or a preparation thereof and at least one other feed or food ingredient selected from the group consisting of proteins, carbohydrates, fats, other probiotics, prebiotics, enzymes, vitamins, immunomodulators, milk replacers, minerals, amino acids, anticoccidial agents, acid-based products, drugs and combinations thereof, preferably manganese or thiamine.

[0047] Therefore, another subject of the present invention is also a pharmaceutical composition, which comprises the strain and / or preparation of the present invention as mentioned above, and a pharmaceutically acceptable carrier.

[0048] A particular subject of the present invention is also a method for enhancing the health of a human 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 flora in a human, wherein the strain and / or the preparation according to the invention is administered to the human.

[0049] Therefore, another subject of the present invention is also the use of the strains and / or preparations according to the invention for enhancing human health and / or improving the general physical condition of humans and / or increasing disease resistance in humans and / or increasing the immune response in humans and / or establishing or maintaining a healthy intestinal flora in humans, wherein the strains and / or preparations according to the invention are administered to humans.

[0050] Another aspect of the present invention relates to a pharmaceutical or non-pharmaceutical composition, which further comprises a targeted release formulation for delayed release or enteric release or colonic release. The targeted release formulation according to the present invention is a formulation that ensures that the components of the preparation according to the present invention are delivered to specific targets in the body. Preferred formulations of this type of preparation promote intestinal or colonic delivery in the lower small intestine or large intestine. The targeted release formulation 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.

[0051] According to the present invention, colon-specific delivery system is a delivery system that directly targets a substance or drug to the colon. The advantage of colon-specific delivery system is that it has a local effect in the case of conditions such as ulcerative colitis, Crohn's disease, irritable bowel syndrome and cancer. In these cases, targeted delivery of drugs to the colon ensures direct treatment at this site with lower dosages and less systemic side effects. In addition to local treatment, the colon can also be used as an entrance for drugs to enter the systemic circulation, such as molecules (such as proteins and peptides) that are degraded / poorly absorbed in the upper intestinal tract can be better absorbed by the milder environment of the colon. Colon-specific drug delivery is considered to be beneficial for the treatment of colon-related diseases and oral delivery of protein and peptide drugs. Typically, each colon-specific drug delivery system is designed based on one of the following mechanisms (with varying degrees of success): 1. coating with pH-dependent polymers, 2. coating with biodegradable polymers that are not dependent on pH, and 3. delivery systems based on colon bacterial metabolic activity.

[0052] Enteric coating is a barrier applied to oral drugs to prevent them from dissolving or disintegrating in the gastric environment. Most enteric coatings work by providing a surface that is stable at the strongly acidic pH in the stomach but rapidly decomposes at a higher pH (alkaline pH). For example, they will not dissolve in the gastric acid (pH ~ 3) of the stomach, but they will begin to dissolve in the environment of the distal small intestine (pH range of proximal to distal small intestine is ~ 5.6 to 7.4). Colon-targeted (drug) delivery systems are designed to selectively release drugs in response to the colon environment without prematurely releasing drugs in the upper gastrointestinal (GI) tract.

[0053] Colon-specific delivery systems may include pH-dependent drug delivery systems because the pH of the colon is relatively higher than that of the upper gastrointestinal (GI) tract. Therefore, colon-targeted delivery systems are designed by using pH-dependent polymers such as cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP) 50 and 55, copolymers of methacrylic acid and methyl methacrylate (e.g., S100, L. FS and P4135F).

[0054] Therefore, in an advantageous configuration, the colon-specific delivery system comprises a coating comprising at least one pH-dependent polymer or biodegradable polymer, which is preferably selected from methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, cellulose acetate trimellitate, sodium alginate, and zein.

[0055] As coating, a polymer obtained by polymerizing 10 to 30% by weight of methyl methacrylate, 50 to 70% by weight of methyl acrylate and 5 to 15% by weight of methacrylic acid is preferably used.

[0056] The disclosed polymer dispersion may preferably contain 15 to 50 wt% of a polymer polymerized from 20 to 30 wt% of methyl methacrylate, 60 to 70 wt% of methyl acrylate, and 8 to 12 wt% of methacrylic acid. The most preferred polymer is polymerized from 25 wt% of methyl methacrylate, 65 wt% of methyl acrylate, and 10 wt% of methacrylic acid.

