IT202200022464A1 - Bacteria Strains to Support Mood and Cognitive Function, Their Compositions and Related Uses - Google Patents

Description

Description of the invention entitled:

"Bacterial Strains for Supporting Mood and Cognitive Function, Their Compositions and Related Uses"

The present invention relates to bacterial strains belonging to the genus Bifidobacterium and Lactobacillus, their combination and their use in therapy, in particular for mood regulation and cognitive function support. The invention also relates to pharmaceutical compositions, nutraceuticals and food supplements containing at least one of these strains belonging to the genus Bifidobacterium and Lactobacillus. Furthermore, the present invention relates to selected bacterial strains belonging to the species Bifidobacterium and Lactobacillus, as well as mixtures and compositions thereof, for use in a method of treatment, preferably preventive or curative, of mood regulation and cognitive function support, preferably for use in a method of treatment of brain disorders, including the treatment and/or prevention of mood disorders, such as anxiety and depression, and for the improvement of cognitive function and the treatment and prevention of cognitive decline, such as in Alzheimer's disease and dementia.

Mood disorders and cognitive decline are serious medical conditions that affect a large portion of the population.

Mood disorders, such as major depressive disorder (MDD) and bipolar disorder (BD), are a heterogeneous group of psychiatric illnesses that affect emotional, cognitive, and behavioral domains.

Despite advances in drug development, the majority of people treated for a mood disorder do not achieve complete symptomatic remission and functional recovery, and overall the prevalence of mood disorders, especially treatment-resistant mood disorders, is increasing globally. Furthermore, guideline-recommended pharmacological treatments were not developed based on disease pathophysiology and their impact on the neurobiology of these conditions remains incomplete. Another factor to consider is that a substantial proportion of patients are unable to tolerate existing medications. Regarding the pathophysiology of these disorders, recent evidence suggests that immune-inflammatory, oxidative stress, and metabolic mechanisms are directly involved: for example, peripheral mediators of inflammatory dysregulation have been substantially implicated during mood and euthymic episodes in both MDD and BD. Furthermore, approximately 60% of patients with anxiety and depression present with impaired bowel function, such as in irritable bowel syndrome. The genes in the brain and the gut are quite similar, especially in relation to neuronal synapse formation, so some gene mutations can lead to abnormalities in both the brain and the gut. The gut microbiome is considered a key component of the gut-brain axis, and there is ample evidence that it can influence symptoms of depression and anxiety. Specifically, the gut is controlled by intrinsic and extrinsic factors: the intrinsic factor is the gut neural system, which in turn is also controlled by extrinsic factors, including vagal and sacral parasympathetic nerves and visceral sympathetic nerves. The humoral connection includes the hypothalamic-pituitary-adrenal axis, responsible for regulating the stress response, and gut endocrine cells, which secrete neuropeptides and gut peptides that act locally and through afferents of the vagus nerve and spinal cord or the blood-brain barrier to act on the brain. The analysis of the gut-brain microbiota axis and the effects that microbiota bacteria can have on the functioning of the central nervous system has shown that, with regard to psychiatric disorders, an interesting approach is the integration of probiotics; probiotics are live bacteria that colonize the gastrointestinal tract and exert a beneficial effect on the health of the host. In particular, probiotics can regulate the constitution of the intestinal microbiota: some authors have shown that the administration of foods containing probiotics to a patient suffering from anxiety led to an increase in total sleep time in the third week, useful for alleviating chronic stress, and to a constitutional change of the microbiota after 2-3 weeks with an increase in Lactobacillus. In general, probiotics can reduce proinflammatory cytokines and oxidative stress in humans, which are related to mood disorders, and can alleviate the symptoms of depression and anxiety: other authors have found that rats administered probiotics performed better than those administered a placebo and similar to those administered diazepam, the reference standard substance, in performing a test on the degree of anxiety. One study indicates that behavioral and neurological changes induced by chronic stress can be alleviated through intervention with Bifidobacterium longum and others found that consumption of probiotics containing Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium bifidum for a period of eight weeks led to a significant reduction in depressive symptoms. Additionally, one study suggests that daily administration of L. plantarum can lead to a decrease in depressive symptoms, cortical arousal, and fatigue levels, as well as improved deep sleep quality.

Cognitive functions are the most complex properties of the nervous system, as they are responsible for rational perception, cognition and interaction with the external world. They are important for the performance of some highly intelligent complex tasks and also in the most ordinary daily activities. Cognitive decline is a growing public health concern. It refers to the deterioration of cognitive abilities at various levels and is very common in a variety of conditions, including aging exerted by oxidative stress and neuroinflammation, which is one of the main factors responsible for the most common neurodegenerative diseases. These factors act mainly by causing disruptions in synaptic transmission and neuronal dysfunction. Most industrialized countries are facing a rapid increase in the percentage of older people considered to be at high risk of neurological diseases; therefore, the development of effective preventive and therapeutic strategies targeting neurodegenerative disorders should be considered a public health priority to promote healthy aging in the population.

In this context, there is an urgent need to find treatments that support cognitive functions. In recent decades, the gut microbiome has emerged as a critical factor influencing neurophysiological and psychophysiological functions, including cognition, emotional neurotransmission, and neurodevelopment. In particular, the gut microbiota undergoes significant transitions in its composition and function during aging, and these alterations may influence health and age-related diseases. Recently, the emerging concept of the gut-brain axis, which refers to a bidirectional relationship between the gut and the brain, has linked the gut microbiota to age-related neurodegenerative diseases, such as Alzheimer's disease, and related cognitive dysfunction. The gut-brain interaction involves a complex network of endocrinological, immunological, and neural mediators, which has been considered a critical target for the manipulation of brain health and neurodegenerative diseases. Among the various microorganisms that colonize the gastrointestinal tract, probiotics, which are transient entities that act on different pathways, could be used to repopulate the intestinal flora and restore the physiology of the organism. For example, it has been shown that the consumption of probiotics increases the expression of brain-derived neurotrophic factor (BDNF), a growth factor crucial for brain plasticity, memory and neuronal health, which is abnormally reduced in patients with depression; or treatment with Lactobacillus plantarum has shown anti-Alzheimer's properties against D-galactose-induced Alzheimer's disease, and there are still scientific articles on the efficacy of Lactobacillus paracasei in improving memory function through nerve growth factor-mediated neurogenesis.

