FIELD OF THE INVENTION

The invention relates to a new synbiotic composition for use in preventing development of and/or treating neurodegenerative disorders. More particularly, the present invention relates to a synbiotic composition comprising a specific combination of bacterial strains and fibers which can be used in the treatment and/or in the prevention of Alzheimer's disease.

BACKGROUND OF THE INVENTION

Food companies have increased their activities to identify food products which will provide beneficial health effects on the consumer in addition to providing essential nutrients named functional foods. Examples are viable microorganisms, so called probiotics which have shown specific health benefits on gut health and which are included in food product. Other food products with health benefits are foods containing dietary fibers and/or prebiotic carbohydrates.

Ageing is associated with impaired cognitive function. Most aging individuals die from atherosclerosis, cancer, or dementia; but in the oldest old, loss of muscle strength resulting in frailty is the limiting factor for an individual's chances of living an independent life until death.

Alzheimer's disease is a devastating neurodegenerative disorder that impairs patients' memory with disastrous socio-economic consequences. It affects 60 million of the world population above the age of 65 years and, as life expectancy increase, is expected to affect 100 million by 2025. It is neuropathologically characterized by the formation of insoluble protein aggregates - or amyloids - that accumulate in Alzheimer patient's brains. The presence of amyloids in the Alzheimer brain is an early and critical event that leads to the death of neurons and eventually dementia. Thus, arguing in favor of this hypothesis, the most rational strategy for an AD therapy would be to retard Αβ aggregation. To date, there is no cure for this devastating neurodegenerative disorder. Both clinical and epidemiological evidence suggest that, among other non-genetic factors, modification of lifestyle factors such as nutrition may be crucial to the development of AD neuropathology. One potential target against non-healthy nutrition is the gut microflora community or microbiota. Diet is a well-characterized modulator of intestinal microflora. However, daily meal ingestion constitutes an uncontrolled way to modulate microflora. Much of the evidence regarding the role of fermentable gut substrates on host metabolism stem from studies with inulin showing benefits on glucose metabolism and weight regulation in animal experimental models.

The need for preventive strategies is thus urgent. Diet based prevention is recognized as the most efficient strategy to modulate gut microbiota in the combat of life style related disease, and epidemiological studies support that high intake of fibers and legumes is beneficial in the prevention and management of healthy ageing.

The human gastro-intestinal (Gl) tract hosts billions of bacteria, which triggers many pleiotropic effects including the immune system homeostasis, food processing, production and secretion of vitamins, modification of bile acid and communication with the Central Nervous System (CNS). Perturbation of the microbial community in the gastro-intestinal tract may contribute to an impaired homeostasis associated with disease states, such as neurological disorders. As aforementioned, both genetic and environmental factors are important in the etiology of AD. A potentially important environmental factor is abnormal intestinal flora that often interacts with other factors such as intestinal permeability and transport of toxic substances. WO 2012/159023 relates to a method of assessing the presence or the risk of development of hepatic encephalopathy (HE) in a patient with liver disease, comprising a step of analyzing gut microflora in order to determine a gut microbiome signature for said patient. The presence of Roseburia in stool samples is indicated as a bacteria associated with better cognition. However, the examples of WO 2012/159023 show that there is decreased abundance of autochtonous Roseburia bacteria in HE patient as compared to non-HE patients (example 2, table 1 1 , p.44).

WO2013/176774 relates to microbiome markers and therapies for autism spectrum disorders. Bacteriae of interest belong to Prevotella, Coprococcus, Prevotellaceae or Veillonellaceae. Verrucomicrobiae are mentioned on p.12-13 and Table 6, while their different abundance in the gut microflora in neurotypical and autistic groups did not show statiscal significance. Bacteriae of the genus Roseburia are cited in Tables 7 and 8 (p. 13-15) while no information is given on their significance. Akkermansia is mentioned as one of the top five more abundant genera in both neurotypical and autistic groups (paragraph [0043]) and the prevalence of Akkermansia in several autistic subjects was noted, though a correlation between the abundance of Akkermansia and the severity of gastrointestinal problems was not found (paragraph [0057]). WO2015/181449 discloses a method for diagnosis, treatment or prevention of Parkinson's disease comprising determining the relative abundance of one or more microbial taxa, in particular Prevotelleceae, in a gut microbiota sample. The abundance of Verrucomicrobiaceae was determined and showed that it was associated with the severity of non-motor symptoms and in particular constipation (Table 5 and p.22).

