Composition, bacterial consortia and uses for preventing and / or treating neurodegenerative diseases, mental illnesses or psychiatric disorders

Abstract

The present invention describes a composition based on bacterial consortia and uses thereof for preventing and / or treating neurodegenerative diseases, mental illnesses or psychiatric disorders.

Description

[0001] COMPOSITION, BACTERIAL CONSORTIUMS, AND USES FOR THE PREVENTION AND / OR TREATMENT OF DISEASES

[0002] NEURODEGENERATIVE, MENTAL DISEASES OR PSYCHIATRIC DISORDERS

[0003] DESCRIPTIVE MEMORY

[0004] FIELD OF INVENTION:

[0005] The present invention relates, in general terms, to a psychobiotic composition and its uses for the prevention and / or treatment of neurodegenerative diseases, mental illnesses or psychiatric disorders.

[0006] BIOLOGICAL DEPOSITS:

[0007] This application contains references to deposits of biological material. The following biological materials have been deposited with the Chilean Collection of Microbial Genetic Resources (CChRGM) in Chillán, Chile, and bear the following designations, accession numbers, and deposit dates: Lacticaseibacillus paracasei, M38 (RGM 3511, deposited September 12, 2023); and Levilactobacillus brevis; G2 (RGM 3512, deposited September 12, 2023). BACKGROUND OF THE INVENTION:

[0008] The term probiotic is defined as “live organisms which, when administered in adequate amounts, confer a health benefit on the host” (Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO), 2001).

[0009] The term “psychobiotics” is a term used to describe exogenous interventions that aim to manipulate gut-brain signaling, mediated by bacteria, to improve mood, cognition, and anxiety (Dinan, Stanton, & Cryan, 2013). This includes probiotics, prebiotics, and other treatments targeting the gut microbiome. Psychobiotics show their psychotherapeutic effects by producing or stimulating the production of various neurotransmitters, short-chain fatty acids, neurohormones, and anti-inflammatory cytokines (Sharma et al., 2021). Psychobiotics have been reported to reduce neuronal aging, inflammation, oxidative stress, and cortisol levels, and increase synaptic plasticity and levels of neurotransmitters and antioxidants (Dhyani et al., 2023).

[0010] The term "microbial consortium" refers to a bacterial community where its members live symbiotically. That is, they have the ability to interact within a community (Du et al., 2020). Microbial consortium engineering involves the rational design of communities of microorganisms that interact and are capable of producing desired behaviors (Bittihn et al., 2018).

[0011] The term “psychobiotic composition” refers to a mixture of bacterial strains that confer mental health benefits to the host when taken appropriately. Psychobiotic strains affect functions related to the central nervous system (CNS) and behavior (Dhyani et al., 2023).

[0012] For example, international patent application WO 2022 / 224273 describes a psychobiotic composition comprising a combination of twelve probiotic strains selected from the genera Saccharomyces, Bifidobacterium, Lactobacillus and Streptococcus, wherein the composition is effective in restoring the gut-brain axis, and in treating conditions associated with autism or autism spectrum disorders, wherein the composition comprises a combination of twelve specific probiotic strains.

[0013] International patent application WO 2016 / 065419 discloses methods and compositions for use in the treatment of depression, anxiety, or a depressive or anxiety-related disorder. Embodiments describe the administration of oratic acid or a salt thereof, or the administration of one or more probiotic microorganisms, selected from at least one Lactobacillus species, at least one Lactococcus species, at least one Bifidobacterium species, at least one Streptococcus species, and / or at least one yeast.

[0014] Additionally, international patent application WO 2022 / 003059 teaches an intestinal microbiota composition, comprising at least one species of Lactobacillus lactis or Lactobacillus helveticus (family Lactobacillaceae), or of genii Mega monas (family Selenomonadaceae), Anaerovibrio (family Selenomonadaceae) or Roseburia (family Lachnospiraceae) of the phylum Firmicutes, for use in the prevention and / or treatment of a mental disorder with memory impairment in a subject, such as Parkinson's disease or Alzheimer's disease.On the other hand, international patent application WO 2018 / 234994 describes a composition comprising a mixture comprising an effective amount of at least one bacterial strain and / or at least one derivative thereof for use in a method for the curative treatment and / or preventive treatment of the symptoms and / or disorders related to at least one pathology that can be selected from a neurodegenerative disease, wherein said at least one bacterial strain belongs to the genus selected from the group comprising or, alternatively, consisting of Lactobacillus, Bifidobacterium, Lactococcus and Streptococcus.

[0015] However, none of the prior art documents teach a composition for the prevention and / or treatment of neurodegenerative diseases, mental illnesses or psychiatric disorders, comprising one or more microorganisms selected from the bacterial phyla Bacteroidota, Bacillota, Actinomycetota, and / or combinations thereof.

[0016] SUMMARY OF THE INVENTION:

[0017] The present invention relates to psychobiotic and probiotic microorganisms, compositions comprising said microorganisms, and their use for the treatment and prevention of neurodegenerative diseases, mental illnesses or psychiatric disorders.

