Food- or feedstuff composition with effect on neurodegenerative diseases

Abstract

The present invention relates to a food- or feedstuff composition comprising a B. subtilis strain and a Ginkgo biloba extract, such composition for use as a medicament as well as such composition for use in treating or preventing neurodegenerative diseases, in particular Alzheimer's disease as well as the use of such composition as a food supplement.

Description

[0001] Food- or Feedstuff Composition with Effect on Neurodegenerative Diseases

[0002] The present invention relates to a food- or feedstuff composition comprising a B. subtilis strain and a Ginkgo biloba extract, such composition for use as a medicament as well as such composition for use in treating or preventing neurodegenerative diseases, in particular Alzheimer's disease as well as the use of such composition as a food supplement.

[0003] There are several risk factors which have been identified for neurodegenerative diseases such as obesity, cardiovascular impairment, and diabetes. However, when getting older the risk of cognitive decline or developing a neurodegenerative disease like Alzheimer’s disease is increasing exponentially. With an aging population this is leading to a significant number of people affected by cognitive diseases. This increase is a challenge for the patients, their families, and the health care system. It is known that neurodegenerative diseases such as Alzheimer’s disease and dementia can be found in people already 20 years before symptoms start. Therefore, a focus is on the reduction of risk factors developing neurodegenerative diseases and by that have the potential to prevent and / or cure such diseases.

[0004] New developments have found a connection between the gut and the brain that can be influenced by the gut microflora. The current invention concerns a composition comprising a B. subtilis strain and a Ginkgo biloba extract that shows effects in postponing neurodegenerative decline and the Alzheimer’s status in Caenorhabditis elegans.

[0005] In the state of art it is described that B. subtilis (NCIB3610 (DSM 10) and JH642) alone fed to C. elegans types N2, CF1038 and PS3551 showed different life prolonging effects compared to C. elegans fed with typical E. coli OP50 bacteria due to biofilm formation and the production of nitric oxide and the quorumsensing pentapeptide CSF. When all of these genes (biofilm formation, NO, CSF) were deactivated the lifespan of C. elegans was decreased (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 researched on the Anti-Alzheimer’s effects of B. subtilis NCIB3610 (DSM 10) on certain C. elegans types due to biofilm formation and the production of the quorum-sensing pentapeptide CSF. C. elegans mutants CL2120 and GMC101 that express A-beta- proteins in the muscle cells of the worms fed with the B. subtilis showed delayed neuronal deterioration, slower paralysis and performed better in behavioral tests. Equal effects could be observed in the C. elegans mutant CL2355 with pan-neuronal expressed A-beta-proteins (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)). Furthermore, B. subtilis strains NCIB3610 (DSM 10), 168 (DSM 23778) and JH642 showed a protective effect against a-synuclein aggregation in the C. elegans mutant NL5901 , expressing human a-synuclein (Goya, M et al. Probiotic Bacillus subtilis Protects against a-Synuclein Aggregation in C. elegans. Cell Rep, 30(2), 367- 38O.e367. https: / / doi.Org / 10.1016 / j.celrep.2019.12.078 (2020)). The biofilm producing capability of the described B. subtilis NCIB3610 (DSM 10) strain was tested in the biofilm promoting MSgg medium as well as in a liquid standard medium NGM only. To ensure the biofilm formation and therefore the bacterial colonization of a probiotic ingredient it needed to be tested under colon similar conditions. Therefore, it is necessary to provide a probiotic strain, which is able to reduce the risk to develop neurodegenerative diseases by microbiome modulation under colon similar conditions.

[0006] The use of B. subtilis strains as probiotic ingredient in the feed industry has been disclosed before in the state of the art. The function of probiotics (also called “direct-fed microbials” or “DFM”) is to influence the gut microflora in a positive way by supporting the growth of beneficial bacteria and / or the suppression of the growth of pathogenic bacteria.