In light of the above, it is clear that it would be useful to identify new probiotic-based treatments for the urological and gynecological disorders described above, as an alternative to conventional phytotherapeutic and pharmacological treatments.

Following extensive research and development, the Applicant has surprisingly discovered that specific bacterial strains of the genus Bifidobacterium and Lactobacillus are able to exert significant beneficial activity in brain disorders, particularly mood disorders, and are also effective in improving brain function.

In particular, the Applicant surprisingly discovered that specific bacterial strains belonging to the genus Bifidobacterium and Lactobacillus, selected from the group comprising or, alternatively, consisting of:

- Lactobacillus paracasei "TJB8", deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 33129 on 17 May 2019;

- Bifidobacterium bifidum "novaBBF7", registered with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with registration number DSM 34336 from 26 July 2022; And

- Bifidobacterium longum "novaBLG2", deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 34339 on 26 July 2022; and mixtures thereof,

They are able to perform an important beneficial activity in brain disorders, especially in mood disorders, and are also effective in improving cognitive function.

All bacterial strains mentioned in the present invention have been deposited in accordance with the provisions of the Budapest Treaty. The Depositing Party of the bacterial strains described and/or claimed in the present patent application and the owner thereof express, from now on, their consent to make all said strains available for the entire duration of the patent.

Bacterial strains belonging to the species Lactobacillus paracasei have been reclassified with the nomenclature Lacticaseibacillus paracasei.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the dose-response study on SHSY-5Y cells for the evaluation of cell viability. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs other concentrations.

Figure 2 shows cell viability, TEER value and butyric acid quantification. Panel A shows cell viability assessed by MTT. Panel B shows TEER analysis and panel C shows butyric acid quantification assessed by ELISA kit. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs other concentrations.

Figure 3 shows cell viability, JC-1 and lipid peroxidation assays. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs L-Glu; #p<0.05 vs single agents.

Figure 4 shows the activity of BAX and Cyto-C. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs L-Glu; #p<0.05 vs individual agents.

Figure 5 shows the activity of IL6 and IL10. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs L-Glu; # and bar p<0.05 vs individual agents.

Figure 6 shows M2 activity and Acetylcholine production. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs L-Glu; the bar and # p<0.05 vs single agent.

Figure 7 shows BDNF and GDNF activity and serotonin production. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs L-Glu; #p<0.05 vs single agents.

Figure 8 shows the dose-response study on SHSY-5Y cells for the evaluation of cell viability. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs other concentrations.

Figure 9 shows cell viability, TEER value and butyric acid quantification. Panel A shows cell viability assessed by MTT. Panel B shows TEER analysis and panel C shows butyric acid quantification assessed by ELISA kit. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs other concentrations.

Figure 10 shows cell viability, ERK/MAPK and PI3K/AKT analysis. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs H2O2 ; #p<0.05 vs single agents.

Figure 11 shows ROS production and SOD and iNOS analysis. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs H2O2 ; #p<0.05 vs single agents.

Figure 12 shows the analysis of JC-1, p53 and BAX activity. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs H2O2 ; #p<0.05 vs single agents.

Figure 13 shows the analysis of APP and APOE. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs H2O2 ; #p<0.05 vs single agents.

Figure 14 shows the analysis of mitochondrial metabolism and lipid peroxidation. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs Fe<3+ >; bar p<0.05 vs single agent.

Figure 15 shows the molecular pathways involved in neurodegeneration. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs Fe<3+ >; bar p<0.05 vs single agent.

DESCRIPTION OF THE INVENTION

According to one of its aspects, the present invention relates to a bacterial strain selected from the group comprising or, alternatively, consisting of:

- Lactobacillus paracasei "TJB8", deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 33129 on 17 May 2019;

- Bifidobacterium bifidum "novaBBF7", registered with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with registration number DSM 34336 from 26 July 2022;

- Bifidobacterium longum "novaBLG2" deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with deposit number DSM 34339 from 26 July 2022;

and their mixtures.

Bacterial strains belonging to the species Lactobacillus paracasei have been reclassified with the nomenclature Lacticaseibacillus paracasei.

Preferably, the present invention relates to a pharmaceutical or nutraceutical association or combination or food supplement comprising or, alternatively, consisting of at least one of the above bacterial strains, as follows:

- Lactobacillus paracasei "TJB8" DSM 33129, preferably in freeze-dried form, or - Bifidobacterium bifidum "novaBBF7" DSM 34336, preferably in freeze-dried form, or - Bifidobacterium longum "novaBLG2" DSM 34339, preferably in freeze-dried form. Preferably, the present invention relates to a pharmaceutical or nutraceutical or food supplement association or combination comprising or, alternatively, consisting of at least two of the above bacterial strains, as follows:

(i) Lactobacillus paracasei "TJB8" DSM 33129 and Bifidobacterium bifidum "novaBBF7" DSM 34336; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example, 1:3 to 3:1; or 1:2 to 2:1; or 1:1; preferably all strains of bacteria are in freeze-dried form.

(ii) Lactobacillus paracasei "TJB8" DSM 33129 and Bifidobacterium longum "novaBLG2" DSM 34339; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example, 1:3 to 3:1; or 1:2 to 2:1; or 1:1; preferably all strains of bacteria are in freeze-dried form.

iii) Bifidobacterium bifidum "novaBBF7" DSM 34336 and Bifidobacterium longum "novaBLG2" DSM 34339; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example, 1:3 to 3:1; or 1:2 to 2:1; or 1:1; preferably all strains of bacteria are in freeze-dried form.

Preferably, the present invention relates to a pharmaceutical or nutraceutical association or combination or food supplement comprising or, alternatively, consisting of three of the above-mentioned bacterial strains, as follows:

(iv) Lactobacillus paracasei "TJB8" DSM 33129, Bifidobacterium bifidum "novaBBF7" DSM 34336 and Bifidobacterium longum "novaBLG2" DSM 34339; preferably in a weight ratio of 1:1:2, or 1:2:1, or 2:1:1, or 1:1:1, preferably all bacterial strains are in freeze-dried form.