[0018] BRIEF DESCRIPTION OF THE FIGURES:

[0019] Figure 1 shows the monoculture growth of the strains that make up the potential GABA-producing consortia on glucose, glutamate, and inulin (measured as OD620nm) for 48 h. Figure 2 shows the growth kinetics of the consortia in the bioreactors, measured as OD620nm. Curves R1 in blue (dots) and R2 in orange (squares) correspond to the growth of reactor 1 and reactor 2, respectively. Bova: Bacteroides ovatus BEI 3_8_47FA, Bt: Bacteroides thetaiotaomicron VPI 5482, Bvulg: Phocaeicola vulgatus CL09T03C04, D3: Bifidobacterium adolescentis D3, G2: Levilactobacillus brevis G2 deposited under accession number RGM 3512, Ls: Lachnoclostridium symbiosum WAL 14673, M38: Lacticaseibacillus paracasei M38 deposited under accession number RGM 3511.

[0020] Figure 3 shows a survival curve of Drosophila melanogaster flies from the different experimental groups in a Drosophila melanogaster model. Panel A presents the survival curve of all experimental groups. Panel B shows the survival curves of the control group and the control group with consortium, which exhibited significant differences (p < 0.05).

[0021] Figure 4 shows a graph with the results of the climbing test for the different experimental groups of Drosophila flies fed or not with the microbial consortia, tested in a D. melanogaster model. Figure 4A and Figure 4B present the results of the climbing test performed at 10 and 25 days of age respectively. The “x” axis of the bar graphs shows the experimental groups and the “y” axis the climbing index, where a higher number indicates a better climbing ability. The cross α-synuclein x control w(cs) corresponds to the control group, α-synuclein x Elav-Gal4 to the EP group, α-synuclein x control w(cs) + G2BtM38 indicates the control group that received the microbial consortium and α-synuclein x Elav-Gal4 + G2BtM38 corresponds to the EP group that received the consortium.

[0022] Figure 5 shows an analysis determining the number of dopaminergic neurons in the four experimental groups after 25 days of testing. Figure 5A corresponds to a representative confocal microscopy image of the control group showing the immunofluorescence of the protein thyroxine hydroxylase (TH), which corresponds to the rate-limiting enzyme in the synthesis of dopamine, and therefore a marker of this neuronal type, in brains of each experimental group. Panel B shows the quantification of dopaminergic neurons in samples from each experimental group. Each experimental group consisted of an n of eight brains.

[0023] Figure 6 shows a three-dimensional PCA plot of the experimental groups in the 1H NMR metabolomics study. 10-day-old control flies (green), 10-day-old Parkinson's flies (red), 10-day-old control flies receiving consortium (blue), and 10-day-old Parkinson's flies receiving consortium (yellow). 6 independent samples were used for each experimental group, which are represented as dots in the figure. 2 X(cum) = 0.77 and Q 2 (cum) = 0.3, UV scale.

[0024] Figure 7 shows the representation of central metabolism and its main metabolic alterations in Drosophila Parkinsoniana with and without treatment. Panel A shows the main metabolites that were found to be significantly altered in the PD group compared to the control group. Panel B shows the metabolites that presented significant differences in the PD group that received the consortium compared to the control group. In red, those metabolites that had significant differences in the PD group compared to the control group and that after treatment returned to their basal state (they did not present statistically significant differences with the control group). Panel C shows the main metabolites that were found to be significantly altered in the PD group with the consortium compared to the Parkinson group. The direction of the arrows indicates whether there was an increase or decrease in the metabolite.The metabolites demarcated in a rectangle are biomarkers in Parkinson's patients reported in the literature.

[0025] Figure 8 shows a 16S ribosomal gene sequence analysis of the microbiome obtained from D. melanogaster gut samples from the four experimental groups. A shows the taxonomic classification at the genus level. The x -axis represents the different groups. The number corresponds to the biological replicate. The y -axis shows the abundance of each genus, expressed as a percentage and detailed in the color legend to the right of the graph. B shows the Shannon diversity index calculated for each of the experimental groups.

[0026] DETAILED DESCRIPTION OF THE INVENTION:

[0027] The present invention relates to psychobiotic and probiotic microorganisms, compositions comprising said microorganisms, and their use for the treatment and prevention of neurodegenerative diseases, and mental illnesses or psychiatric disorders. Neurodegenerative diseases may be selected from, but are not limited to, Parkinson's, Alzheimer's, Amyotrophic Lateral Sclerosis, Huntington's Disease, Friedreich's Ataxia, Spinal Muscular Atrophy, Dementia with Lewy bodies. Mental illnesses or psychiatric disorders may be selected from, but are not limited to, depression, anxiety, and autism spectrum disorders (ASD).

[0028] In one of the embodiments of the present invention, a composition is described for the prevention and / or treatment of neurodegenerative diseases, and mental illnesses or psychiatric disorders, which comprises one or more microorganisms selected from the bacterial phyla, Bacteroidota, Bacillota, Actinomycetota, and / or combinations thereof, and because it also comprises pharmaceutically acceptable excipients.

[0029] Preferably, the bacteria are selected from but are not limited to the genera Bifidobacterium, Bacteroides, Clostridium, Coprococcus, Enterococcus, Eubacterium, Faecalibacterium, Levilactobacillus, Lacticaseibacillus, Lactobacillus, Propionibacterium, Streptococcus, Roseburia, and / or combinations thereof.