[0057] A 30% by weight aqueous dispersion of a polymer obtained by polymerization of 25% by weight of methyl methacrylate, 65% by weight of methyl acrylate and 10% by weight of methacrylic acid corresponds to the commercial product biotic.

[0058] The percentage of monomers totals 100%. The amount of functional polymer applied is 2-30 mg / cm 2 , preferably 5-20 mg / cm 2 .

[0059] In this context, another preferred subject matter is the use of the pharmaceutical composition as described above as a medicament. The present invention provides a therapeutic composition for preventing or treating Alzheimer's disease.

[0060] The composition of the present invention, in particular feed, food and pharmaceutical composition and drinking water or feeding water, preferably comprises the strain of the present invention and is present in an amount of about 1×10 3 About 2×10 12 CFU / g feed or CFU / ml water are administered to animals at a rate of about 1×10 3 or about 1×10 4 or about 1×10 5 or about 1×10 6 or about 1×10 7 or about 1×10 8or about 1×10 9 or about 1×10 10 or about 1×10 11 or about 1×10 12 CFU / g feed or CFU / ml water are administered to animals at a rate of preferably about 1×10 4 About 1×10 10 CFU / g feed or CFU / ml water are administered to animals, more preferably 1×10 4 Up to 1×10 7 The amount of CFU / g feed or CFU / ml water was administered to the animals.

[0061] Accordingly, the preferred amount of the strains and / or preparations of the invention in the feed, food and water compositions of the invention preferably ranges from 0.1% to 10% by weight, more preferably from 0.2% to 5% by weight, in particular from 0.3% to 3% by weight.

[0062] The food or feed composition according to the invention also comprises a dietary supplement in the form of a pill, capsule, tablet or liquid.

[0063] In addition, the present invention also relates to a method for improving the health status of a subject in need thereof, such as a human or an animal, in particular the intestinal health status, comprising administering the Bacillus subtilis strain or preparation according to the present invention to the human or the animal.

[0064] The bacterial strain of the present invention and preparation can be obtained by culturing the bacterial strain of the present invention according to methods well known in the art, including by using culture medium and other methods as described in, for example, US 6,060,051, EP0287699 or US2014 / 0010792. Conventional large-scale microbial culture methods include deep fermentation, solid-state fermentation or liquid surface culture. When the fermentation is close to the end, as the nutrients are exhausted, the cells of the bacterial strain begin to transition from the growth phase to the sporulation phase, so that the final product of the fermentation is mainly spores, metabolites and residual fermentation medium. Spore formation is part of the natural life cycle of these bacterial strains, usually initiated by cell response to nutrient restriction. Fermentation is configured to obtain a high level of Bacillus subtilis cells of colony forming units and promote sporulation. The bacterial cells, spores and metabolites in the culture medium produced by fermentation can be used directly, or concentrated by conventional industrial methods such as centrifugation, tangential flow filtration, deep filtration and evaporation. The concentrated fermented broth can be washed, for example, by a diafiltration process to remove residual fermented broth and metabolites.

[0065] The fermentation broth or fermentation broth concentrate can 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 dry product can be further processed, for example by grinding or granulation, to obtain a specific particle size or physical form. The carrier as described above can also be added after drying.

[0066] The preparation of the strain of the present invention may be a cell-free preparation, or a preparation containing cell fragments, or a preparation containing a mixture of intact cells and cell fragments.

[0067] The cell-free preparation of bacterial strain of the present invention can be obtained for example by centrifugation and / or filtration of fermentation liquid.According to the technology used, these cell-free preparations may not be completely without cells, but may still comprise a relatively small amount of cells.Because the cell secretes compounds such as metabolites, enzymes and / or peptides into the surrounding culture medium, the supernatant of the cell comprises a mixture of such compounds, particularly metabolites, enzymes and / or peptides secreted by the cells.Therefore, in a preferred embodiment of the present invention, the prepared product of the bacterial strain is the supernatant of fermentation liquid.