Preferably, the present invention relates to a pharmaceutical or nutraceutical composition or a food supplement comprising or, alternatively, consisting of at least one of the bacterial strains described above: Lactobacillus paracasei "TJB8" DSM 33129, preferably in lyophilized form, or Bifidobacterium bifidum "novaBBF7" DSM 34336, preferably in lyophilized form, or Bifidobacterium longum "novaBLG2" DSM 34339, preferably in lyophilized form; and, optionally, together with at least one excipient and/or a physiologically and/or pharmaceutically acceptable carrier.

Preferably, the present invention relates to a pharmaceutical, nutraceutical or food supplement composition comprising or, alternatively, consisting of the association or combination of i), or ii), or iii), as indicated above; and optionally together with at least one excipient and/or a physiologically and/or pharmaceutically acceptable carrier.

Preferably, the present invention relates to a pharmaceutical or nutraceutical composition or food supplement comprising or, alternatively, consisting of the association or combination of iv), as indicated above; and, optionally, together with at least one excipient and/or a physiologically and/or pharmaceutically acceptable carrier. According to another of its aspects, the present invention relates to the use of at least one of the aforementioned bacterial strains and/or mixtures thereof i), or ii), or iii), or iv), as indicated above, in therapy and, more specifically, in brain disorders, including the treatment and/or prevention of mood disorders, such as anxiety and depression, and to improve cognitive function and treat and prevent cognitive decline, such as in Alzheimer's disease and senile dementia. According to another of its aspects, the present invention relates to the use of a pharmaceutical or nutraceutical composition or food supplement association or composition in therapy and, more specifically, in brain disorders, including the treatment and/or prevention of mood disorders, such as anxiety and depression, and to improve cognitive function and treat and prevent cognitive decline, such as in Alzheimer's disease and senile dementia. specifically, in brain disorders, including treatment to treat and/or prevent mood disorders, such as anxiety and depression, and to improve cognitive function and treat and prevent cognitive decline, such as in Alzheimer's disease and dementia. Preferably, the present invention relates to a composition comprising (w/w):

- from 1 mg to 100 mg of Lactobacillus paracasei TJB8, preferably from 5 mg to 50 mg, more preferably from 20 mg to 30 mg, preferably in lyophilized form, and/or

- 1 mg to 100 mg of Bifidobacterium bifidum novaBBF7, preferably 5 mg to 50 mg, more preferably 20 mg to 30 mg, preferably in lyophilized form, and/or

- 1 mg to 100 mg of Bifidobacterium longum novaBLG2, preferably 5 mg to 50 mg, more preferably 10 mg to 20 mg, preferably in lyophilized form.

Preferably, the present invention relates to a composition comprising 5 to 20 mg of Lactobacillus paracasei TJB8, 5 to 20 mg of Bifidobacterium bifidum novaBBF7 and 1 to 10 mg of Bifidobacterium longum novaBLG2, preferably in lyophilized form, more preferably 10 mg of Lactobacillus paracasei TJB8, 10 mg of Bifidobacterium bifidum novaBBF7 and 5 mg of Bifidobacterium longum novaBLG2, preferably in lyophilized form.

The compositions of the invention may comprise one or more physiologically and/or pharmacologically acceptable excipients and/or carriers.

Preferably, the bacterial strain Lactobacillus paracasei TJB8 is administered in a daily dose in the range of 1.0x10<6 >CFU/day to 1.0x10<12 >CFU/day; preferably, from 1.0x10<8 >CFU/day to 1.0x10<10 >CFU/day, such as 3.0x10<9 >CFU/day.

Preferably, the bacterial strain Bifidobacterium bifidum novaBBF7 is administered in a daily dose in the range of 1.0x10<6 >CFU/day to 1.0x10<12 >CFU/day; preferably, from 1.0x10<8 >CFU/day to 1.0x10<10 >CFU/day, such as 1.0x10<9 >CFU/day.

Preferably, the bacterial strain Bifidobacterium longum novaBLG2 is administered in a daily dose in the range of 1.0x10<6 >CFU/day to 1.0x10<12 >CFU/day; preferably, from 1.0x10<8 >CFU/day to 1.0x10<10 >CFU/day, such as 0.5x10<9 >CFU/day (CFU = Colony Forming Units).

The compositions can be administered once or more times a day, preferably once a day.

The strains of the invention may be present in the composition as live and viable bacteria, dead bacteria or their cellular components, cellular extracts, lysates or tyndallized.

According to one embodiment, the at least one physiologically and/or pharmacologically acceptable excipient may include prebiotics. According to one embodiment, the at least one physiologically and/or pharmacologically acceptable excipient may include a monosaccharide, a disaccharide, a polysaccharide, soluble and/or insoluble fiber, GOS, FOS, inulin, maltodextrin, and mixtures thereof.

According to one embodiment, the at least one physiologically and/or pharmacologically acceptable excipient may include partially hydrolysed guar gum (PHGG).

According to different embodiments, the physiologically and/or pharmacologically acceptable excipient can be selected from the group consisting of: inulin, a fructooligosaccharide (FOS), maltodextrin and mixtures thereof.

The compositions according to the invention are preferably compositions for oral use, advantageously in the form of capsules. However, other pharmaceutical forms may be used.

The strains of the invention have been subjected to several experimental tests, reported below, to study the interaction between the microbiome and the brain.

In the Figures, the combination of the invention is indicated as MIX.

The combination of the invention has been shown to exert an unexpected synergistic activity, as will be evident from the experimental tests that follow.

Mood disorders

First, assays were performed to investigate the role of the microbiome in the development of mood disorders, especially the mechanisms leading to anxiety and depression. A dose-response study was performed on an in vitro model using SHSY-5Y cells to evaluate the safety of different probiotics and substances, as follows:

? Bifidobacterium Bifidum novaBBF7 (hereinafter also referred to as B.

Bifidum) in the range from 5mg/ml to 30mg/ml (corresponding to 0.5x10<9>-3x10<9 >CFU)

? Bifidobacterium Longum novaBLG2 (hereinafter also referred to as B.