[0030] More preferably, the bacteria are selected from but are not limited to the species Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium bifidum, Bifidobacterium longum subsp. Infantis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Clostridium perfringens, Clostridium butyricum, Coprococcus eutactus, Enterococcus faecalis, Enterococcus faecium, Eubacterium biforme, Eubacterium rectale, Faecalibacterium prausnitzii, Levilactobacillus brevis, Lacticaseibacillus paracasei, Lactobacillus plantarum, Lactobacillus plantarum subsp. Plantarum, Propionibacterium acnes, Streptococcus mitis, Streptococcus salivarius, Roseburia intestinalis, and / or combinations thereof.In one embodiment of the present invention, a microbial consortium called G2M38 was evaluated, which is made up of two species, isolated from the intestinal microbiome of Chilean subjects, which reached an individual growth of at least 2 OD620 nm at the time of forming the consortium. In particular, said consortium includes, but is not limited to, the mixture of two species, comprising the bacterial strains Levilactobacillus brevis G2 deposited under accession number RGM 3512; and Lacticaseibacillus paracasei M38 deposited under accession number RGM 3511 , in a ratio between 1:99 and 99:1 , more preferably the ratio is between 1:50 and 50:1 , more preferably the ratio is between 1:25 and 25:1 , more preferably the ratio is between 1:10 and 10:1 , more preferably the ratio is between 1:5 and 5:1 , more preferably the ratio is 1:1 .

[0031] L. brevis is a microaerophilic, heterofermentative, lactic acid bacterium that has been isolated from different environments such as food and beverages, and probiotic strains of this species have been described that confer several potential health benefits. The scientific classification of L. brevis is: Domain: Bacteria Phylum: Bacillota Class: Bacilli Order: Lactobacillales, Family: Lactobacillaceae, Genus: Levilactobacillus, Species: Levilactobacillus brevis.

[0032] L. paracasei is a homofermentative lactic acid bacterium that has been isolated from various habitats, such as the oral cavity, cereals, vegetables, meats, and dairy products. Some strains are currently used as probiotics. The scientific classification of L. paracasei is: Domain: Bacteria, Phylum: Bacillota, Class: Bacilli, Order: Lactobacillales, Family: Lactobacillaceae, Genus: Lacticasibacillus, Species: Lacticaseibacillus paracasei. In one embodiment of the present invention, the psychobiotic composition further comprises pharmaceutically acceptable excipients. The term "excipient" is defined as any material or substance that, when included in the psychobiotic composition, is added to the active ingredients to enable their preparation and stability, modify their organoleptic properties, or determine the physicochemical properties of the psychobiotic composition and its bioavailability.

[0033] Thus, excipients may bind the components (e.g., starches, sugars, or cellulose), sweeten, color, protect the active ingredient (e.g., to isolate it from air and / or moisture), fill a tablet, capsule, or any other presentation, or have a disintegrating function to facilitate the dissolution of the components, without excluding other excipients not mentioned in this section. The "pharmaceutically acceptable" excipient must allow the components or compounds of the pharmaceutical composition to function, that is, be compatible with the strains of the present invention.

[0034] In one embodiment of the present invention, the composition is available to be supplied in both solid and liquid forms, including tablets, granules, powders, capsules and solutions, emulsions and suspensions.

[0035] In one embodiment of the present invention, the composition is in the form of a beverage, syrup, food or food supplement.

[0036] In one embodiment of the present invention, the composition provides a method for preparing a medicament useful for preventing and / or treating neurodegenerative diseases and / or mental illnesses or psychiatric disorders in a subject, more particularly a mammal, and more preferably a human. In the present invention, the term "medicament" may include a supplement, a food, a nutraceutical, a dietary supplement, and / or combinations thereof.

[0037] In one of the embodiments of the present invention, the neurodegenerative disease is selected from Parkinson's Disease, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease, Friedreich's Ataxia, Spinal Muscular Atrophy, Dementia with Lewy bodies,

[0038] In one embodiment of the present invention, the mental illnesses or psychiatric disorders are selected from depression, anxiety, and autism spectrum disorders (ASD).

[0039] The application of the microbial consortium, as detailed in the following examples section, was carried out in an animal model of Parkinson's disease in D. melanogaster, where healthy and diseased groups were treated. The microbial consortium statistically significantly increased the survival of the healthy group. Furthermore, the application of the consortium did not have a negative impact on the survival of either group. The evaluation of the locomotor capacity of the individuals showed that in the Parkinson's group that received the consortium, at both 10 and 25 days, the climbing index was significantly higher than in the Parkinson's group that did not receive the consortium. Furthermore, the diseased group that received the consortium did not present significant differences in the climbing index with respect to the control group and the control group with the consortium.Regarding the number of dopaminergic neurons, it was observed that in both the healthy and Parkinson's groups there was a tendency toward an increase in the number of dopaminergic neurons after administration of the microbial consortium, which was not significant.

[0040] The metabolomic study in the heads of the different experimental groups showed metabolic alterations related to energy, amino acid, and neurotransmitter metabolism in the Parkinson's group compared to the control group. Treatment with the microbial consortium significantly reduced these alterations and the metabolic differences with the healthy group. Furthermore, it reduced the level of biomarkers reported in the literature as being associated with the disease in patients with Parkinson's.