[0068] The composition of the cell fragments comprising the bacterial strain can be obtained by applying technology known to those skilled in the art to make cell rupture, for example, by mechanical means or by applying high pressure. According to the degree of applied force, a composition comprising only ruptured cells or a composition comprising a mixture of cell fragments and intact cells is obtained. The homogenization of the cell can be achieved, for example, by utilizing a French cell crusher, an ultrasonic disruptor (sonicator), a homogenizer, a microfluidizer, a ball mill, a rod mill, a gravel mill, a bead mill, a high-pressure roller mill, a vertical shaft impactor, an industrial mixer, a high shear mixer, a paddle mixer and / or a polytron homogenizer. A suitable alternative is an enzyme and / or chemical treatment of the cell.

[0069] The cell-free preparations of the invention also include preparations obtained by first disrupting the cells by applying the techniques as described above and subsequently removing the cell debris and remaining intact cells. The removal of cell debris and remaining intact cells can be performed in particular by centrifugation and / or filtration.

[0070] The preparation of the strain of the invention may comprise as active compound at least one metabolite, preferably a mixture of metabolites, and / or at least one enzyme, and / or at least one peptide, and / or a combination thereof, the enzyme being selected from proteases, in particular nattokinase, subtilisin, xylanase and / or cellulase.

[0071] For example, a preparation containing an effective metabolite mixture as contained in the strain of the present invention and / or as contained in the cell preparation as mentioned above can be obtained according to the method described in U.S. Patent No. 6,060,051. In particular, the preparation can be obtained by precipitating the metabolites contained in the preparation as mentioned above using an organic solvent such as ethyl acetate, and then re-dissolving the precipitated metabolites in an appropriate solvent. The metabolites can then be purified by size exclusion filtration, which groups the metabolites into different fractions based on the molecular weight cutoff.

[0072] According to the invention it is always preferred that in embodiments of the invention an effective amount of the strain and / or preparation of the invention is used. The term "effective amount" refers to an amount which produces at least one beneficial effect on the animal and / or the environment compared to an animal which has not been administered the strain and / or preparation and / or composition of the invention but has otherwise been administered the same diet (including feed and other compounds), in particular with regard to the characteristics already mentioned above.

[0073] In the case of therapeutic applications, it is preferred to use a therapeutic amount of the strain and / or preparation of the present invention. The term "therapeutic amount" refers to an amount sufficient to improve, reverse or prevent a disease state in a human or animal. One skilled in the art can easily determine the optimal dosage level for various animals by evaluating, in particular, the ability of the composition to (i) inhibit or reduce pathogenic bacteria in the intestine at different doses, (ii) increase or maintain the level of beneficial bacteria, and / or (iii) enhance the health of a human or animal, particularly intestinal health, at different doses.

[0074] Example

[0075] method

[0076] 1. Biofilm Assay

[0077] Substances produced by Bacillus subtilis DSM10 under biofilm formation have been linked to anti-aging effects and have been shown to be associated with life extension in wild-type strains of Caenorhabditis elegans and delayed onset of Alzheimer's disease mutants in Caenorhabditis elegans (Donato, Ayala et al., 2017; Cogliati, Clementi et al., 2020).

[0078] Test strain Bacillus subtilis DSM 34350 and reference strain Bacillus subtilis DSM10 were revitalized by plating 50 μl of glycerol cryo stock on tryptone soy agar (TSA) and incubating at 37° C. for 24 hours. Cell material from the plates was used to inoculate a pre-culture of 10 ml of tryptone soy broth (TSB) in a 100 ml Erlenmeyer culture bottle. The culture bottle was incubated at 200 rpm and 37° C. for 16 hours.

[0079] Biofilm assays were performed under colonic conditions in human simulated colonic environment medium (SCEM) and in TSB as a control, containing 6.25 g / l Bacto tryptone (BD), 2.6 g / l D-glucose, 0.88 g / l NaCl, 2.7 g / l KHCO3, 0.43 g / l KH2PO4, 1.7 g / l NaHCO3 and 4 g / l No. 3 bile salts, pH 7. 200 μl SCEM medium in 96-well plates was inoculated with a preculture of the test strain to an optical density OD600 of 0.2 in 6 replicates. The plates were incubated at 37°C without shaking to allow biofilm formation. After 24 hours, the absorbance at 600 nm of each well was measured on a Tecan Spark device to determine the general growth capacity in the corresponding medium. The culture is then removed from the wells by inverting the plate and pouring the contents into a waste container. The wells are washed twice by filling each well completely with sterile water, inverting the plate, and pouring out the contents. The biofilm still attached to the wells is stained by adding 250 μl of 0.1% crystal violet solution and incubating for 30 minutes at room temperature (RT). Afterwards, all wells are washed three or four times with sterile water until the staining can no longer be washed off. The plate is allowed to dry overnight at room temperature, and then the stained biofilm is dissolved by adding 225 μl of 99.9% ethanol and incubating for 15 minutes at RT. In the TecanSpark device, the absorbance of the stained biofilm is measured at 595 nm (100 flashes, 100 ms interval). If the absorbance exceeds the linear range of the device, the sample is diluted in ethanol and measured again.