WO2013/050792 discloses the use of Roseburia hominis for treating inflammatory, immune or intestinal disorders by immunoregulation. WO2016/203221 discloses compositions containing bacteria of the Roseburia genus (in particular Roseburia hominis) for preventing or treating inflammatory and autoimmune diseases, in particular when mediated by the IL-17 and the Th 17 pathways. WO2014/201037 discloses the treatment of diseases related to the immune system (autoimmune diseases and allergies) in a mammal by enhancing the growth or activity of at least one bacterial species in the intestinal and/or oropharyngeal and/or vaginal microbiota of said mammal. In particular, the bacterial strain is selected from Lachnospiraceae, Clostridiaceae, Bacteroidaceae, Bifidobacteriaceae and Enterobacteriaceae. WO2014/201037 teaches that the enhancement of bacterial growth decreases the differentiation of T lymphocytes involved in allergic, inflammatory and auto-immune diseases (Th 2, Th 1 andTh 17). The examples showing activity are carried out with Bifidobacterium sp. in a diabetes model.

The involvement of gut microbiota in the development of Abeta amyloid pathology was shown in a germ-free APP transgenic mouse model (T. Harach et al., 2017, Scientifc Reports 7, Article number : 41802).

A review of the trend of ongoing research in the field of probiotics and prebiotics is given in Y. Dixit et al., J. Food Microbiol. Saf. Hyg., 2016, 1 , 1 -13.

SUMMARY OF THE INVENTION

The invention provides a novel synbiotic composition comprising mixtures of probiotics and prebiotics, as well as their use for the effective prevention and/or treatment of Alzheimer's disease, without unwanted side effects. The synbiotic composition comprises a mixture of beneficial microbes (probiotics) and a combination of fibers (prebiotics) suitable for oral ingestion.

The invention relates to the unique finding that bacterial species belonging to the Roseburia genus have beneficial effects on host health, in particular when associated to bacterial species belonging to the Verrucomicrobiale family. The species belonging to Roseburia and Verrucomicrobiale can be used in a synbiotic composition, as a probiotic, together with dietary fibers, as a prebiotic. This association makes it possible to ensure the bacterial viability and beneficial activity on the host gut microbiota, by providing the bacteriae with substrates that stimulate their growth and/or activity.

The invention thus relates to a synbiotic product composition comprising a probiotic composition, which comprises at least one Roseburia species and at least one Verrucomicrobiale species, and a prebiotic mixture, which comprises at least one dietary fiber, and to its use for the prevention and/or treatment of Alzheimer's disease. The invention further relates to a pharmaceutical composition containing said symbiotic composition and a pharmaceutically acceptable vehicle.

The invention also relates to relates to a probiotic composition comprising at least one Roseburia species and at least one Verrucomicrobiale species, in particular an Akkermansia species, to a pharmaceutical composition containing it, and to its use for the prevention and/or treatment of Alzheimer's disease.

As even a mild alteration of gut microbiota (GMB) is related to a lower performance in cognitive tests, the present invention describes the concept linking benefits of GMB modulation to neurodegenerative diseases related to pathological ageing. Also, the synbiotic composition of the invention provides neuroprotective, as well as reduced beta-amyloid and plaque accumulation, thereby preventing against Alzheimer's disease.

DEFINITIONS Probiotics : viable microorganisms which, when administered in sufficient numbers, confer health benefits to the host. Prebiotics : non digestible selectively fermentable ingredients that allow specific changes in the composition and/or activity of the resident microflora by selectively augmenting the probiotic bacteria.

Synbiotics: is the term used for a composition which comprises probiotics (non-pathogenic microorganisms) in addition to prebiotics (natural substrate for the aforementioned microorganisms)

Amyloid: Amyloids are insoluble fibrillar protein aggregates that share specific structural traits. Amyloids arise from at least 20 misfolded proteins and polypeptides present naturally in the body. These inappropriately folded structures alter their proper configuration such that they erroneously interact with one another or other cell components forming insoluble fibrils. Amyloids have been associated with the pathology of more than 20 serious human diseases in that, abnormal accumulation of amyloid fibrils in organs may lead to amyloidosis, and may play a role in various neurodegenerative disorders Microbiota/Microflora: The term "microbiota or microflora" refers to the whole microbial community found in the gastro-intestinal tract of a higher organism, including bacteria, archaea, yeasts, and various parasites.