[0041] Analysis of the gut microbiome composition of the experimental groups revealed that the microbial consortium significantly increased species diversity in the Parkinson's group, favoring the presence of beneficial species.

[0042] Based on these results, it is possible to conclude that the present invention provides a potential formulation for the prevention and / or treatment of neurodegenerative diseases, mental illnesses or psychiatric disorders.

[0043] In the present invention, the term "prevention" means avoiding the onset of a disease or pathological symptomatology in an individual, particularly when the individual is predisposed to developing said pathology, but has not yet been diagnosed. In the present invention, said disease corresponds to a neurodegenerative disease, or a mental illness or psychiatric disorder. Neurodegenerative diseases can be selected from, but are not limited to: Parkinson's, Alzheimer's, Amyotrophic Lateral Sclerosis, Huntington's Disease, Friedreich's Ataxia, Spinal Muscular Atrophy, Dementia with Lewy bodies, to mention a few. Mental illnesses or psychiatric disorders can be selected from, but are not limited to, depression, anxiety, and autism spectrum disorders (ASD), to mention a few.

[0044] In the present invention, the term "treating" or "treatment" refers to reducing the disease and its symptoms, i.e., halting its progression; alleviating the disease, i.e., causing the regression of the disease or pathological condition; alleviating or reducing at least one of the symptoms or signs of the disease or pathological condition; and / or stabilizing the disease or pathological condition in an individual. In the present invention, said disease to be remedied corresponds to a neurodegenerative disease, or a mental illness or psychiatric disorder. Neurodegenerative diseases may be selected from, but are not limited to: Parkinson's, Alzheimer's, Amyotrophic Lateral Sclerosis, Huntington's Disease, Friedreich's Ataxia, Spinal Muscular Atrophy, Dementia with Lewy Bodies, to name a few.Mental illnesses or psychiatric disorders can be selected, but are not limited to, depression, anxiety, and autism spectrum disorders (ASD), to name a few.

[0045] EXAMPLES:

[0046] Microorganisms used:

[0047] For the design of the microbial consortia, the list of microorganisms from bacteria isolated from the intestinal microbiome of Chilean subjects was considered, particularly bacteria belonging to the bacterial phyla Actinomycetota, Bacteroidota and Bacillota (Table 1, Table 2 and Table 3). Table 1: Bacterial strains from Chilean fecal isolates.

[0048] Bacterial phylum Strains Abbreviation

[0049] Actinomycetota Bifidobacterium adolescentis D3 Ba_D3

[0050] Bifidobacterium adolescentis R2 BA_R2

[0051] Bifidobacterium longum D4 BI_D4

[0052] Bifidobacterium longum E1 BI_E1

[0053] Bifidobacterium longum M 12 BI_M 12

[0054] Bifidobacterium longum S3 BI_S3

[0055] Propionibacterium acnes D5 Pa_D5

[0056] Bacteroidetes Bacteroides fragilis C1 Bf_C1

[0057] Bacteroides fragilis J4 Bf_J4

[0058] Bacteroides thetaiotaomicron S5 Bt_S5

[0059] Phocaeicola vulgatus S1 (formerly Bacteroides vulgatus) Bv_S1

[0060] Bacillus Clostridium perfringens A3 Cp_A3

[0061] Enterococcus faecalis H 1 Ef_H 1

[0062] Enterococcus faecium G1 Efa_G1

[0063] Levilactobacillus brevis G2 (formerly Lactobacillus brevis) Lb_G2

[0064] Lacticaseibacillus paracasei M38 (formerly Lactobacillus paracasei) Lc_M38 Streptococcus mitis LM4 Sm_LM4

[0065] Streptococcus salivarius LM5 Ss_LM5 Tabla 2: Bacteria producing GABA, database of Virtual Metabolic Human.

[0066] Filo bacterialon Onions Abbreviation

[0067] Actinomycetota Bifidobacterium adolescentis ATCC 15703 Ba_ATCC15703

[0068] Bifidobacterium angularum DSM 20098 Ban_DSM20098

[0069] Bifidobacterium dentium ATCC 27678 Bd_ATCC27678

[0070] Bifidobacterium bifidum BGN4 Bf_ BGN4

[0071] Bifidobacterium bifidum NCIMB 41171 Bf_ NCIMB 41171

[0072] Bifidobacterium bifidum PRL2010 Bf_ PRL2010

[0073] Bifidobacterium bifidum S17 Bf S17

[0074] Bifidobacterium longum subsp. Infantis 157F-NC Bi_157F-NC

[0075] Bifidobacterium longum subsp. InfantisMCC 15697 Bi_ATCC15697

[0076] Levilactobacillus brevis ATCC367 Lb_ATCC367 Bacillota Lactiplantibacillus plantarum subsp. Plantarum

[0077] ATCC 14917 (formerly Lactobacillus plantarum) Lp ATCC14917 Lactiplantibacillus plantarum JDM1 (formerly

[0078] Lactobacillus plantarum) Lp_JDM1

[0079] Lactiplantibacillus plantarum WCFS1 (formerly Lactobacillus plantarum) Lp_WCFS1

[0080] Streptococcus salivarius JIM8777 Ss_JIM8777

[0081] Lachnoclostridium WAL 14673 Ls WAL 14673

[0082] Table 3: Butyrate-producing bacteria, from the Virtual Metabolic Human database.