Longum) in the range from 1mg/ml to 30mg/ml (corresponding to 1x10<8>-3x10<9 >CFU)

? Lactobacillus Paracasei TJB8 (hereinafter also referred to as L. Paracasei) in the range of 3?g/ml to 20mg/ml (corresponding to 1x10<6>-6x10<9 >CFU) ? a strain of L. Helveticus TJA4 deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 33128 dated 17 May 2019 in the range of 1.1?g/ml to 17mg/ml (corresponding to 1.1x10<5>-1.7x10<9 >CFU)

? a strain of L. Plantarum TJA7 deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 33132 on 17 May 2019 in the range from 100?g/ml to 2.5mg/ml (corresponding to 2.5x10<8>-1x10<9 >CFU)

? a strain of B. Psychaerophilum Q5 deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with deposit number DSM 33131 as of 2 July 2019 in the range from 3?g/ml to 20mg/ml (corresponding to 3x10<5>-2x10<9 >CFU)

All substances were prepared directly in the stimulation medium.

The best concentration of each probiotic (based on conversion from CFU to mg/ml per day) was maintained for all subsequent experiments.

Based on the data obtained, the best concentration of each substance was tested for the intestinal barrier or to modulate the mediators released by probiotics. In this context, a gut/brain co-culture model was established by seeding intestinal CaCo-2 cells in the apical part of the Transwell? system and stimulating the cells with B. Bifidum 10 mg/ml, L. Longum 5 mg/ml, L. Paracasei 10mg/ml, alone and in combination. Samples metabolized by intestinal cells were used to stimulate brain epithelium (SHSY-5Y cells) placed in the basolateral compartment for 48 hours to analyze mitochondrial metabolism. Then, further experiments were performed to evaluate the muscarinic receptor activity.

Materials and methods

Cell Cultures

SH-SY5Y cells (ATCC, ) were cultured in a mixture of Dulbecco's Modified Eagle Medium F12 and Dulbecco's Modified Eagle Medium in a 1:1 ratio, supplemented with 10% fetal bovine serum, 2 mM HEPES, 2 mM L-Glutamine and 1% penicillin/streptomycin.

. Cells are maintained in an incubator at 37?C with 5% CO2 and 95% humidity. The CaCo-2 cell line, provided by the American Type Culture Collection

, was grown in DMEM/F12 containing 10% FBS, 2 mM l-glutamine and 1% penicillinstreptomycin at 37?C in a 5% CO2 incubator.

The cells were treated with different concentrations of six different probiotics to find the minimum effective dose to use in the final formulation:

? B. Bifidum novaBBF7, 5 mg/mL to 30 mg/mL

? B. Longum novaBLG2, 1 mg/mL to 30 mg/mL

? L. Plantarum TJA7, 2.5 mg/mL to 30 mg/mL

? B. Psychaerophilum Q5, 3 ?g/mL to 20 mg/ mL

? L. Paracasei TJB8, from 3 ?g/mL to 20 mg/mL

? L. Helveticus TJA4, from 1 ?g/mL to 17 mg/mL

The probiotics with the dosages selected for the final formulation were also tested after pre-treatment with H2O2100 ?M, Fe<3+ >75 ?M and Glutamate 8mM for 30 minutes to understand their role in the regeneration of brain damage.

Gut-brain axis

To test the interaction between CaCo-2 cells and SHSY-5Y cells, the Transwell co-culture system was used. SHSY-5Y cells were pretreated to induce hypoxia-reoxygenation injury as previously described. A semipermeable membrane with 0.4 μm pores (BD Biosciences) was used to separate the two chambers filled with DMEM medium. CaCo-2 cells were seeded in the upper chamber, while SHSY-5Y cells were seeded in the lower chamber. Cell viability assays, quantification of ROS production, and evaluation of mitochondrial metabolism during brain degenerative process were performed on the cells.

MTT Test

At the end of the experiment, the MTT assay was performed as described in the literature to determine cell viability after stimulations. Cells were incubated in DMEM without phenol red 0% FBS with 1% MTT dye for 2 hours in a 37?C incubator, then cell viability was determined by measuring absorbance using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 570 nm with correction at 690 nm. The results were obtained by comparing them with control cells (100% viable).

Acetylcholine ELISA Kit

To determine the Ach level, the Choline/Ach Quantification Kit (Merck, Milan, Italy) was used, as reported in the literature. Briefly, blood was centrifuged at 4,000 rpm for 10 min at 4°C to obtain the serum fraction. Twenty microliters of serum sample were mixed with 30 μL of choline assay buffer and centrifuged at 13,000 rpm for 10 min. 50 μL of each sample was added to 50 μL of reaction mixture in 96-well plates on a horizontal shaker for 30 min at room temperature protected from light. The Ach concentration was determined by measuring the absorbance using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 570 nm and calculated by comparing the results with choline standards (0–5 nmol).

Quantification of brain-derived neurotrophic factor (BDNF) was measured with the Rat BDNF Elisa Kit, following the manufacturer's instructions. Briefly, biotinylated detection antibody was added to each well and the plate was incubated for 1 h at room temperature. Then, after 45 min of incubation with HRP-conjugated streptavidin, TMB substrate solution was added for 30 min and then the reaction was stopped by adding stop solution. BDNF concentration was determined by measuring absorbance using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm and calculated by comparing the results with the BDNF standard curve.