Microflora modulation: Refers to a change in the representation of bacteria in a microbiological community of a particular individual. Microflora can be modulated by compounds that induce the growth and/or activity of commensal microorganisms that contribute to the well-being of their host. These coumpounds called prebiotics are present in diet or in the gastro-intestinal tract and are typically but not exclusively non-digestible fibers compounds that stimulate the growth and/or activity of beneficial bacteria that colonize the large bowel by acting as substrate for them. Microflora can also be modulated by microorganisms. These live micro-organisms called probiotics, when administered adequately, confer a health benefit to the host. Gastro-intestinal microflora can be also modulated by antibiotics. These molecules refer to any substance produced by a living microorganism which is antagonistic to the growth and/or division of other living microorganisms. Nutritional approach leading to modulation of gut microbiota by a combination of probiotics and prebiotics are commonly referred as synbiotics.

Microbiome analysis: Techniques for characterizing the microbiome include use of nucleic acid and/or proteins. Nucleic acid analysis includes analysis of, DNA, RNA, mRNA, rRNA, and/or tRNA, and can be accomplished using, but not limited to pyrosequencing, qPCR, RT-qPCR, clone libraries, DGGE, T-RFLP, ARISA, micro arrays, FIFH, dot-blot hybridization, next generation sequencing, DNA mapping devices and any other DNA hybridization methods that will detect a specific sequence.

Proteome analysis: Protein analysis can be performed by 2-Dimensional

Gel Electrophoresis, 2-Dimensional Difference Gel Electrophoresis (2D-DIGE),

MALDI TOFMS, (2D-) LC-ESI-MS/MS, AQUA, and iTRAQ.

These characterizations are combined with statistical analysis (Linear discriminant analysis effect size (LEfSE, www.huttenhower.sph.harvard.edu/galaxy/)) to precisely determine the players within the microbiome. Rigorous bioinformatics analysis provides accurate characteristics and distributions of intestinal microflora between individuals.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a synbiotic composition comprising probiotics and prebiotics, and its use in preventing and/or treating Alzheimer's disease (AD), as well as pharmaceutical compositions comprising this synbiotic composition.

The synbiotic composition comprising probiotics and prebiotics of the present invention supplies a specific bacterial equilibrium to the gastro-intestinal (Gl) tract. This synbiotic composition alters the bacterial profile in the Gl tract and makes it possible to prevent or cure neurodegenerative disorders.

The synbiotic composition of the invention, comprises i) a probiotic composition, which comprises at least one Roseburia species, and ii) a prebiotic mixture, which comprises at least one dietary fiber.

Preferably, said probiotic composition comprises at least one Roseburia species and at least one or more additional microbial species, for example at least one Verrumicrobiale species. More preferably, said probiotic composition comprises at least one Roseburia species and at least one Verrumicrobiale species.

In a preferred embodiment, the probiotic composition of the invention comprises only at least one Roseburia species and at least one or more Verrumicrobiales species, as microbial species. The probiotic composition comprising at least one Roseburia species and at least one Verrucomicrobiale species, as defined above, is also an object of the invention.

A preferred Verrumicrobiale species is selected from Akkermansia, in particular Akkermansia muciniphila. Preferred species of Roseburia include Roseburia intestinalis, Roseburia cecicola, Roseburia faecis, Roseburia hominis and Roseburia inulinivorans.

Upon performing previous analysis comparing gut microbiota profiles of Alzheimer patients with healthy subjects, the inventors surprisingly found that both bacteriae of the Roseburia species and Verrumicrobiale species, in particular Akkermansia were in lower content or lacking from biological samples of AD patients, in comparison to healthy subjects .

The inventors have now shown that administration of the synbiotic composition comprising probiotics and prebiotics according to the invention into a mouse model of AD leads to a reduction of cerebral levels of soluble Abeta42, which is the most pathogenic Abeta isoform (Figure 1A), as well as cerebral plaques (Figure 1 B). Reduction of Abeta 42 levels occurs in both young and aged animals. Furthermore, oral administration of a synbiotic composition comprising prebiotics and probiotics according to the invention induces reduction in cortical Abeta 42 levels. Advantageously, the synbiotic composition of the invention reduces cerebral Abeta 42 in a dose-dependent manner (Figure 2).

Unexpectedly, it has thus been found that the modulation of non-animal elements could make it possible to act on brain pathologies in an animal model.