[0007] Many neurodegenerative diseases do not have one clear pathway in the body but are multifactorial disease. The invention is targeting multiple pathways and shows a positive influence and an in-situ production of several metabolites.

[0008] The B. subtilis strain DSM 34350 strain showed efficient biofilm formation in human simulated-colonic- environment medium (SCEM), whereas the B. subtilis NCIB3610 (DSM 10), 168 (DSM 23778) and JH642 described by the state of the art had a similar growth rate but were not able to build a biofilm under human colon conditions. Also, the B. subtilis strain DSM 34350 strain showed significantly higher fibrinolytic enzyme activity under human colon conditions compared to the described strains. Fibrinolytic enzymes such as Nattokinase are able to break down protein plaques such as amyloid fibrils that accumulate in the brain and other organs of Alzheimer patients. Therefore, a higher fibrinolytic activity is thought to be beneficial concerning the prevention of amyloid plaque formation.

[0009] Further tests in different types of C. elegans confirmed significant differences. It could be shown that paralysis in C. elegans mutant GMC101 could be significantly postponed, that means the lifespan could be prolonged compared to worms eating E. coli OP50 or B. subtilis NCIB3610 (DSM 10).

[0010] New insights could be gained in another experiment where B. subtilis DSM 34350 was fed to C. elegans to examine aging related neurodegeneration. Here also significant life prolonging effects could be observed.

[0011] Bacillus subtilis DSM 34350 has been identified by targeted screening of naturally occurring isolates. It has been deposited with the Leibniz-lnstitut 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 Purpose of Patent Procedure under the Accession Number DSM 34350 in the name of Evonik Operations GmbH.

[0012] The Bacillus subtilis strain as deposited under DSM 34350 at the DSMZ exhibits the following characterizing sequences: a) a 16S rDNA sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 1 ; b) a yqfD sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 2; c) a gyrB sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 3; d) an rpoB sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 4; e) a groEL sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 5.

[0013] The Bacillus subtilis strain DSM 34350 is preferably able to grow under anaerobic conditions. Further, it is preferably able to grow under human colon conditions.

[0014] The Bacillus subtilis strain DSM 34350 is characterized by being able to build a biofilm under colon conditions after 24h at 37°C, preferably with a biofilm intensity of at least 0.1 , determined by measurement of absorption at 565 nm. It is preferred, when the biofilm is built in SCEM medium or in TSB medium at pH 7.

[0015] In particular, the Bacillus subtilis strain DSM 34350 is characterized by having a fibrinolytic activity under colon conditions, preferably a nattokinase activity, preferably determined by an average halo diameter of at least 20 mm produced on fibrin agar plates after 24 h.

[0016] The Bacillus subtilis strain DSM 34350 is also characterized by an ability to produce short chain fatty acids under colon conditions, preferably acetate, butyrate and lactate, preferably more than 0.1 g / l or more than 0.2 g / l after 26 h.

[0017] The objective of the present invention to provide an improved food- or feedstuff composition comprising among other a probiotic strain, which is able to reduce the risk to develop neurodegenerative diseases by microbiome modulation under colon similar conditions.

[0018] Therefore, the present invention concerns a composition comprising a Bacillus subtilis strain DSM 34350 as described before and a Ginkgo biloba extract.

[0019] The cells of the Bacillus subtilis strain DSM 34350 may be present in the compositions of the current invention as spores which are dormant, as vegetative cells which are growing, as transition state cells which are transitioning from growth phase to sporulation phase, or as a combination of at least two, in particular all of these types of cells. In a preferred embodiment, the composition of the current invention comprises mainly or only spores.

[0020] The Ginkgo biloba extract comprised in the composition according to the present invention essentially comprises flavone glycosides and terpene lactones, in particular 18 - 27% by weight flavone glycosides and 5 - 7 % by weight terpene lactones, preferably 24% flavone glycosides and 6% terpene lactones. The flavone glycosides primarily are quercetin, kaempferol and isorhamnetin; the terpene lactones essentially comprise 2.8-3.4% ginkgolides A, B and C, and 2.6-3.2% bilobalide.