ELISA kit for human HTR1A (5-hydroxytryptamine receptor 1A)

The concentration of HTR1A was determined using the Human HTR1A (5-hydroxytryptamine receptor 1A) ELISA kit according to the instructions. Briefly, 100 μL of each sample was added to each well and the plate was incubated at 37°C for 90 minutes. At the end of the incubation time, the material in each well was removed and the wells were washed twice with wash buffer. 100 μL of biotin-labeled antibody working solution was added to the above wells and the plate was incubated at 37°C for 60 minutes. At the end of the incubation time, the solution in each well was removed and the wells were washed three times with wash buffer. Then, 100 μL of SABC working solution was added to each well and the plate was incubated at 37°C for 60 minutes. was incubated at 37?C for 30 minutes. At the end, the wells were washed five times and 90 ?L of TMB substrate was added to each well. After 10-20 minutes, 50 ?L of stop solution was added to each well and the plate was immediately read at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve relating the color intensity (O.D.) to the concentration of the standards (range 0.156 ng/mL to 10 ng/mL) was plotted. GDNF ELISA Kit

GDNF activity was determined using an ELISA kit (MyBioSource, Cambridge, UK) according to the manufacturer's instructions. Briefly, 100 μL of each standard or sample was added to each well and the plate was incubated for 90 minutes at 37°C. The liquid was then removed and 100 μL of biotin-conjugated anti-human DGNF Ab was added. After 1 hour at 37°C, the liquid was removed and the plate was washed 3 times before adding 100 μL of ABC working solution for 30 minutes at 37°C. After 5 washes, 90 μL of Color Development Reagent was added and the plate was incubated for 15 minutes at 37°C. Finally, 100 ?L of stop solution was added and the samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm; the concentration is expressed as ng/mL against a standard curve (range 31.2 pg/mL to 2000 pg/mL) and the results are expressed as percentage (%) compared to the control (line 0).

Oxygen consumption and mitochondrial membrane potential

Oxygen consumption and mitochondrial membrane potential were measured immediately and simultaneously according to the manufacturer's instructions, using an Oxygen Consumption/Mitomembrane Potential Dual Assay Kit (Cayman Chemical Company; Ann Arbor, MI, USA). Briefly, 100 μL of JC-1 staining solution was added and the plate was incubated for 15 min at 37°C. Then the plate was centrifuged for 5 min at 400 × g at room temperature. After removal of the supernatant, 200 μL of assay buffer was added and the plate was centrifuged again as before. These two steps were repeated again. Finally, 100 μL of assay buffer was added. Healthy cells with mainly JC-1 J-aggregates can be measured with excitation and emission wavelengths of 540 and 570 nm, respectively, while apoptotic or unhealthy cells with mainly JC-1 monomers can be detected with excitation/emission of 485/535 nm using a fluorescence spectrometer (Infinite 200 Pro MPlex, Tecan). Results are expressed as mean ± SD (%) compared to control cells.

Interleukin 6 ELISA Kit

The concentration of interleukin 6 was determined by ELISA kit (FineTest) according to the instructions. Briefly, 100 μL of each sample was added to each well and the plate was incubated at 37 μC for 90 minutes. At the end of the incubation time, the material in each well was removed and the wells were washed twice with wash buffer. 100 μL of biotin-labeled antibody working solution was added to the above wells and the plate was incubated at 37 μC for 60 minutes. At the end of the incubation, the solution in each well was removed and the wells were washed three times with wash buffer. Then, 100 μL of HRP-Streptavidin conjugate was added to each well and the plate was incubated at 37 μC for 30 minutes. Finally, the wells were washed five times and 90 μL of TMB substrate was added to each well. After 10-20 minutes, 50 μL of stop solution was added to each well and the plate was immediately read at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve relating the color intensity (O.D.) to the concentration of the standards (range 4.688-300 pg/ml) was plotted.

ELISA Kit for Interleukin 10

The concentration of interleukin 10 was determined by ELISA kit (FineTest) according to the instructions. Briefly, 100 μL of each sample was added to each well and the plate was incubated at 37 μC for 90 minutes. At the end of the incubation time, the material in each well was removed and the wells were washed twice with wash buffer. 100 μL of biotin-labeled antibody working solution was added to the above wells and the plate was incubated at 37 μC for 60 minutes. At the end of the incubation, the solution in each well was removed and the wells were washed three times with wash buffer. Then, 100 μL of HRP-Streptavidin conjugate was added to each well and the plate was incubated at 37 μC for 30 minutes. Finally, the wells were washed five times and 90 μL of TMB substrate was added to each well. After 10-20 minutes, 50 μL of stop solution was added to each well and the plate was immediately read at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve relating the color intensity (O.D.) to the concentration of the standards (range 7.813-500 pg/ml) was plotted.

ELISA assay for BAX

BAX activity was determined using an ELISA kit (MyBiosource, San Diego, CA, USA) that analyzes BAX in cell lysates, according to the manufacturer's instructions. Briefly, 100 μL of sample or standard was added to each well and incubated at 37°C for 90 min. Then, after washing steps, 100 μL of biotinylated antibody was added to each well and incubated for 60 min at 37°C. Next, each well was washed and 100 μL of SABC working solution was added for 30 min at 37°C. Then, 90 μL of TMB substrate was added and incubated for 15–30 min at 37°C. Samples were analyzed using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as pg/mL relative to a standard curve (range 0 to 2000 pg/mL) and the results are expressed as a percentage (%) relative to the control (line 0).

ELISA Kit for Muscarinic Receptor M1

M1 activity was determined using an ELISA kit (Novus Biologicals, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, 100 μL of each standard or sample was added to each well and the plate was incubated for 90 minutes at 37°C. Then the liquid was removed and 100 μL of biotinylated detection Ab was added. After 1 hour at 37°C, the liquid was removed and the plate was washed 3 times before adding 100 μL of HRP conjugate for 30 minutes at 37°C. After 5 washes, 90 μL of Substrate Reagent was added and the plate was incubated for 15 minutes at 37°C. Finally, 50 ?L of stop solution was added and the samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm; the concentration is expressed as ng/mL relative to a standard curve (range 0.31 ng/mL to 20 ng/mL) and the results are expressed as percentage (%) relative to the control (line 0).

ELISA Kit for Muscarinic Receptor M3

The activity of M3 was determined using an ELISA kit

according to the manufacturer's instructions. Briefly, 100 μL of each standard or sample was added to each well and the plate was incubated for 90 min at 37°C. Then the liquid was removed and 100 μL of biotinylated detection Ab was added. After 1 h at 37°C, the liquid was removed and the plate was washed 3 times before adding 100 μL of HRP conjugate for 30 min at 37°C. After 5 washes, 90 μL of Substrate Reagent was added and the plate was incubated for 15 min at 37°C. Finally, 50 μL of stop solution was added and the samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm; the concentration was expressed as ng/mL against a standard curve (range 0.188 ng/mL to 20 ng/mL) and results are expressed as a percentage (%) relative to the control (line 0).