In particular, it has been found that the composition comprising at least one Roseburia species and a Verrumicrobiale species, in particular at least one Akkermansia species, preferably Akkermansia muciniphila, in combination with dietary fibers possesses beneficial effects with respect to the treatment and/or prevention of neurodegenerative disorders, in particular Alzheimer's disease.

Advantageously, affecting the gut microbiota composition by increasing the proportion of at least one Roseburia species, and, preferably, at least one Verrucomicrobiale species, and providing fibers resulted in increased gut fermentation activity (breath hydrogen (H2) as a test marker), increased concentrations of SCFA and improved behaviour in a mouse model of AD.

The prebiotics which are part of the symbiotic composition of the invention can be selected from dietary fibers, namely carbohydrates including three or more monomers that resist digestion and absorption in the small intestine, and are completely or partially fermented in the colon by gut bacteria. Dietary fibers can be selected from natural or synthetic fibers, purified fibers or mixtures thereof. As understood in the present description, dietary fibers relate to non-starch derived indigestible polysaccharides such as beta-glucans, arabinoxylans, cellulose, oligosaccharides, fructans, pectin, guar gum. Indigestible substrates that are closely associated with indigestible polysaccharides are also included in the definition (e.g., phenolic compounds, lignin and phytosterols).

For example, dietary fibers from cereals or vegetables such as from barley, rye, wheat, oats, vegetable seeds, beta-glucans, guar gum, lignin, lignans and oligosaccharides such as galacto-oligosaccharides and fructo-oligosaccharides, or else chemically modified starch can be used. Examples of resistant starch (RS) are vegetable starches such as retrograded starch, botanically encapsulated starch, starch and cyclodextrins.

According to the invention, dietary fibers can be selected, for example, from β-glucans (such as chitine and cellulose), Hemicelluose (Hexoses, pentoses, lignine, xanthum gum and resistant starch) or else from arabinoxylose, fructans, for example galacto-oligosaccharides (GOS), and inulin, or mixtures thereof.

A combination of galacto-oligosaccharides (GOS) and inulin is preferred.

In particular, the amount of aforementioned prebiotic is between 10% and 50% w/w of the synbiotic composition. Advantageously, the combination of probiotic (bacteriae) and their substrate

(prebiotic) enhances the beneficial impact of the microorganisms on health as the included substrate (fibers) will continuously provide an energy source. Consequently, in combination with fibers contained in ingested meals, fibers of the synbiotic composition of the invention serves as an energy booster for exogenous and endogenous fermenters.

The single or combined use of the synbiotic composition of the invention, comprising at least one Roseburia species and a Verrumicrobiale species, in particular at least one Akkermansia species, can also, for example be provided as dry powder and packed in disposable containers to be added to meals, e.g. breakfast cereals.

Preferably, the invention provides a synbiotic composition in a dosage form containing 10 ⁶ to 10 ¹¹ viable colony forming units (CFU) per dose. In yet another embodiment, the synbiotic composition of the invention comprises less than 60% probiotics on a weight-percentage basis. In another embodiment, the synbiotic composition comprises at least 20%, in particular at least 40%, notably 50% of the aforementioned probiotics on a weight-percentage basis.

If more than one bacterial species is present, the species may be present in differing amounts or equal amounts, on a weight percentage basis, with respect to each other. For example, a synbiotic composition according to the invention can comprise 50% probiotic on a weight percentage basis, i.e., a 500 mg dose of a synbiotic composition according to the invention can comprise 250 mg of a probiotic composition. The probiotic composition, in turn, can consist of 30wt% Roseburia intestinalis, 20wt% Roseburia cecicola, and 50wt% Akkermansia muciniphila.

According to a preferred embodiment, the invention relates to a synbiotic composition, comprising (i) a probiotic composition, wherein the probiotic composition comprises at least one Roseburia species and at least one Verrumicrobiale species ; and (ii) a prebiotic mixture, wherein the prebiotic mixture comprises galacto-oligosaccharides (GOS) and inulin.

A preferred synbiotic composition according to the invention comprises a bacterial combination of at least one Roseburia species and at least one Akkermansia species, as a probiotic, and inulin and GOS, as a prebiotic. Preferred species of Roseburia include Roseburia intestinalis, Roseburia cecicola, Roseburia faecis, Roseburia hominis and Roseburia inulinivorans.

A preferred species of Akkermansia is Akkermansia muciniphila.