[0021] The composition according to the present invention preferably comprises between 1x108and 1x1011CFU (Colony Forming Units) of the probiotic Bacillus strain and Gingko extract ranging from 50-500 mg. In a further embodiment of the present invention the composition comprises between 1-100 mg of the probiotic Bacillus strain and Gingko extract ranging from 50-500 mg.

[0022] CFU (Colony Forming Units) is determined according to DIN EN ISO 4833-2 (May 2014).

[0023] The composition according to the present invention may be comprised in a capsule containing a daily dose of 1x108to 1x1010CFU Bacillus strain corresponding to 1-40 mg Bacillus strain powder and 100 - 300 mg Gingko extract.

[0024] The composition according to the present invention may further comprise at least one feed or food ingredient selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immune modulators, milk replacers, minerals, amino acids, coccidiostats, acid-based products, medicines, and combinations thereof, preferably manganese and thiamin.

[0025] The methods and uses of the composition of the present invention may be therapeutic or non-therapeutic.

[0026] A further aspect of the present invention is the inventive composition as described before for use as a medicament or medical food or supplement, in particular for use in treating or preventing neurodegenerative disorders and diseases. The neurodegenerative disease may be Alzheimer's disease.

[0027] The inventive composition as described before may also be used as a food- or feedstuff composition or as a functional food ingredient.

[0028] In a preferred embodiment of the current invention, the composition of the present invention is administered orally to animals or human beings.

[0029] Thus, a further subject of the current invention are compositions, such as feedstuffs, foodstuffs, drinking and rearing water as well as therapeutic compositions, comprising the B. subtilis strain DSM 34350 and a Ginkgo biloba extract according to the current invention. A further subject of the current invention is also the use the composition of the current invention as a probiotic ingredient (DFM) in feed or food products. Preferred foodstuffs are dairy products, in particular yoghurt, cheese, milk, butter and quark.

[0030] The cells of the strain B. subtilis DSM 34350 may be present, in particular in the compositions of the current invention, as spores (which are dormant), as vegetative cells (which are growing), as transition state cells (which are transitioning from growth phase to sporulation phase) or as a combination of at least two, in particular all of these types of cells. In a preferred embodiment, the composition of the current invention comprises mainly or only spores.

[0031] Another aspect of the present invention is directed to a pharmaceutical or non-pharmaceutical composition, in particular a dosage form comprising the composition according to the present invention which further comprises a targeted-release formulation for delayed release or enteric or colonic release. A targeted-release formulation may be a formulation which ensures the delivery of the composition according to the present invention to a specific target in the body. A preferred formulation of such preparations promotes enteral or colonic delivery in the lower small intestine or in the large intestine. The targeted-release formulation can be obtained by adding enteric polymers to the matrix of the dosage form, or by adding a coating to the dosage form, preferably an enteric coating.

[0032] A colon-specific delivery system is a delivery system, which targets the substance or drug directly to the colon. The advantage of a colon-specific delivery system is the local action, in case of disorders like ulcerative colitis, Crohn’s disease, irritable bowel syndrome, and carcinomas. Targeted drug delivery to the colon in these cases ensures direct treatment at the site with lower dosing and fewer systemic side effects. In addition to local therapy colon can also be utilized as the portal entry of the drugs into systemic circulation for example molecules that are degraded / poorly absorbed in upper gut such as proteins and peptides may be better absorbed from the more benign environment of the colon. Colon-specific drug delivery is considered beneficial in the treatment of colon-related diseases and the oral delivery of protein and peptide drugs. Generally, each colon-specific drug delivery system has been designed based on one of the following mechanisms with varying degrees of success; 1 . Coating with pH dependent polymers, 2. Coating with pH independent biodegradable polymers and 3. Delivery systems based on the metabolic activity of colonic bacteria.