ELISA Kit for Muscarinic Receptor M5

The activity of M3 was determined using an ELISA kit

according to the manufacturer's instructions. Briefly, 100 μL of each standard or sample was added to each well and the plate was incubated for 90 min at 37°C. Then the liquid was removed and 100 μL of biotinylated detection Ab was added. After 1 h at 37°C, the liquid was removed and the plate was washed 3 times before adding 100 μL of HRP conjugate for 30 min at 37°C. After 5 washes, 90 μL of Substrate Reagent was added and the plate was incubated for 15 min at 37°C. Finally, 50 μL of stop solution was added and the samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm; the concentration was 0.05 μL. expressed as ng/mL against a standard curve (range 0.156 ng/mL to 10ng/mL) and results are expressed as a percentage (%) compared to the control (line 0).

Results

In recent years, evidence on the use of probiotics for the benefit of psychiatric disorders has increased, as demonstrated by the increase in the number of clinical trials; considering the evidence on the interaction between the microbiome and the brain and the role of the microbiome in the development of mood disorders, the present study aimed to investigate the role of different probiotics in improving cellular mechanisms related to cognitive functions. To this end, the first step consisted of a 24-hour dose-response study to determine the best concentrations of probiotics to use to create a new formulation. As shown in Figure 1, B. Bifidum, B. Longum and L. Paracasei were shown to have the most beneficial effect on cell viability compared to the control and the other probiotics tested (p<0.05); in particular, the concentrations with the greatest effect were found to be B. Bifidum 10mg/ml, B. Longum 5mg/ml and L. Paracasei 10mg/ml with the strongest effects on cell viability (p<0.05). Considering the results obtained in this first phase, these concentrations were used for the subsequent experiments (Figure 1).

Before conducting further analyses in brain epithelia, further investigations were performed to gain more insight into physiological absorption in an EMA and FDA validated intestinal barrier model. To demonstrate the ability of the new formulation to maintain proper cell physiology and not induce cell damage or irritability, cell viability, TEER values, and metabolite production were assessed. As shown in Figure 2, cell viability showed an increase in mitochondrial metabolism for all tested agents compared to control; the greatest effect (p<0.05) was observed with the combination of all probiotics. TEER analyses were then performed to confirm the integrity of the intestinal monolayer. Transepithelial electrical resistance (TEER) values of 510 ? 10 !*cm<2 >for CaCo2 intestinal cells are in agreement with the results of the literature and suggest that the cells form an intact monolayer after the treatments, maintaining a correct barrier integrity. In confirmation of this, all the tested probiotics were able to cross the intestinal barrier reaching the plasma environment (p < 0.05), in particular this was observed by the analysis of the presence of butyric acid at the basolateral level. Specifically, it was observed that the combination of the invention is able to amplify the effect of the individual agents, with consequent increase in absorption through the intestinal barrier; specifically, the combination of the invention increases the production of short-chain fatty acids by 25% compared to B. Bifidum, by 69% compared to B. Longum and by 80% compared to L. Paracasei. Based on these results, it can be concluded that the combination of the invention increases the production of short-chain fatty acids by 25% compared to B. Bifidum, by 69% compared to B. Longum and by 80% compared to L. Paracasei. hypothesize that the combination of the invention and all the selected agents are able to positively influence intestinal permeability without side effects, thus demonstrating the safety of the product and suggesting that the probiotic formulation is able to directly influence the target organ (Figure 2).

Based on the known involvement of glutamate in modulating mood, a pre-treatment with 8 mM glutamate was used to simulate a condition of depression or more generally of mood disorders, as reported in the literature. As shown in figure 3, pre-treatment with 8 mM glutamate alone induced a significant reduction in cell viability. On the contrary, treatment with probiotics was able to reverse this trend and, as previously reported, the best results were shown by the combination of the invention with an increase in viability of 1.9% compared to L-Glu alone, 12% compared to B. Bifidum, 60% compared to B. Longum and 5% compared to L. Paracasei. The effect of this pre-treatment on the formation of a proton gradient across the inner mitochondrial membrane was also evaluated. Again, probiotics alone had a positive effect, inducing a significant increase in JC-1 fluorescence (p<0.05), while the combination of the invention was able to reverse the dissipation of mitochondrial potential compared to pre-treatment with L-Glu, shifting the fluorescence signal from green to red and thus indicating an increase in membrane potential. L-Glu-induced oxidative stress damage was also analyzed; from this analysis it emerged that the combination of the invention was able to reduce lipid peroxidation better than single probiotics, in particular the combination of the invention reduced peroxidation by 3-fold compared to L-Glu alone, by 33% compared to B. Bifidum, by 13% compared to B. Longum and by 60% compared to L. Paracasei. These results indicate that the probiotic combination attenuates L-Glu-induced cellular damage by reducing oxidative stress and modulating mitochondrial fitness (Figure 3).

Based on the results obtained on the ability of Probiotics and the combination of the invention in particular to counteract cellular damage, the effect of Probiotics against apoptosis was also analyzed, since anxiety and depression are conditions known to cause high levels of cell death. The activity of BAX, a proapoptotic protein, and of cytochrome C, an activating marker of the apoptotic process with no possibility of switching it off, was then analyzed. L-Glu increased both the activity of BAX and Cytochrome C compared to the control (p<0.05); on the contrary, treatment with probiotics inhibited the increase of these two apoptotic markers inducing a reduction in damage from exposure to glutamate. As expected, the combination of the invention was able to modulate both BAX and Cytochrome C, confirming their active role in maintaining the homeostasis of neuronal cells. In particular, the mixture was able to modulate both BAX and Cytochrome C, confirming their active role in maintaining the homeostasis of neuronal cells. was able to reduce BAX activity by 28% compared to B. Bifidum, by 60% compared to B. Longum and by 56% compared to L. Paracasei and to reduce Cytochrome C activity by 60% compared to B. Bifidum, by 72% compared to B. Longum and by 55% compared to L. Paracasei. These data indicate that the combination of the invention provides neuroprotection by dramatically enhancing the overexpression of BAX and Cytochrome C (Figure 4).