In particular, the synbiotic composition of the invention contains Roseburia intestinalis and Akkermansia muciniphila. Culture conditions for Akkermansia muciniphila are disclosed, for example, in

M. Derrien et al., Int J Syst Evol, Microbiol. 2004 Sep; 54(Pt 5):1469-76. PubMed PMID: 15388697.

Culture conditions for Roseburia intestinalis are disclosed, for example, in SH Duncan et al., Int J Syst Evol Microbiol. 2002 Sep; 52(Pt 5):1615-20. PubMed PMID: 12361264.

The invention also relates to a pharmaceutical composition comprising a symbiotic composition as disclosed above and a pharmaceutically acceptable vehicle. Said symbiotic composition may be capsulated or lyophilised by freeze drying which is well known in the art.

The symbiotic composition can be a stable emulsion, where, in particular, the at least one Roseburia species and the at least one Verrumicrobiale species, are encapsulated with prebiotics in emulsion droplets.

Preferably, the Verrumicrobiale species is an Akkermansia species.

Preferably, said pharmaceutical composition is in the form of enterically coated vegetable capsule, namely including an enteric coating when one or more of the probiotic organisms in the encapsulated formulation is non-viable under acidic conditions (e.g., the acidic conditions of the stomach).

In another embodiment, the synbiotic composition of the invention is a composition which is liquid at room temperature. In other embodiments, the pharmaceutical composition comprising a symbiotic composition is for example, in the form of tablets, capsules, powders, granules, solutions, emulsions, oral suspensions, drops, syrups, etc.. Advantageously, regardless of the galenic form, the pharmaceutical composition comprising a symbiotic composition is stable for at least one year when stored at temperatures between approximately 2 and 10 degrees Celsius.

A preferred pharmaceutical composition according to the invention comprises inulin and GOS blended with a specially selected bacterial combination of at least one species belonging to the Roseburia genus and at least one Verrumicrobiale species, preferably an Akkermansia species. In one embodiment, the final blend consists of about 2 billion colonies forming units (CFU)/capsule.

The invention also relates to a pharmaceutical composition containing a probiotic composition comprising at least one Roseburia species and at least one Verrucomicrobiale species, as defined above.

All preferred features described above for the synbiotic composition also apply to the pharmaceutical composition. The invention further relates to a synbiotic composition as disclosed above for use in preventing and/or treating Alzheimer's disease.

In particular, the invention relates to a synbiotic composition as disclosed above for use in preventing and/or treating Alzheimer's disease, wherein the levels of cerebral soluble Abeta42 are decreased and/or the number of cerebral amyloid plaques is reduced.

The invention further relates to a method for preventing and/or treating Alzheimer's disease (AD), comprising a step of administering the symbiotic composition as described above to a subject in need thereof . When used for prevention and/or treatment of Alzheimer's disease, a typical treatment regimen is one capsule, taken one time or two times daily.

For example, the duration of the treatment can be at least one month, preferably at least 3 months.

The dosage regimen will be adjusted as a function of the patient's needs, in particular taking account of the fact that prevention or treatment of AD is considered, and, in the case of treatment, of the stage of development of AD.

The invention further relates to a probiotic composition comprising at least one Roseburia species and at least one Verrucomicrobiale species, as defined above, for use in preventing and/or treating Alzheimer's disease. The invention further relates to a method for preventing and/or treating

Alzheimer's disease (AD), comprising a step of administering a probiotic composition comprising at least one Roseburia species and at least one Verrucomicrobiale species, as defined above to a subject in need thereof .

Preferred species of Roseburia include Roseburia intestinalis, Roseburia cecicola, Roseburia faecis, Roseburia hominis and Roseburia inulinivorans.

A preferred Verrucomicrobiale species is Akkermansia, preferably Akkermansia muciniphila. In yet another embodiment, the invention provides a method for carrying out a home-determination of the efficacy of a synbiotic composition as described above. Said method can comprise the steps of: providing instructions for administering a synbiotic composition over a predetermined time period to a subject and a questionnaire for the subject to self-record the effects of the administration of said synbiotic composition; collecting the self-recorded data from the questionnaire after the predetermined time period has passed; and evaluating the self-recorded data.

The questionnaire may be provided on-line.