[0033] An enteric coating is a barrier applied on oral medication that prevents its dissolution or disintegration in the gastric environment. Most enteric coatings work by presenting a surface that is stable at the intensely acidic pH found in the stomach but breaks down rapidly at a higher pH (alkaline pH). For example, they will not dissolve in the gastric acids of the stomach (pH ~3), but they will start to dissolve in the environment present in the distal small intestine (pH range proximal to distal small intestine is ~5.6 to 7.4). Colon targeted (drug) delivery systems are designed to selectively release a drug in response to the colonic environment without premature drug release in the upper Gl tract.

[0034] The colon-specific delivery system can comprise a pH-dependent drug delivery system, since the colon exhibits a relatively higher pH than the upper Gl tract. Accordingly, a colon-targeted delivery system is designed by using pH-dependent polymers such as cellulose acetate phthalates (CAP), hydroxypropyl methyl-cellulose phthalate (HPMCP) 50 and 55, copolymers of methacrylic acid and methyl methacrylate

[0035] (e.g., Eudragit® S 100, Eudragit® L, Eudragit® FS, and Eudragit® P4135 F).

[0036] Therefore, in an advantageous configuration, the colon-specific delivery system comprises a coating comprising at least one pH dependent polymer or biodegradable polymer, preferably selected from methyl acrylate-methacrylic acid copolymers, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, shellac, cellulose acetate trimellitate, sodium alginate, zein.

[0037] As a coating it is preferred to use a polymer polymerized from 10 to 30 % by weight methyl methacrylate, 50 to 70 % by weight methyl acrylate and 5 to 15 % by weight methacrylic acid.

[0038] The polymer dispersion may preferably comprise 15 to 50 % by weight of a polymer polymerized from 20 to 30 % by weight methyl methacrylate, 60 to 70 % by weight methyl acrylate and 8 to 12 % by weight methacrylic acid. Most preferred the polymer is polymerized from 25 % by weight methyl methacrylate, 65 % by weight methyl acrylate and 10 % by weight methacrylic acid.

[0039] 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® biotic.

[0040] The percentages of the monomers add up to 100 %. The functional polymer is applied in amounts of 2- 30 mg / cm2, preferably 5-20 mg / cm2.

[0041] The food- or feedstuff composition according to the present invention does also include dietary supplements in the form of a pill, capsule, tablet or liquid.

[0042] The strain Bacillus subtilis DSM 34350 may be obtained by culturing according to methods well known in the art, including by using the media and other methods as described for example in US 6,060,051 , EP0287699 or US2014 / 0010792. Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, the cells of the strains begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of these strains and is generally initiated by the cell in response to nutrient limitation. Fermentation is configured to obtain high levels of colony forming units of the Bacillus subtilis cells and to promote sporulation. The bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation. The concentrated fermentation broth may be washed, for example via a diafiltration process, to remove residual fermentation broth and metabolites.

[0043] The fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation. The resulting dry products may be further processed, such as by milling or granulation, to achieve a specific particle size or physical format. Carriers, as described above, may also be added post-drying. Preparations of the strain of the current invention may be cell-free preparations or preparations containing cell debris or preparations containing a mixture of intact cells and cell debris.

[0044] Cell-free preparations of the Bacillus subtilis strain can be obtained for example by centrifugation and / or filtration of fermentation broth. Depending on the technique used, these cell-free preparations may not be completely devoid of cells, but may still comprise a smaller amount of cells. As the cells secret compounds like metabolites, enzymes and / or peptides into the surrounding medium, the supernatant of the cells comprises a mixture of such compounds, in particular metabolites, enzymes and / or peptides, as secreted by the cells. Thus, in a preferred embodiment of the invention, the preparation of the strains is a supernatant of the fermentation broth.

[0045] Compositions comprising cell debris of the strains may be obtained by rupturing the cells applying techniques as known to those of skill in the art, for example by mechanical means or by applying high pressure. Depending on the degree of feree applied, a composition comprising only ruptured cells or a composition comprising a mixture of cell debris and intact cells is obtained. Homogenization of the cells may be realized for example by utilizing a French cell press, sonicator, 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. Suitable alternatives are enzymatic and / or chemical treatment of the cells.