[0083] Filo bacterialon Onions Abbreviation

[0084] Clostridium butyricum DSM 10702 Cb_ DSM 10702

[0085] Coprococcus eutactus ATCC 27759 Ce_ATCC 27759

[0086] D ... . Eubacterium biforme DSM 3989 Eb_ DSM3989 Bacillota

[0087] Eubacterium rectal ATCC33656 Er_ATCC33656

[0088] Faecalibacterium prausnitzii KLE1255 Fp_KLE1255

[0089] Roseburia intestinalis L182 Ri_L182 Bacterial fungi and cultivation media:

[0090] The microorganisms that formed the different consortia were obtained from the Systems Microbiology Laboratory (Professor Daniel Garrido) at the School of Engineering of the Pontifical Catholic University of Chile. They were grown in Reinforced Clostridial Medium (RCM, Becton-Dickinson, Franklin Lakes, NJ) and Man, Rogosa, and Sharpe medium (MRS, Becton-Dickinson, Franklin Lakes, NJ), supplemented with 1.0 g / L and 0.5 g / L of L-cysteine ​​(Loba Chemie, India), respectively. The bacteria were incubated at 37°C for 48 h in anaerobic jars (Anaerocult, Merck, Darmstadt, Germany) with anaerobic patches (Gaspak EM, Becton-Dickinson, Franklin Lakes, NJ, USA) until use.

[0091] Monoculture growth evaluation: The growth of potential bacteria that would form the consortia in monoculture was evaluated with both inulin and glucose as carbon sources. Figure 1 shows the growth (OD620 nm) of the bacteria in monoculture with glucose and inulin as carbon sources for 48 h.

[0092] Growth of microbial consortia in batch bioreactors:

[0093] For the growth of microbial consortia, bacteria were cultured and inoculated (initial OD620nm of 1 ) in a 250 mL batch bioreactor connected to a MyControl system (Mini-bio ApplikonBiotechnology, Netherlands). 210 mL of optimized mZMB culture medium was used, supplemented with 10 g / L inulin, 0.5 g / L L-cysteine, and 0.1% monosodium glutamate. The bioreactor was maintained at 37°C, pH 5.5, stirring at 90 rpm, and anaerobic environment by nitrogen injection (99.99% purity) to simulate the conditions of the proximal colon. Samples were taken every 2 h for 24 h and were analyzed, where it was detected that the biological replicas of each of the consortia had a similar behavior, which is why the growth kinetics of the consortia for the reactors did not have significant differences.Similarly, the values ​​obtained for the specific growth rate in the biological replicates of each consortium were very similar. Overall, it was observed that all consortia had a lag phase of approximately 10 h. The stationary phase was reached between 4 and 10 h in all consortia, and growth close to 2 OD620 nm was achieved (Figure 2).

[0094] Strain selection and preparation of the microbial consortium.* After experimental characterization of the bacteria in monoculture and the microbial consortia, a consortium was selected for evaluation in a model of Parkinson's disease (PD) in D. melanogaster. The selection criterion considered the production of a metabolite of interest by the consortium. The selected consortium consisted of two species, Levilactobacillus brevis G2 deposited under accession number RGM 3512; and Lacticaseibacillus paracasei M38 deposited under accession number RGM 3511. To prepare the formulation, the bacteria from the selected consortium were grown (inoculum 10% v / v) in their respective complex media (MRS-cys or RCM-cys) for 48 hours at 37°C under anaerobic conditions.Cultures were then centrifuged at 7871 xg for 2 min and pellets were resuspended in sterile 1X PBS supplemented with inulin (1% v / v) as a carbon source and monosodium glutamate (0.1% v / v).

[0095] Evaluation of the neuroprotective effect of the microbial consortium in a Drosophila melanogaster model

[0096] Different Drosophila lines were used for this study, Table 4 shows the experimental groups that were used.

[0097] Table 4: Experimental groups of Drosophila melanogaster.

[0098] Group Treatment Justification

[0099] Control Axenic D. melanogaster + diet Negative control

[0100] (standard group

[0101] Control)

[0102] Parkinson's disease Axenic D. melanogaster with Parkinson's disease Control

[0103] (EP Group) + standard Parkinson's diet

[0104] Control+ axenic D. melanogaster with consortium Experimental group to have

[0105] Selected consortium + standard diet an approach on selected safety and toxicity of the selected consortium Parkinson + axenic D. melanogaster with consortium Experimental group to evaluate selected consortium and Parkinson + standard diet the effect of the selected consortium on the phenotype (Group EP + Parkinson's consortium)

[0106] The neuroprotective effect of microbial consortia was evaluated in D. melanogaster. A genetic model of PD was developed based on the overexpression of the mutated α-synuclein protein in neurons, whose aggregation in neurons is associated with the development of the disease. To generate the Parkinson's line, virgin Elav-GAL4 females were crossed with UAS-α Syn males. The UAS-Syn line expresses the A30P mutant of α-synuclein under UAS control (also called responder). Additionally, the Elav-GAL4 line (called driver, which expresses the GAL4 transcription factor under a neuronal promoter (Elav)) is also required. Thus, the Parkinson's line was obtained from the cross of virgin Elav-GAL4 females with UAS-Syn males. Four experimental groups were used: the control group and PD with and without administration of the consortium.Survival, locomotor capacity, and the number of dopaminergic neurons were assessed in these groups. In the same groups of flies in which locomotion was assessed, the metabolic profile of each experimental group was determined in the head. Finally, the bacterial genera in the gut microbiome of each experimental group were characterized.