Preferably, the predetermined time period is at least one month, preferably at least 3 months. Each individual will be provided with a score sheet query about the impact of the synbiotic composition on their overall health and brain status. The query can be directed to both quantitative and qualitative parameters, and can include, for example, questions relating to :

- health status before treatment with the synbiotic composition, namely overall health, intestinal health, brain health, current treatment with other therapeutic agents (such as, for example, antibiotics, hormones, agents for aromatherapy, agents for phytotherapy, live biotherapeutics etc.),

- health status after treatment with the synbiotic composition,

- subject compliance with treatment, and - usual daily activities of the subject.

With respect to intestinal health before or after treatment, the questionnaire can, for example, include queries relating to the occurrence of diarrhea, constipation, intestinal discomfort, intestinal flu, nausea symptoms, appetite etc.

With respect to brain health before or after treatment, the questionnaire can, for example, include queries relating to memory problems, cognitive impairments, stress, anxiety, sleepiness, migraine, libido problems, etc. With respect to usual daily activities of the subject before or after treatment, the questionnaire can, for example, include queries relating to shopping or travelling activity, in particular shopping or travelling alone, meal or hot beverage preparation, games of skill, remembering information, etc. Answers to each query can consist in a yes/no response or in a score value.

The questionnaire may include other queries and vary in function of the subject.

The invention further relates to an in vitro method for determining the probability of a subject developing or having Alzheimer's disease, comprising the steps of: a) measuring the relative abundance of at least one Roseburia species and at least one Verrucomicrobiales species in a biological sample obtained from a subject, and b) determining the probability of the subject developing or having Alzheimer's disease based on the relative abundance measured in step a), where a high relative abundance of at least one Roseburia species and at least one Verrucomicrobiales species indicates a low probability of the subject developing or having Alzheimer's disease.

Alternatively, the invention relates to an in vitro method for determining the probability of a subject developing or having Alzheimer's disease, comprising the steps of: a) measuring the relative abundance of at least one Roseburia species and at least one Verrucomicrobiales species in a biological sample obtained from a subject, and b) determining the probability of the subject developing or having Alzheimer's disease based on the relative abundance measured in step a), where a low relative abundance of at least one Roseburia species and at least one Verrucomicrobiale species indicates a high probability of the subject developing or having Alzheimer's disease.

The biological sample can be, for example a faecal sample (stool) or a mouth swab. Preferably, the subject is human.

The relative abundance of the at least one Roseburia species and at least one Verrucomicrobiale species can be measured by bacterial genomic extraction and PCR amplification of the V3-4 region of bacterial 16S ribosomal RNA (rRNA) genes.

In the present description "relative abundance" is understood as the number of bacteria of a particular taxonomic level (from phylum to species) as a percentage of the total number of bacteria in a biological sample. This relative abundance can be assessed, for example, by measuring the percentage of 16S rRNA gene sequences present in the sample which are assigned to these bacteria. It can be measured by any appropriate technique known by the skilled artisan, such as 454 pyrosequencing and quantitative PCR of these specific bacterial 16S rRNA gene markers or quantitative PCR of any gene specific for a bacterial group.

Relative abundance of at least one Roseburia species and at least one Verrucomicrobiales species can be determined by methods used in the field, as shown, for example, in N. Vogt et al., Nature, Scientific reports, 2017, 7:13537. Preferably, the determination of step b) is performed by comparing the relative abundance measured in step a) to a predetermined threshold. Said predetermined threshold is determined beforehand, in view of the amount of Roseburia species and Verrucomicrobiales species in the tested samples.

Preferred species of Roseburia include Roseburia intestinalis, Roseburia cecicola, Roseburia faecis, Roseburia hominis and Roseburia inulinivorans.

A preferred species of Verrumicrobiale is an Akkermansia species, more preferably Akkermansia muciniphila. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 describes the impact of the synbiotic composition of the invention on the level of cerebral Αβ42 (Figure 1A) and on cerebral plaques (Figure 1 B). Αβ42 is a pathogenic peptide involved in Alzheimer's disease as the main component of the amyloid plaques and on cerebral Αβ plaques in APPPS1 mice, a mouse model of Alzheimer.

Figure 2 describes the dose-dependent effect of the synbiotic composition of the invention on the level of cerebral Αβ42 in APPPS1 mice.

The invention is illustrated by the following examples. Example 1 : Synbiotic composition

The ingredients of the synbiotic composition are shown in Table 1 .

Table 1

Total CFU/capsule : 2 billion (2x 10 ⁹ ) CFU The ingredients are in powder form.