[0080] 2. Fibrinolysis Assay

[0081] The fibrinolytic assay is used to evaluate the fibrinolytic activity of enzymes (such as nattokinase) of the test strains under human colonic conditions. Fibrinolytic activity is required for the breakdown of protein plugs, which are insoluble, misfolded amyloid fibrils that accumulate in the brain and other organs of patients with Alzheimer's disease (Hsu, Lee et al., 2009; Fadl, Ahmed et al., 2013).

[0082] The solutions used were fibrinogen stock solution (0.6 g / 100 ml in 50 mM sodium phosphate buffer at pH 7.4), thrombin stock solution (10 μg / ml in 50 mM sodium phosphate buffer at pH 7.4), agarose solution (2% agarose in 50 mM sodium phosphate buffer at pH 7.4) and plasmin stock solution (10 μg / ml in 100 mM sodium phosphate, 25% glycerol at pH 7.3). Before each test, 5 ml of fibrinogen stock solution, 5 ml of agarose solution and 0.1 ml of thrombin stock solution were mixed in a petri dish and allowed to polymerize at room temperature for 1 hour before use to prepare a fresh fibrin agar plate.

[0083] Test strain Bacillus subtilis DSM 34350 and control strain Bacillus subtilis DSM10 were revitalized by coating 50 μl of glycerol cryogenic stock on TSA and incubating at 37°C for 24 hours. Pre-culture medium of 10 ml SCEM and 10 ml TSB (as control) in 100 ml Erlenmeyer culture bottle was inoculated with cell material from the plate. The culture bottle was incubated at 200 rpm and 37°C for 16 hours. The culture was diluted to OD 600 of 2 in SCEM, and 50 μl of the diluted culture was applied to a hole of 9 mm in diameter cut into fibrin agar. Pure SCEM was used as a negative control, and 50 μl of plasmin stock was used as a positive control. The plate was incubated for 24 hours, and the diameter of the transparent empty circle was measured, which is directly proportional to the fibrinolytic activity of the test strain.

[0084] 3. Short-chain fatty acids (SCFA) production

[0085] The production of SCFAs by the gut microbiota is associated with multiple beneficial effects on the host and has also been described to regulate metabolic adaptability of the brain (Erny, Dokalis et al., 2021). Reduced amounts of SCFAs have been associated with the induction of inflammatory processes in vivo, thereby affecting the permeability of the blood-brain barrier and leading to neuroinflammation (Eicher and Mohajeri 2022). Mainly butyrate and acetate have been documented to have positive effects on brain health, while lactate produced by Bacillus subtilis strains can serve as a substrate for cross-feeding commensal bacteria to produce butyrate in the human gut.

[0086] The test strain DSM34350 was rejuvenated by plating 50 μl of glycerol cryostock on TSA and incubating at 37°C for 24 hours. Cell material from the plate was used to inoculate a pre-culture of 10 ml of TSB in a 100 ml Erlenmeyer flask. The flask was incubated at 200 rpm and 37°C for 16 hours.

[0087] The short chain fatty acid (SCFA) production of strain DSM 34350 was tested in SCEM under human colon conditions. 1000 μl SCEM medium in 48-well plates was inoculated with a pre-culture of the test strain to an optical density OD 600 of 0.2 in triplicate. The plates were incubated at 37°C and 400 rpm under anaerobic conditions for 26 hours. SCFA acetic acid and lactic acid were quantified by high performance liquid chromatography.