[0107] Survival assay:

[0108] To assess fly longevity, survival assays were performed. Experiments were conducted with newborn males from each group in triplicate, with 20 males per biological replicate. Flies were transferred every two days to a new vial, and the number of dead flies was recorded. Flies were kept at 25°C throughout the experiment, and the experimental groups were defined as Control, EP, Control + consortium, and EP + consortium.

[0109] Figure 3 shows the results obtained. Overall, there were no statistically significant differences in survival between the different groups (Figure 3, panel A). However, the consortium significantly increased the survival of the treated control group compared to the untreated control group (Figure 3, panel B). Therefore, the application of the consortium not only did not have a negative impact on the survival of D. melanogaster but actually prolonged its survival.

[0110] Climbing test:

[0111] To evaluate the flies' locomotor ability, a climbing test was performed based on their negative geotactic behavior. For this test, newborn females were transferred in groups of 15 flies to vials containing standard food. The test was conducted at 10 and 25 days of age in triplicate, and each experimental group consisted of a total of 135 flies.

[0112] Prior to testing, the flies were kept for 15 minutes at a temperature of 23°C and in daylight to acclimatize to the environmental conditions of the test. The flies were kept inside the climbing device's tube for one minute to acclimatize, after which they were lowered to the bottom of the tube by a series of successive taps on a smooth surface covered with a mat to cushion the impact and prevent severe trauma to the flies. After the taps, the flies fell to the bottom of the tube, got up, and climbed the walls of the tubes again, reaching a second tube placed next to them. After 30 seconds, the flies were switched to a clean tube and the taps were repeated. This process was repeated six times at 30-second intervals for each condition.A climbing index was calculated by determining the number of flies that did not reach the second tube in each of the 6 test tubes and using the formula: climbing index. where #Fn corresponds to the number of flies in the tube, "n" (being 0 in the initial tube and 5 in the last tube), and #FT is the total number of flies.

[0113] Figure 4 shows the results of the climbing test performed on the different experimental groups. The experimental groups are presented on the “x” axis of the bar graphs, and the climbing index is shown on the “y” axis. The cross α-synuclein x control w(cs) corresponds to the genetic control group, α-synuclein x Elav-Gal4 to the EP group, α-synuclein x control w(cs) + G2BÍM38 indicates the genetic control group that received the microbial consortium, and α-synuclein x Elav-Gal4 + G2BtM38 corresponds to the EP group that received the consortium.

[0114] As can be seen in panel A of Figure 4, the climbing index of the EP group was significantly lower than that of the control group. That is, at 10 days, flies in the EP model that express the mutant form of α-Synuclein A30P in neurons show a premature loss of climbing ability compared to their genetic control. At 25 days (panel B), the EP group also showed a reduction in their climbing ability compared to the control group, and both groups showed lower climbing indices compared to 10 days. Regarding the groups that received the consortium, it could be noted that at both 10 and 25 days, the climbing index of the EP group that received treatment was significantly higher than that of the group with the disease that did not receive the consortium. Moreover, the EP group that received the consortium did not show significant differences in climbing index compared to the control group and the control group with the consortium.

[0115] Immunofluorescence of dopaminergic neurons:

[0116] The number of dopaminergic neurons was assessed by immunostaining with anti-TH antibody followed by confocal microscopy. For this, 25-day-old adult females were used. The experimental groups were considered Control, EP, Control + consortium and EP + consortium. The flies were anesthetized with CO2 and their brains were dissected in 1X PBS. Subsequently, the brains were fixed with 4% paraformaldehyde at room temperature for 15 minutes with shaking at 70 rpm followed by 5 washes of 5 minutes with 1X PBS supplemented with triton (0.03%) (PBST). A blocking solution (PBST + 1% goat serum) was applied for 1 h, and then the brains were incubated with the primary antibody generated in rabbit (Rabbit anti-TH), which was diluted 1:500 in the blocking solution and added to the brain samples, which were kept at 4°C overnight. The brains were then washed 4 times with PBST for 7 minutes per wash.The samples were then incubated for 2 h with the secondary antibody against rabbit antibodies conjugated to the fluorescent molecule Fit-C (Rabbit fit-C), which was diluted 1:300 in the blocking solution. The samples were then washed three times for 7 min each in PBST buffer. Finally, they were placed in 50% glycerol for hydration and storage at 4°C until mounting in Vectashiel medium.

[0117] Images were taken using an Olympus FV1000 confocal microscope (Shinjuku, Tokyo, Japan). A total of 10 images were taken per experimental group at 25 days of age. Images were processed, and the total number of dopaminergic neurons was counted using the open-source program ImageJ.