[0046] Cell-free preparations comprise also preparations which are obtained by first rupturing the cells by applying techniques as mentioned before and subsequently removing the cell debris and the remaining intact cells. Removing of the cell debris and remaining intact cells can be carried out in particular by centrifugation and / or filtration.

[0047] Description of Figure 1

[0048] Figure 1 shows the age-dependent development of neurodegenerative damage graphically and statistically evaluated. It shows in particular the proportion of morphological abnormalities (damage) of the cholinergic neurons of the nematodes. Synchronized nematodes of the LX929 strain were fed with E. coli OP50, B. subtilis DSM10, B. subtilis DSM 34350 ± Ginkgo Preparation (Ginkgo Preparation; 0.83 mg / ml), or B. subtilis 168 ± Ginkgo Preparation (Ginkgo Preparation; 0.83 mg / ml) from the L1 arrest (day 0). The morphology of the cholinergic neurons was determined on days 3, 5, 10, and 15. For evaluation, a classification based on morphology was performed into the categories healthy (normal, a), mild (mild, b), and severe (severe, c) signs of neurodegeneration, (d) The neurodegeneration index is intended to graphically represent the severity of the morphological abnormalities. For this purpose, fixed numerical values were assigned to the described categories (normal=0, mild= 1 , and severe=2), and the mean of the sum of the factors of the frequency of occurrence and the numerical value was calculated. The mean values from three independent experiments are shown. The statistical analysis was performed using oneway ANOVA with Dunnett's multiple comparison test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 vs. B. subtilis 168. Description of the Sequences

[0049] SEQ ID NO: 1 16S rDNA of B. subtilis DSM 34350;

[0050] SEQ ID NO: 2 yqfD gene of B. subtilis DSM 34350;

[0051] SEQ ID NO: 3 gyrB gene of B. subtilis DSM 34350;

[0052] SEQ ID NO: 4 rpoB gene of B. subtilis DSM 34350;

[0053] SEQ ID NO: 5 groEL gene of B. subtilis DSM 34350.

[0054] Experimental Part

[0055] Materials and Methods

[0056] To prove beneficial effect of combining a gingko preparation with a probiotic Bacillus subtilis on reducing cholinergic neurodegeneration in vivo, Caenorhabditis elegans was chosen as a model organism. Different B. subtilis strains with and without the ginkgo preparation were fed to the nematodes and the degeneration their neuros were monitored.

[0057] Test strains and products

[0058] The C. elegans strain LX929, which expresses green fluorescent protein (GFP) in cholinergic neurons, was obtained from the Caenorhabditis Genetics Center (CGC; University of Minnesota, USA).

[0059] The standard bacterial feeding strain E. coli strain OP50 was also obtained from the CGC. The B. subtilis control strains DSM10 and 168 were obtained from the DSMZ and tested together with the probiotic strain B. subtilis DSM 34350. Bacillus subtilis DSM 34350 has been deposited with the Leibniz-I nstitut 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 Purpose of Patent Procedure under the Accession Number DSM 34350 in the name of Evonik Operations GmbH.

[0060] The Ginkgo biloba extract used for experiments contains approximately 24% flavone glycosides (primarily quercetin, kaempferol and isorhamnetin) and 6% terpene lactones (2.8-3.4% ginkgolides A, B and C, and 2.6-3.2% bilobalide). A powdered gingko preparation was solved in sterile water and applied together with the individual probiotic strains.

[0061] Preparation of bacterial cultures

[0062] Bacterial cultures were grown in LB medium at 37°C and 100 rpm for 24 hours. NGM agar plates were inoculated with bacteria at an OD600 of ~1 and used for feeding C. elegans during the experiment.