[0088] 4. Analysis of Anti-Alzheimer's Disease Effects in Caenorhabditis elegans

[0089] The in vivo anti-Alzheimer's effects of Bacillus subtilis DSM10 and DSM 34350 were tested in the transgenic C. elegans line GMC101. This mutant expresses the full-length human A-beta-1-42 peptide, which constructs the extracellular insoluble amyloid plaques found in human Alzheimer's disease brains. The peptide is produced in the body wall muscle cells of the larvae and is under the control of a temperature-sensitive promoter. Shifting stage 4 larvae or young adult animals from 20°C to 25°C results in expression of the peptide in the muscle cells and rapid paralysis (inability to move) of the larvae.

[0090] For testing strains, Bacillus subtilis strains and Escherichia coli OP50 (a standard non-probiotic C. elegans food strain) were cultured in TSB medium at 37°C for 24 hours (200 rpm) and harvested by centrifugation at 3000 rpm for 15 minutes. For each probiotic, the supernatant was carefully removed and the pellet was resuspended in S medium buffer (5.85 g / l NaCl, 1 g / l K2HPO4, 6 g KH2PO4, H2O, 1 m / L cholesterol (5 mg / ml in ethanol), 3 ml / l 1 M CaCl2, 3 ml / l 1 M MgSO4, 10 ml / l 1 M potassium citrate, 10 ml / l trace metal solution [1.86 g disodium EDTA, 0.69 g FeSO4×7H2O, 0.2 g MnCl2×4H2O, 0.29 g ZnSO4×7H2O, 0.025 g CuSO4×5H2O, add H2O to 1 L] and 0.6 ml / l TWEEN 20) to reach OD600. 50 μl of the resuspended culture was plated on 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 CaCl2, 1 ml / l 1 M MgSO4 and 25 ml / l 1 M (pH 6.0) KPO4 and allowed to dry overnight at room temperature.

[0091] Larvae were maintained on NGM agar plates containing the target bacterial strain from the L1 larval stage to the L4 larval stage at 20°C. Larvae were then transferred to probiotic or feeding strain plates and incubated at 25°C to induce amyloid expression. Larval colonies were transferred to newly inoculated plates containing the target strain every other day. Five plates (n=5) were tested for each strain, with 10 larvae per plate.

[0092] The larval population was observed once a day under a stereomicroscope. Before each motility monitoring, the plate containing the larvae was tapped three times on the laboratory bench to ensure uniform stimulation in the population. After this first stimulation, three parameters were continuously recorded:

[0093] Spontaneous movement in the absence of further stimulation (within 30 seconds, the larva is observed to have the ability to move). If not observed:

[0094] · Head or tail irritation (gently touch the head and tail once with a larval pick)

[0095] If no:

[0096] Paralysis (no response after three additional stimuli)

[0097] The motility endpoints obtained daily for each plate containing 10 larvae were used to calculate the percentage of larvae showing (1) spontaneous movement, (2) head / tail movement, (3) paralysis. The percentages obtained in each condition were compared with the control (larvae fed with OP50 bacteria). The time point D0 corresponds to the time point just before the transfer of the population at 25°C: at this time point, the entire population was considered to be 100% motile. In all experiments, statistical analysis was performed by 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 statistical values ​​after bonferroni multiple test / $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols representing statistical values ​​after ANOVA-2, both of which explain the variation between conditions). GraphPad Prism 5 was used for all statistical analyses.

[0098] 5. Identification of compounds with biofilm-enhancing effects

[0099] Since the biofilm-forming ability of Bacillus subtilis has been associated with anti-aging effects, supplemental ingredients with potential biofilm-enhancing effects in strain DSM34350 were tested.

[0100] The test strain Bacillus subtilis DSM 34350 was rejuvenated by plating 50 μl of a glycerol cryostock on Tryptone Soy Agar (TSA) and incubating at 37° C. for 24 hours. Cell material from the plate was used to inoculate a pre-culture of 10 ml of Tryptone Soy Broth (TSB) in a 100 ml Erlenmeyer flask. The flask was incubated at 200 rpm and 37° C. for 16 hours.

[0101] For biofilms, the assays were performed in SCEM under colonic conditions with and without the test substances manganese, thiamine, and a combination of phenylalanine and tryptophan. To test the substances, 0.015-0.04 mg / ml of manganese in the form of manganese sulfate tetrahydrate, 0.68 μg / ml of thiamine in the form of thiamine × HCl, and a combination of phenylalanine and tryptophan at 0.05 mg / ml each were added individually to the SCEM medium and the biofilm forming capacity of each additive compared to the SCEM control was considered.