Capsule composition: Maltodextrin, magnesium stearate, hydroxypropyl methyl cellulose, methacrylic acid, methyl methacrylate copolymer.

The capsule weight may be 2 to 3 times higher, for example from 1 g to 2 g, while maintaining the same percentage by weight of probiotic mix and prebiotic mix. Example 2: Efficacy of a synbiotic composition in an Alzheimer mouse model

Preclinical demonstration of the efficacy of a synbiotic composition containing Roseburia intestinalis and Akkermansia muciniphila as probiotic mix, and galacto- oligosaccharides (GOS) and inulin as prebiotic mix was performed as shown below by dietary intervention in an Alzheimer mouse model by selective modulation of gastro-intestinal microbiota and positive impact on Abeta amyloidosis (n= 5/group). Data are shown on Figure 1 as mean ± SEM Statistical differences between control and synbiotic composition-treated mice ^** :p<0.01 ;

The dose-dependent-effect is shown in Figure 2.

a) APPPS1 mice

APPPS1 transgenic mice (Radde R. et al., EMBO Rep. 2006, (9):940-6) were maintained at animal core specific pathogen free facility. There was unlimited access to autoclaved food and water. APPPS1 animals co-express the KM670/671 NL Swedish mutation of human amyloid precursor protein (APP) and the L166P mutation of human presenilin 1 (PS1 ) under the control of the Thy-1 promoter, and show age- dependent accumulation of parenchymal Αβ plaques with minimal vascular Αβ amyloid that is restricted to the pial vessels'! 4. APPPS1 mice were generated on a C57BL/6 background. Both male and female APPPS1 mice as well as age-matched control wild-type (WT) littermates were used. APPPS1 mice and wildtype littermate were housed together in grouped cages (n = 5 mice/cage) until analyzed. Throughout the study these mice are referred to as conventionally-raised (CONVR) APPPS1 and WT mice.

b) Preparation of the symbiotic composition

Akkermansia muciniphila BAA35 was grown in anaerobic condition using the following protocol and medium: At Day 1 , glass bottles used for liquid cultures were placed in the oven (3h at 180°C or 120°C overnight). Then the glassware were put under an anaerobic tent for at least 2 days prior being used.

At Day 3, media were split into anaerobic glass bottles (10ml in each) sealed with a rubber lid. At this stage, media were taken out of the anaerobic tent and placed them overnight at 37°C in order and check their sterility condition.

At Day 4, media can be used to culture bacteria or can be stored in the dark at 4°C.

The Anaerobic Akkermansia-Media is made of 18,5 g/l Brain Heart Infusion, 15 g/l Trypticase soy broth, 5 g/l yeast extract, 2,5 g/l K2HPO4, 1 g/ml Hemin and 0,5 g/l Glucose. All reagents were mixed via sterile filtration prior to autoclave. The following compound were autoclaved separately: 0,4 g/l Na2CO3 autoclave, 0,5 g/l Cystein hydrochloride, 0,5 g/ml Menadione filter sterile, 3 % FCS (complement- inactivated ^* ) filter sterile 0,025 % Mucin (gastric mucin from porcine stomach,) separately 2 ml of 2.5% in water (100x) The medium was pre-reduced in anaerobic tent for 2 days prior to be aliquoted

Roseburia intestinalis L82 is an aerobic strain and it grows in RUMEN BACTERIA MEDIUM made of Mineral solution. This buffers contains 0.30 g K ₂ HPO ₄ , 2.00 g Trypticase peptone (BD BBL), 0.50 g Yeast extract (OXOID), 3.10 ml Volatile fatty acid mixture, Haemin solution (0.05% w/v, see medium 104), 0.50 g Glycerol, Na-resazurin solution (0.1 % w/v), 4.00 g Na ₂ CO ₃ , D-Glucose 0.50 g, Maltose 0.50 g, Cellobiose 0.50 g, Starch, soluble 0.50 g, L-Cysteine-HCI x H ₂ O 0.25 g, Na ₂ S x 9 H ₂ O 0.25 g and distilled water was added qsp 1000ml.