[0063] Preparation of C. elegans For experiments, seven C. elegans larvae in L4 state were transferred to a 90 mm NGM agar plate with 1 ml OP50. After 5 days, eggs were collected and synchronized. Eggs were washed with distilled water, treated with a sodium hypochlorite solution to avoid bacterial contamination, and washed again. The eggs were then held in a salt buffer with added magnesium chloride to allow hatching of larvae but to prevent further development (L1 arrest). After 24 hours, the larvae were washed and transferred to treatment plates. The nematodes underwent three more larval stages (L2-L4) before reaching adulthood. After 48 hours, the nematodes reached the L4 stage and were sexually mature. After another 24 hours, the nematodes were adults, and the dendrites and axons were clearly visible.

[0064] Both the incubation chamber, where the nematodes are kept, and the laboratory, where the worms are manipulated and analyzed, maintain a constant temperature of 20°C. All chemicals, solutions, and consumables used have either been purchased as sterile or have been sterilized by heat / steam pressure.

[0065] Investigation of products on cholinergic neurodegeneration

[0066] For the experiments, the nematodes were divided into six groups and fed with B. subtilis control strains DSM10 and 168 (± gingko preparation (0.83 mg / ml)), or the probiotic B. subtilis strain DSM 34350 (± gingko preparation (0.83 mg / ml)) starting from L1 -stage (day 0). Microscopic images to monitor degeneration of the GFP-labelled cholinergic neurons were taken on days 3 (first day of the adult stage), 5, 10, and 15 after L1 arrest.

[0067] The analyses were performed using fluorescence microscopy from the third day after L1 arrest. For each treatment, approximately 30 nematodes were anesthetized with sodium azide and transferred to an object slide. The GFP signal was detected using a filter pair for 472 / 30 nm (excitation) and 520 / 35 nm (emission). The images were taken in the area between the pharynx and vulva of the nematodes, where typically 3-5 nerve strands were found. The nematodes were randomly chosen.

[0068] Each product and strain were tested in three independent experiments.

[0069] Analysis

[0070] The analysis of the images was performed by grouping the nerve strands into three categories based on their morphological appearance in the fluorescence microscope. The categories were healthy or normal nerve strands, nerve strands with knots, and nerve strands with severe and advanced degeneration characterized by a break or tear.

[0071] A neurodegenerative index was calculated for transforming the data for graphical representation. Fixed numerical values were assigned to the described categories (normal=0, mild= 1 , and severe=2), and the mean of the sum of the factors of frequency of occurrence and numerical value was calculated. in which xO = number of healthy nerve strands at day n x1(n) = number of mildly degenerated strands at day n x2(n) = number of severely degenerated strands at day n

[0072] The statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001 vs. B. subtills 168.

[0073] Results

[0074] The average neurodegenerative index over the three independent replicates is depicted in Table 1 with the standard deviation over the replicates following in Table 2. The statistical analysis was performed in comparison to the group of nematodes fed with the control strain B. subtills 168 and results are shown in Table 3. Figure 1 shows the age-dependent development of neurodegenerative damage graphically and statistically evaluated.

[0075] Based on the neurodegeneration index the statistically significant neuroprotective effect of feeding with the probiotic strain DSM 34350 is clearly demonstrated; this was observed at all time points considered. Supplementation with the ginkgo preparation resulted in a lower damage of the GFP-labeled cholinergic neurons when supplemented with both B. subtills 168 and DSM 34350 compared to each B. subtills alone.

[0076] Supplementation with the Ginkgo blloba preparation, which is postulated to have a preventive effect against age-associated cognitive impairments, resulted in a further reduction of neurodegenerative damage in the nematodes. Interestingly, this neuroprotective effect of ginkgo was observed both when fed with the B. subtills control strain 168 and when fed with the already neuroprotective bacterial strain B. subtills DSM 34350 (Table 1). This suggests differences in the molecular mechanisms of probiotic B. subtills bacteria and Ginkgo blloba preparations that underlie the observed neuroprotective effects and confirm the benefit of combining both probiotic B. subtills and ginkgo.