[0102] 200 μl of each culture medium variant in a 96-well plate was inoculated with a preculture of the test strain to an optical density OD600 of 0.2, in 6 replicates. The plate was incubated at 37 ° C without shaking to allow biofilm formation. After 24 hours, the absorbance at 600 nm of each well was measured on a Tecan Spark device to determine the general growth capacity in the corresponding culture medium. The culture was then removed from the well by inverting the plate and pouring the contents into a waste container. The wells were washed twice by filling each well completely with sterile water, inverting the plate, and pouring out the contents. The biofilm still attached to the well was stained by adding 250 μl of 0.1% crystal violet solution and incubating at room temperature (RT) for 30 minutes. Afterwards, all wells were washed three or four times with sterile water until the staining could no longer be washed off. The plate was allowed to dry overnight at room temperature, and the stained biofilm was then dissolved by adding 225 μl of 99.9% ethanol and incubating at RT for 15 minutes. The absorbance of the stained biofilm was measured at 595 nm in a TecanSpark device (100 flashes, 100 ms interval). If the absorbance exceeded the linear range of the device, the sample was diluted in ethanol and measured again.

[0103] 6. Comparison with other strains in the context of neurodegenerative disease treatment

[0104] In order to compare the strain DSM 34350 according to the invention with other prior art strains described in the context of the treatment of neurodegenerative diseases, the following strains were tested in the above-mentioned biofilm formation and fibrinolysis assays (1. and 2.) under colonic conditions (SCEM medium):

[0105] Table 1: Prior art strains of Bacillus subtilis (DSM10 and JH642) were selected for comparison with DSM 34350 in fibrinolysis and biofilm formation assays, supplemented with other benchmark strains with high genome-wide average nucleotide identity (gANI) (DSM1090, SMY, PY79 and DSM23778).

[0106]

[0107] The biofilm assay was performed with 6 technical replicates and the fibrinolysis assay was performed with 3 technical replicates. Statistical analysis was performed by using one-way ANOVA.

[0108] result

[0109] 1. Biofilm formation under colonic conditions

[0110] Bacillus subtilis DSM10 and DSM 34350 were tested for biofilm formation under colon-like conditions using a crystal violet-based biofilm assay. General growth, expressed as absorbance at 600 nm, and biofilm intensity after crystal violet staining were measured in TSB and SCEM media (Table 2). Although both strains showed growth in both media, only DSM 34350 was able to establish biofilms in SCEM media. In complete media (TSB), both strains built biofilms, but DSM 34350 made the biofilm intensity more than 6 times higher.

[0111] Table 2: Growth and biofilm formation of Bacillus subtilis DSM 34350 and Bacillus subtilis DSM 10 in TSB and SCEM media. Values ​​are the average of 6 replicates.

[0112]

[0113] 2. Nattokinase activity under colonic conditions

[0114] The fibrinolytic activity of enzymes (such as nattokinase) of Bacillus subtilis DSM 10 and DSM 34350 was tested under colon-like conditions by using a plate-based fibrinolytic assay. The average clearance of the two strains grown in TSB as a control and in SCEM after 24 hours was measured, which is proportional to the fibrinolytic activity (Table 3). The fibrinolytic activity of Bacillus subtilis DSM 34350 in both media was significantly higher than that of DSM10. In SCEM, the activity of Bacillus subtilis DSM 34350 increased compared to that in TSB, and resulted in an increase in the size of the empty circle by nearly 10 mm compared to that of Bacillus subtilis DSM10.

[0115] Table 3: Nattokinase activity of Bacillus subtilis DSM 34350 and Bacillus subtilis DSM10 in TSB and SCEM medium, expressed as the average diameter of the empty circle produced on fibrin agar plates.

[0116]

[0117] 3. Produce short-chain fatty acids (SCFA)

[0118] Bacillus subtilis DSM 34350 was cultivated under anaerobic conditions in SCEM and the production of the short-chain fatty acids acetate and DL-lactic acid was quantified after 26 h. The strain was able to produce both SCFAs under conditions similar to those in the human colon, thus providing potential substrates for other commensal microorganisms that cross-feed in the human gut to produce butyrate (Table 4).