Except glucose, maltose, cellobiose, soluble starch, cysteine and sulfide, all ingredients were dissolved in carbonate then inoculated to medium with 100% CO ₂ gas for 30 - 45 min to make it anoxic. Carbonate was added and the medium was equilibrated with the CO ₂ gas to pH 6.8. The medium was distributed under 100% CO ₂ gas atmosphere into an anoxic Hungate-type tubes or serum vials prior to autoclave. Thereafter, glucose, maltose, cellobiose, soluble starch, cysteine and sulfide were from sterile anoxic stock solutions prepared under 100% N ₂ gas atmosphere. Finally pH of complete medium was adjusted to 6.7 - 6.8. To complete the buffers, a mineral solution was prepared as follows: 12.00 g of KH ₂ PO ₄ NaCI (NH ₄ )2SO ₄ were mixed with 1 .60 g CaCI ₂ x 2 H ₂ O and 2.50 g MgSO ₄ x 7 H2O and filled up with distilled water 1000.00 ml. As an initial mimicking of the gastro-intestinal fermentative part, a volatile fatty acid mixture made of: 548.50 ml pure Acetic acid, 193.50 ml Propionic acid Butyric acid, 32.25 ml n-Valeric acid, 32.25 ml, 32.25ml iso- Butyric acid and 32.25 ml DL-2-Methyl butyric acid iso-Valeric acid was added.

After having cultured the bacteria separately, they have been washed twice with PBS 1 X (4500 rpm for 10 min/each time) and at the end pooled in 1 :1 ratio. 25 mg of Galactooligosaccharides (GOS) and 25mg of inulin powders were mixed to the pooled probiotics. The combination of the aforementioned bacteria and fibers constitutes the symbiotic composition. The procedure has been performed under axenic conditions in order to avoid external unwanted contaminations. Furthermore, the cleanness of the gavage inoculum has been checked by 16S rRNA sequencing on the left over from the gavage inoculum.

c) Inoculation protocol

The synbiotic composition obtained as described above was administered to APPPS1 mice by oral gavage at 2*10 ^{1 1} colony forming units per ml (For the single dose experiment described in Figure 1 ). 200 microliters were administered to the animals once or three times a day for 3 consecutive months. For dose dependent effect of the synbiotic composition , 2*10 ¹⁰ and 2*10 ^{1 1} cfu/ml were administered to APPPS1 mice once or three times a day during 3 consecutive months.

d) ELISA

Brain protein extracts and plasma were diluted to the fourth into sampling medium provided by the manufacturer and a final of 25 μΙ volume was loaded into a 96 well plates. Αβ38, Αβ40 and Αβ42 measurements were performed according to the manufacturer's instructions (Peptide Panel 1 (6E10) Kit (1 Plate) V- PLEX™K15200E-1 Mesoscale Discovery, Gaithersburg). All levels of Αβ38, Αβ40 and Αβ42 were normalized against total protein amount. For cytokine measurements, total brain homogenates were centrifuged at 14'000 rpm, 30 min, 4 °C and supernatants were analyzed using the mouse pro-inflammatory panel 1 V-plex plate (Mesoscale Discovery) according to the manufacturer's instructions and normalized against total protein content.

e) Quantification of lba-1 -positive immunoreactivitv

Neocortical microglia immunoreactivity was quantified in hemi-brain sections immunostained for activated microglia (with the lba-1 antibody). Acquisition of images from lba-1 -stained sections was similar to that described for ThT quantification. Subsequently, a threshold of 1 10 with a variance of 10% was applied to each image using Image J. This threshold was selected in order to best display the microglia while minimizing the background noise. After visual inspection, ROI were selected such that staining artefacts on the section was manually removed by cropping. The microglia were quantified using the analysis particles tool embedded in ImageJ. In order to consider both the single microglia and the microglial loads, the pixel size was not specified and the circularity parameter was set to 0.0-1 .0.

f) Statistical analysis (Histology)

Data represent the means ± standard errors of the means. Statistical analysis was performed using ANOVA followed by Hom-Sidak's test for multiple comparisons and unpaired t test followed by Welch's correction. q) Statistical analysis (ELISA)

Graphpad Prism 6 software was used to perform statistical analysis. Data represent the means ± standard errors of the means. Statistical analysis was performed using ANOVA followed by Hom-Sidak's test for multiple comparisons.

h) Results

The results show that a single dose of the synbiotic composition of the invention, when administered during 3 months to APPPS1 mice, either young or aged, decreases the levels of cerebral soluble Abeta and reduces the number of cerebral amyloid plaques (see Figure 1 ).

Furthermore, the synbiotic composition of the invention reduces cerebral soluble Abeta 42 in a dose-dependent manner when administered for 3 consecutive months.