Furthermore, it is known that the condition of major depression is accompanied by a significant activation of immuno-inflammatory markers, such as proinflammatory cytokines that can lead to the disruption of brain regulation and signaling mechanisms involving behavioral and emotional aspects. In this context, IL6 and IL10 were found to be significantly elevated in patients with mood disorders. As shown in Figure 5, exposure to L-Glu improved the production of cytokines, increasing the inflammatory process compared to the control (p<0.05). On the contrary, probiotics are able to improve this condition by reducing the production of inflammatory cytokines, suggesting that this new formulation should be able to maintain its anti-inflammatory capacity even during depression. The results obtained indicate that the combination of probiotics reverses the inflammation induced by L-Glu, in particular the combination of the invention is able to reverse the inflammation induced by L-Glu. able to reduce IL6 production by 88%, 78% and 80% compared to B. Bifidum, L. Longum and L. Paracasei respectively. Furthermore, the combination of the invention was able to reduce IL10 activity by 84% compared to B. Bifidum, by 40% compared to B. Longum and by 73% compared to L. Paracasei (Figure 5).

Finally, several reports have identified a reduction in M2-AChR density in both bipolar and major depressive disorder patients, demonstrating that the cholinergic system plays an important role in mood regulation. Therefore, a reduced expression of these receptors could increase ACh release and, indeed, an increase in central choline levels was found in depressed patients. As reported in Figure 6, M2-AChRs activity and ACh production were increased after L-Glu treatment, confirming its effects in inducing neuronal damage (p<0.05); on the contrary, probiotic treatment was able to reverse this condition, decreasing both muscarinic receptor activity and ACh production compared to L-Glu (p<0.05); however, the maximum effect was observed after L-Glu treatment. was observed when the probiotics were combined, confirming the cooperative role of these probiotics in modulating mood by acting on the cholinergic system. In particular, the combination of the invention was able to reduce M2 activity by 64%, 32% and 25% compared to B. Bifidum, L. Longum and L. Paracasei respectively. Furthermore, the combination of the invention was able to reduce acetylcholine production by 72% compared to B. Bifidum, by 48% compared to B. Longum and by 40% compared to L. Paracasei. These results indicate that the combination of probiotics acts on the cholinergic system by reversing the mechanisms underlying L-Glu-induced mood disorders thanks to their cooperative effects (Figure 6).

Finally, analyses were conducted on BDNF and GDNF activity and serotonin production, for the implication that neural factors in the pathophysiology of depression may be of gut or brain origin and play essential roles in systemic homeostasis and in the regulation of neural circuit development and plasticity. Specifically, they co-modulate each other in mediating their physiological roles in the regulation of neural circuit development and plasticity. Serotonin stimulates BDNF/GDNF expression, while BDNF/GDNF promotes neurogenesis and neuronal survival than serotonin. The alteration of the serotonin-BDNF signaling mechanism has been implicated in the pathophysiology of depression. This mechanism influences neuronal development, survival and differentiation, acting synergistically on the survival of damaged motor neurons in depressive episodes. As shown in Figure 7, a significantly higher activity was observed elevated GDNF/BDNF activity in L-Glu-treated samples compared to control (p < 0.05). However, the combination of the invention was able to restore the damage by activating the survival pathway and maintaining the balance between BDNF/GDNF activity better than the single agents during L-Glu exposure (p < 0.05). In particular, the combination of the invention was able to increase BDNF activity by 37% compared to B. Bifidum, by 20% compared to B. Longum and by 48% compared to L. Paracasei. Furthermore, the combination of the invention was able to reduce GDNF activity by 52%, 41% and 43% compared to B. Bifidum, L. Longum and L. Paracasei respectively. Finally, the combination of the invention was able to restore the damage by activating the survival pathway and maintaining the balance between BDNF/GDNF activity better than the single agents during L-Glu exposure (p < 0.05). was able to increase serotonin production by 60%, 79% and 65% compared to B. Bifidum, L. Longum and L. Paracasei respectively (Figure 7).

These results are confirmed by the induction of serotonin by the combination of the invention (p < 0.05), the production of which plays a key role in the pathophysiology of stress, triggering a deterioration of synaptic plasticity, neuronal dysfunction and neuroinflammation.

The tests described above have demonstrated that supplementation of the specific strains of the invention can restore proper intestinal metabolism, which is related to maintaining mood.

The presence of SCFA secretion, for example butyric acid, is able to function as second messengers and activate various mechanisms that are altered in conditions of cognitive dysfunction.

It can be hypothesized that maintaining intestinal homeostasis may improve brain activity by decreasing cell loss.

The combination of the invention provides neuroprotection in glutamate-induced cell injury.

The protective effects of the combination were related to the modulation of the expression of apoptosis-related proteins, through the downregulation of the synthesis of proapoptotic BAX and the increase of the expression of antiapoptotic bcl-2.

The analysis of the neuroprotective effects of the combination of the invention in a model of neuronal cell injury, mediated through serotonergic-cholinergic interactions, underlines the importance of said strains in the neuroprotective mechanism leading to mood disorders.

Inhibition of proinflammatory cytokines is the basis of the decrease in neuroinflammation in depression, capable of altering the brain's regulatory and signaling mechanisms that involve behavioral and emotional aspects.

Cognitive Functionality

Assays were also performed to investigate the role of the microbiome in relation to cognitive disorders.

First, a dose-response study was conducted in an in vitro model using SHSY-5Y cells to evaluate the safety of different probiotics and substances, as follows:

<? >Bifidobacterium Bifidum novaBBF7 (hereinafter also referred to as B. Bifidum) in the range of 5 mg/ml to 30 mg/ml (corresponding to 0.5x10<9>-3x10<9 >CFU) ? Bifidobacterium Longum novaBLG2 (hereinafter also referred to as B. Longum) in the range of 1 mg/ml to 30 mg/ml (corresponding to 1x10<8>-3x10<9 >CFU)

? Lactobacillus Paracasei TJB8 (hereinafter also referred to as L. Paracasei) in the range from 3 ?g/ml to 20 mg/ml (corresponding to 1x10<6>-6x10<9 >CFU)

? a strain of L. Helveticus TJA4 in the range of 1.1?g/ml to 17mg/ml (corresponding to 1.1x10<5>-1.7x10<9 >CFU)

? a strain of L. Plantarum TJA7 in the range of 100?g/ml to 2.5mg/ml (corresponding to 2.5x10<8>-1x10<9 >CFU)

<? >a strain of B. Psychaerophilum Q5 in the range of 3?g/ml to 20mg/ml (corresponding to 3x10<5>-2x10<9 >CFU)

All substances were prepared directly in the stimulation medium.