[0119] Table 4: Production of short chain fatty acids acetic acid and DL-lactic acid by Bacillus subtilis DSM 34350 in SCEM under anaerobic conditions

[0120]

[0121] 4. Analysis of Anti-Alzheimer's Disease Effects in Caenorhabditis elegans

[0122] The anti-Alzheimer's disease effects of Bacillus subtilis DSM10 and Bacillus subtilis DSM 34350 were evaluated in the transgenic C. elegans strain GMC101, which expresses human amyloid-β in muscle cells, which results in increased paralysis and decreased spontaneous movement over time.

[0123] Compared with the non-probiotic strain Escherichia coli OP50, feeding GMC101 with Bacillus subtilis DSM10 significantly (p < 0.05) delayed global paralysis ( Figure 1 A). For Bacillus subtilis DSM 34350, the paralysis progression was found to be more significantly delayed (p<0.0001) ( Figure 1 B). For individual time points, a significant reduction in the percentage of paralyzed larvae was found for Bacillus subtilis DSM10 at day 6 and for Bacillus subtilis DSM 34350 at days 4, 6 and 8. After 8 days, less than 65% of the larvae fed with Bacillus subtilis DSM 34350 were paralyzed, while 84% of the larvae fed with E. coli OP50 were paralyzed at the same age.

[0124] Figure 1Results of paralysis assays of C. elegans GMC101 fed with E. coli OP50 compared to C. elegans GMC101 fed with A) Bacillus subtilis DSM10 or B) Bacillus subtilis DSM 34350 maintained at 25°C for 8 days starting from larval stage L4 are shown. Statistical analysis was performed using two-way analysis of variance (ANOVA-2) followed by Bonferroni 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 indicating statistical values ​​after Bonferroni multiple test / $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols indicating statistical values ​​after ANOVA-2, both of which account for variations between conditions).

[0125] Two days later, it was found that the spontaneous movement of the E. coli OP50-fed group was completely stopped ( Figure 2 A and B), which were recorded without any stimulation of the larvae. Both Bacillus subtilis DSM10 and Bacillus subtilis DSM 34350 showed a significant effect, delaying the loss of spontaneous movement by two to three days. Only after 6 days was a complete cessation of spontaneous movement found for both strains. Significant differences at a single time point were not found until the second day for Bacillus subtilis DSM10 and not until the third day for Bacillus subtilis DSM 34350.

[0126] Figure 2 The results of spontaneous movement assays of C. elegans GMC101 fed with E. coli OP50 compared with C. elegans GMC101 fed with B. subtilis DSM10 or B. subtilis DSM 34350 maintained at 25°C for 8 days starting from larval stage L4 are shown. Statistical analysis was performed by using two-way analysis of variance (ANOVA-2) followed by Bonferroni 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 indicating statistical values ​​after Bonferroni multiple test / $p<0.5; $$p<0.01; $$$p<0.001; $$$$p<0.0001 are symbols indicating statistical values ​​after ANOVA-2, both of which account for the variation between conditions).

[0127] To evaluate the significant differences between Bacillus subtilis DSM10 and Bacillus subtilis DSM 34350, a second ANOVA-2 (DSM 34350 vs. DSM10) was performed (Table 5). Bacillus subtilis DSM 34350 significantly improved the motility of larvae compared to Bacillus subtilis DSM10 treatment.

[0128] Table 5: ANOVA-2 results comparing Bacillus subtilis DSM 34350 and Bacillus subtilis DSM10

[0129]

[0130] 5. Identification of compounds with biofilm-enhancing effects

[0131] Since the biofilm forming ability of Bacillus subtilis has been associated with anti-aging effects, supplemental ingredients were tested for potential biofilm enhancing effects in strain DSM 34350. The effects of the substances manganese, thiamine, and a combination of phenylalanine and tryptophan on biofilm formation of DSM 34350 under colonic conditions in SCEM were investigated.