[0077]

[0078] Table 2: Standard deviation of Neurodegenerative Index over three independent replicates determined by the morphology of the cholinergic neurons on days 3, 5, 10, and 15 in synchronized nematodes of the

[0079] LX929 strain fed with B. subtilis DSM10, B. subtilis DSM 34350 ± Ginkgo Preparation or B. subtilis 168 ±

[0080] Ginkgo Preparation from the L 1 arrest. ? | |

[0081] Table 3: Statistical significance of differences in Neurodegenerative index in comparison to B. subtilis 168. Statistical analysis was performed using one-way ANOVA with Dun nett's ultiple comparison test.

[0082] *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 vs. B. subtilis 168. | | j References

[0083] Cogliati, S., V. Clementi, M. Francisco, C. Crespo, F. Arganaraz and R. Grau (2020). "Bacillus subtilis Delays Neurodegeneration and Behavioral Impairment in the Alzheimer's Disease Model Caenorhabditis Elegans." J Alzheimers Dis 73(3): 1035-1052.

[0084] Donato, V., F. R. Ayala, S. Cogliati, C. Bauman, J. G. Costa, C. Lenini and R. Grau (2017). "Bacillus subtilis biofilm extends Caenorhabditis elegans longevity through downregulation of the insulin-like signalling pathway." Nature Communications 8(1): 14332.

[0085] Eicher, T. P. and M. H. Mohajeri (2022). "Overlapping Mechanisms of Action of Brain-Active Bacteria and Bacterial Metabolites in the Pathogenesis of Common Brain Diseases." Nutrients 14(13): 2661.

[0086] Goya, M. E., et al. (2020). "Probiotic Bacillus subtilis Protects against a-Synuclein Aggregation in C. elegans." Cell Rep 30(2): 367-380. e367.

[0087] Erny, D., N. Dokalis, C. Mezo, 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 Aguero, A. J. Macpherson, E. J. Pearce and M. Prinz (2021). "Microbiota-derived acetate enables the metabolic fitness of the brain innate immune system during health and disease." Cell Metabolism 33(11): 2260- 2276.e2267.

[0088] Fadi, N. N., H. H. Ahmed, H. F. Booles and A. H. Sayed (2013). "Serrapeptase and nattokinase intervention for relieving Alzheimer's disease pathophysiology in rat model." Hum Exp Toxicol 32(7): 721- 735.

[0089] 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 Food Chem 57(2): 503-508.

Claims

Claims1 . A composition comprising a Bacillus subtilis strain DSM 34350 and a Ginkgo biloba extract.

2. The composition of claim 1 , wherein the Ginkgo biloba extract essentially comprises flavone glycosides and terpene lactones.

3. The composition of claims 1 or 2, further comprising at least one feed or food ingredient selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immune modulators, milk replacers, minerals, amino acids, coccidiostats, acid-based products, medicines, and combinations thereof, manganese and thiamin.

4. The composition of any of the preceding claims, wherein the Bacillus subtilis strain DSM 34350 is present between of 1x108and 1x1011CFU (Colony Forming Units).

5. The composition of any of the preceding claims, wherein the Gingko extract is present between from 50-500 mg.

6. The composition of any of the claims 1 to 5, wherein the Bacillus subtilis strain DSM 34350 is present between 1-100 mg and the Gingko extract is present between from 50-500 mg.

7. The composition of any of the preceding claims for use as a medicament or medical food or supplement.

8. The composition of any of claims 1 to 6 for use in treating or preventing neurodegenerative disorders and diseases.

9. The composition of claim8, wherein the neurodegenerative disease is Alzheimer's disease.

10. A use of the composition of any of claims 1 to 6 as a food- or feedstuff composition.

11. A use of the composition of any of claims 1 to 6 as functional food ingredient.

12. A capsule comprising a composition according to any of claims 1 to 6.

13. A method of treating or preventing neurodegenerative disorders and diseases comprising the step of: i) providing an effective amount of composition according to of any of claims 1 to 6.

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