[0118] The results at 25 days are shown in Figure 5. Panel A shows a representative confocal microscopy photograph of TH immunofluorescence in the control group. Panel B presents the quantification of dopaminergic neurons for each experimental group. Each experimental group consisted of eight brains.

[0119] As can be seen in Figure 5, the number of dopaminergic neurons in the PD group was significantly lower than in the control group and in the group that received the consortium. It is also possible to see that the PD group that received the treatment did not present significant differences with either control group or the PD group that did not receive treatment. However, in both the control group and the PD group that received the consortium, there was a tendency toward an increase in the number of dopaminergic neurons, which differentiates them from the group with the disease.

[0120] Metabolomics assay:

[0121] To identify overexpressed metabolites and altered metabolic pathways in flies with PD and treated flies compared to controls, a metabolomics assay using 1H nuclear magnetic resonance (1H NMR) was performed. The assay was performed head-to-head for all four experimental groups at 10 days of age. The methodology included sample preparation, 1H NMR analysis, metabolite assignment, and multivariate analysis.

[0122] Figure 6 shows a three-dimensional PCA plot showing that the samples clustered by phenotype. The Parkinson's group that received the consortium (yellow) was further removed from the untreated Parkinson's group (red) and closer to the control group (green).

[0123] Figure 7 shows the main metabolic alterations in Drosophila. Comparison of the metabolic profile of the PD group with respect to the control group (Figure 7, panel A) revealed several metabolic alterations, mainly related to the glycolysis and citric acid cycle (TCA) pathways. Interestingly, when comparing the treated PD group with the control group, it was noted that the consortium restored these metabolic alterations related to energy balance, redox, neurotransmitter and amino acid metabolism (Figure 7, panel B, metabolites in red). Moreover, the consortium reduced the level of biomarkers reported in PD patients (Figure 7, panel C, metabolites boxed in a rectangle).

[0124] Analysis of the 16S rRNA profile of the intestinal microbiome of Drosophila melanogaster.

[0125] To analyze the gut microbiome composition of the four experimental groups, the midgut of 10-day-old Drosophila females was dissected in sterile 1X PBS. Prior to dissection, flies were anesthetized with CO2 and kept on ice for 15 min. During the dissection, the Malpighian tubules and trachea were removed. Fifteen guts were collected in triplicate per condition (45 guts in total), frozen in liquid nitrogen, and stored at -80°C until use. Subsequently, guts were homogenized with 200 uL of PBS using a small mortar and pestle, and DNA was extracted using the Zymo Quick-DNA fecal / soil microbe miniprep kit (D6010, Zymo, Irvine, CA) following the manufacturer's instructions. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq sequencer following the manufacturer's guidelines.Paired-end reads of length 250 were obtained and analyzed using QIIME2 and R software. These were filtered using DADA2 with the denoised-paired tool, which allowed removing chimeras and filtering reads to a minimum length of 240 nucleotides. For taxonomic classification, the GreenGenes 13.8 database trained with the classify-skleam classifier was used. Then, the taxonomic annotation results for Amplicon sequence variants (ASVs) were exported and converted to the Phyloseq format in R. Subsequently, the relative abundance of genera and species was graphed. Finally, Shannon diversities were calculated and plotted according to their significance using t-test to compare significant differences between conditions.

[0126] Taxonomic classification results revealed a predominance of Acetobacter in all groups. Treatment with the consortium resulted in a reduction in the abundance of this genus, which was most noticeable in the EP group, where Staphylococcus, Lactobacillus, and Corynebacterium genera could be distinguished (Figure 8, panel A). Moreover, the consortium produced a shift in the microbiome composition in the control and EP groups, where in particular, the species diversity of the EP group with the consortium was significantly higher than in the untreated control and EP groups.

[0127] In view of the foregoing, those skilled in the art will understand that changes can be made to the specific aspects described and still obtain the same or similar results without departing from the spirit and scope of the invention. Therefore, the specific functional and structural details described herein should not be construed as exhaustive. It should be understood that the entire description of each reference cited herein is incorporated into the description of the present application.

[0128] While this invention has been described in the manner set forth above, it may seem obvious that other alternatives, modifications, or variations would yield the same results. However, we have been able to determine that the subject matter described in this application is fundamental to the success of the invention described. Consequently, the embodiments of the invention are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the invention as defined in the following claims.

[0129] All patents, patent applications, scientific articles and other public documents that, to the applicant's knowledge, constitute the state of the art have been duly cited in this application.

[0130] References cited:

[0131] Bittihn, P, Din, M. O., Tsimhng, L. S., & Hasty, J. (2018). Rational engineering of synthetic microbial systems: from single cells to consortia. Current Opinion in Microbiology, 45, 92-99. https: / / doi.Org / https: / / doi.org / 10.1016 / j.mib.2018.02.009

[0132] Dhyani, R, Goyal, C., Dhull, S. B., Chauhan, A. K., Singh Saharan, B., Harshita, Duhan, J. S., & Goksen, G. (2023). Psychobiotics for Mitigation of Neuro- Degenerative Diseases: Recent Advancements. Molecular nutrition & food research, e2300461. Advance online publication.