[0132] When comparing the strength of the biofilms constructed with DSM 34350, an increase in the values ​​was observed for all tested substances and concentrations compared to the control without added substances (Table 6). For the different concentrations of manganese, an inverse dose effect was observed: with decreasing added manganese, higher biofilm formation was triggered. 0.05 mg / ml thiamine resulted in an increase in biofilm formation comparable to the lowest concentration of manganese, while the combination of phenylalanine and tryptophan only slightly increased biofilm formation compared to the control.

[0133] Table 6: Growth and biofilm formation of Bacillus subtilis DSM 34350 in SCEM medium with and without potential enhancing compounds. Values ​​are the mean of 6 replicates.

[0134]

[0135] In summary, both manganese and thiamine were identified as suitable additives to trigger biofilm formation in DSM 34350, which is associated with anti-aging and anti-Alzheimer's disease effects (Donato, Ayala et al., 2017; Cogliati, Clementi et al., 2020).

[0136] 6. Comparison with other strains in the context of neurodegenerative disease treatment

[0137] In order to compare the strain DSM 34350 according to the present invention with other prior art strains described in the context of the treatment of neurodegenerative diseases, the strains were tested in biofilm formation and fibrinolysis assays under colonic conditions (SCEM medium). The biofilm assay was performed with 6 technical replicates and the fibrinolysis assay was performed with 3 technical replicates. The results are shown in Table 1. Figure 3 and Figure 4 Statistical analysis was performed using one-way ANOVA. Means with the same letters are not significantly different.

[0138] Although strains other than the strain DSM 34350 described in the present invention can also grow under simulated colon conditions ( Figure 3 A), but DSM 34350 showed significantly more biofilm formation compared to all other strains ( Figure 3 B).

[0139] Compared to all other tested strains, the strain DSM 34350 according to the present invention showed significantly higher fibrinolytic activity under simulated colonic conditions ( Figure 4 ).

[0140] References

[0141] Cogliati,S.,V.Clementi,M.Francisco,C.Crespo,F. and R.Grau(2020)."Bacillus subtilis Delays Neurodegeneration and Behavioral Impairmentin the Alzheimer's Disease Model Caenorhabditis Elegans."JAlzheimers Dis 73(3):1035-1052.

[0142] Donato,V.,FRAyala,S.Cogliati,C.Bauman,JGCosta,C. andR.Grau(2017). "Bacillus subtilis biofilm extends Caenorhabditis eleganslongevity through downregulation of the insulin-like signaling pathway." Nature Communications 8(1):14332.

[0143] Eicher,T.P.and M.H.Mohajeri(2022)."Overlapping Mechanisms of Actionof Brain-Active Bacteria and Bacterial Metabolites in the Pathogenesis ofCommon Brain Diseases."Nutrients 14(13):2661.

[0144] Goya,M.E.,et al.(2020)."Probiotic Bacillus subtilis Protects againstα-Synuclein Aggregation in C.elegans."Cell Rep 30(2):367-380.e367.

[0145] Erny,D.,N.Dokalis,C. A.Castoldi,O.Mossad,O.Staszewski,M.Frosch,M.Villa,V.Fuchs,A.Mayer,J.Neuber,J.Sosat,S.Tholen,O.Schilling,A.Vlachos,T.Blank,M.Gomez de Agüero,A.J.Macpherson,E.J.Pearce and M.Prinz(2021)."Microbiota-derived acetate enables the metabolic fitness of the brain innateimmune system during health and disease."Cell Metabolism33(11):2260-2276.e2267.

[0146] Fadl,N.N.,H.H.Ahmed,H.F.Booles and A.H.Sayed(2013)."Serrapeptase andnattokinase intervention for relieving Alzheimer's disease pathophysiology inrat model."Hum Exp Toxicol 32(7):721-735.

[0147] Hsu,R.L.,K.T.Lee,J.H.Wang,L.Y.Lee and R.P.Chen(2009)."Amyloid-degrading ability of nattokinase from Bacillus subtilis natto."J Agric FoodChem 57(2):503-508。

Claims

1. A Bacillus subtilis strain deposited with the DSMZ as DSM 34350 or a preparation thereof.

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

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

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

5. The composition according to any one of claims 3 or 4, further comprising a targeted release formulation for delayed release or intestinal release or colonic release.

6. A composition according to any one of claims 4 or 5 for use as a medicament.

7. The composition according to any one of claims 4 or 5, for use in treating or preventing Alzheimer's disease.

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

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