[0133] Dinan, T. G., Stanton, C., & Cryan, J. F. (2013). Psychobiotics: a novel class of psychotropic. Biological psychiatry, 74(10), 720-726.

[0134] Du, R, Zhao, H., Zhang, H., Wang, R., Huang, J., Tian, Y, Luo, X., Luo, X., Wang, M., Xiang, Y, Qian, L., Chen, Y, Tao, Y, & Lou, C. (2020). De novo design of an intercellular signaling toolbox for multi-channel cell-cell communication and biological computation. Nature communications, 11(1 ), 4226. https: / / doi.Org / 10.1038 / s41467-020-17993-w

[0135] Sharma, R., Gupta, D., Mehrotra, R., & Mago, P. (2021 ). Psychobiotics: The Next- Generation Probiotics for the Brain. Current microbiology, 78(2), 449-463. https: / / doi.Org / 10.1007 / s00284-020-02289-5

Claims

CLAIMS 1. A composition for the prevention and / or treatment of neurodegenerative diseases, mental illnesses or psychiatric disorders, CHARACTERIZED in that it comprises one or more microorganisms selected from the bacterial phyla, Bacteroidota, Bacillota, Actinomycetota, and / or combinations thereof.

2. The composition according to claim 1, CHARACTERIZED in that the bacteria are selected from the genera Bifidobacterium, Bacteroides, Clostridium, Coprococcus, Enterococcus, Eubacterium, Faecalibacterium, Levilactobacillus, Lacticaseibacillus, Lactobacillus, Propionibacterium, Streptococcus, Roseburia, and / or combinations thereof.

3. The composition according to claim 1, CHARACTERIZED because the bacteria are selected from the species Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium bifidum, Bifidobacterium longum subsp. Infantis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Clostridium perfringens, Clostridium butyricum, Coprococcus eutactus, Enterococcus faecalis, Enterococcus faecium, Eubacterium biforme, Eubacterium rectale, Faecalibacterium prausnitzii, Levilactobacillus brevis, Lacticaseibacillus paracasei, Lactobacillus plantarum, Lactobacillus plantarum subsp. Plantarum, Propionibacterium acnes, Streptococcus mitis, Streptococcus salivarius, Roseburia intestinalis, and / or combinations thereof.

4. The composition according to claim 1, CHARACTERIZED in that the bacteria are selected more particularly from the RGM 3511 (Lacticaseibacillus paracasei M38) and RGM 3512 (Levilactobacillus brevis G2) deposits.

5. The composition according to claim 1, CHARACTERIZED in that it also comprises pharmaceutically acceptable excipients.

6. The composition according to claim 1, CHARACTERIZED in that it is available in both solid and liquid form, including tablets, granules, powders, capsules and solutions, emulsions and suspensions.

7. The composition according to claim 1, CHARACTERIZED in that the composition is in the form of a drink, syrup, food or beverage or food supplement.

8. A microbial consortium for the prevention and / or treatment of neurodegenerative diseases, mental illnesses or psychiatric disorders, CHARACTERIZED in that it comprises one or more microorganisms selected from the bacterial phyla Bacteroidota, Bacillota, Actinomycetota, and / or combinations thereof.

9. The microbial consortium according to claim 8, CHARACTERIZED in that the bacteria are selected from the genera Bifidobacterium, Bacteroides, Clostridium, Coprococcus, Enterococcus, Eubacterium, Faecalibacterium, Levilactobacillus, Lacticaseibacillus, Lactobacillus, Propionibacterium, Streptococcus, Roseburia, and / or combinations thereof.

10. The microbial consortium according to claim 8, CHARACTERIZED in that the bacteria are selected from the species Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium bifidum, Bifidobacterium longum subsp. Infantis, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Clostridium perfringens, Clostridium butyricum, Coprococcus eutactus, Enterococcus faecalis, Enterococcus faecium, Eubacterium biforme, Eubacterium rectale, Faecalibacterium prausnitzii, Levilactobacillus brevis, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Lactobacillus plantarum subsp. Plantarum, Propionibacterium acnes, Streptococcus mitis, Streptococcus salivarius, Roseburia intestinalis, and / or combinations thereof.

11. El consorcio microbialo de accordo con la reivindicación 8, CHARACTERIZADO porque preferentemente, las bacteria que se se seleccionan para obtener dicho consorcio microbial psicobiótico, se encuentra depositadas bajo los numeros de acceso RGM 3511 (Lactiseibacillus paracasei M38 y RGM 3512 Levilactobacillus brevis G2).

12. Use of the composition according to claims 1 to 7, CHARACTERIZED in that it serves to prepare a supplement that is useful for the prevention and / or treatment of diseases in a subject.

13. The use according to claim 12, CHARACTERIZED in that the disease may be a neurodegenerative disease, which is selected from Parkinson's Disease, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease, Friedreich's Ataxia, Spinal Muscular Atrophy, Dementia with Lewy bodies.

14. The use according to claim 12, CHARACTERIZED in that the disease may be a mental illness or psychiatric disorder, which is selected from depression, anxiety and autism spectrum disorders (ASD).

15. The use according to claim 12, CHARACTERIZED in that the subject is a human.

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