The best concentration of each probiotic (based on conversion from CFU to mg/ml per day) was maintained for all subsequent experiments.

Based on the data obtained, the best concentration of each substance was tested for the intestinal barrier or to modulate the mediators released by probiotics. In this context, a gut/brain co-culture model was established by seeding intestinal CaCo-2 cells in the apical part of the Transwell? system and stimulating the cells with B. Bifidum 10 mg/ml, B. Longum 5 mg/ml, L. Paracasei 10 mg/ml, alone and in combination. Safety tests and barrier integrity analysis were then performed to avoid irritability, also evaluating the active role of their metabolites in crossing the intestine. Consequently, samples metabolized by intestinal cells were used to stimulate the brain epithelium (SHSY-5Y cells) located in the basolateral compartment, analyzing the main biological activity exerted by probiotics during cognitive dysfunction. More In detail, further experiments were performed to evaluate two different biological aspects involved in brain aging and neurodegeneration: oxidative stress and iron-dependent damage. In fact, the role of oxidative stress was investigated by pretreatment for 30 minutes with 200 ?M H2O2 on neuronal cells. Cell viability, mitochondrial membrane potential and ROS production were analyzed to evaluate the ability of all probiotics, alone or in combination, to prevent or restore the damage caused by oxidative stress with H2O2. Finally, to induce neurodegeneration, cells were pre-treated with 75 μM catalytic iron (Fe<3+>) for 24 h and then treated with all probiotics, alone and in combination, for 24 h to study the protection exerted by the combination, analyzing viability, lipid peroxidation and intracellular pathways activated during iron accumulation that led to cognitive dysfunction.

Materials and methods

Cell Cultures

SH-SY5Y cells were cultured in a mixture of Dulbecco's Modified Eagle Medium F12 and Dulbecco's Modified Eagle Medium in a 1:1 ratio, supplemented with 10% fetal bovine serum, 2 mM HEPES, 2 mM L-Glutamine and 1% penicillin/streptomycin (Merck, Milan, Italy). Cells were maintained in a 37?C incubator at 5% CO2 and 95% humidity. The CaCo-2 cell line, provided by the American Type Culture Collection

, was grown in DMEM/F12 containing 10% FBS, 2 mM l-glutamine and 1% penicillinstreptomycin at 37 ?C in a 5% CO2 incubator.

Gut-brain axis

To test the interaction between CaCo-2 cells and SHSY-5Y cells, the Transwell co-culture system was used. SHSY-5Y cells were pretreated to induce oxidative stress damage as previously described. A semipermeable membrane with a pore size of 0.4 μm (BD Biosciences) was used to separate the two chambers filled with DMEM medium. CaCo-2 cells were seeded in the upper chamber at a density of 50,000 cells/well and reached confluence for 28 days (cell maturation time when intestinal cells produce microvilli); when intestinal cells transformed into a monolayer, SHSY-5Y cells were seeded in the lower chamber at a density of 50,000 cells/well and maintained in culture until confluence was reached.

MTT Test

At the end of the experiment, the MTT assay was performed as described in the literature to determine cell viability after stimulations. Cells were incubated in DMEM without phenol red 0% FBS with 1% MTT dye for 2 hours at 37?C in an incubator, then cell viability was determined by measuring absorbance using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 570 nm with correction at 690 nm. The results were obtained by comparing them with control cells (100% viable).

Intestinal integrity analysis

The TEER values of the inserts were measured every other day continuously for 21 days using EVOM3 and the experiments were started when the TERR reached ?500 ? ?cm2. It is reported in the literature that TEER values ?500 ? 52.9? ?cm2 are recommended for transport study.

Quantification of butyric acid

To quantify probiotic metabolites, butyric acid production was analyzed using an ELISA kit (Cloud-Clone, Wuhan) according to the manufacturer's instructions. The absorbance of each sample was measured after addition of the stop solution at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan), and the OD was interpolated with a standard curve (10,000 pg/mL to pg/mL), expressing the data as mean (pg/mL) versus control.

ROS production

The rate of superoxide anion release was used to examine ROS produced by astrocytes after stimulation. After treatment, 100 μl of cytochrome C was added to all samples (treated and untreated), and 100 μl of superoxide dismutase was also added to another sample for 30 minutes in an incubator (all substances were from Sigma-Aldrich). Absorbance was measured by a spectrometer (Infinite 200 Pro MPlex, Tecan), at 550 nm, and O2 was expressed as nanomoles of cytochrome C reduced per microgram of protein compared to the control in percent (%).

Mitochondrial membrane potential

Mitochondrial membrane potential was analyzed with the Oxygen Consumption/Mito membrane Potential Dual Assay Kit according to the manufacturer's instructions. Mitochondrial membrane potential was measured using JC-1 aggregates at 560/590 nm excitation/emission and monomers at 485/535 nm excitation/emission in a fluorescence spectrometer (Infinite 200 Pro MPlex, Tecan). Results are expressed as (%) relative to control cells.

ELISA Kit for AKT/ERK

AKT/ERK activation was measured with InstantOneTM ELISA

on chondrocyte lysates as reported in the literature. The strips were measured using a spectrometer at 450 nm (Infinite 200 Pro MPlex, Tecan). The results were expressed as mean absorbance (%) compared to the control.

ELISA Kit for P53

P53 activity was measured with the specific ELISA kit (p53 transcription factor assay kit, Cayman Chemical), examining the nuclear extracts obtained at the end of each stimulation following the manufacturer's instructions. Nuclear extraction was obtained with the classical technique using a complete buffer present in the kit. Briefly, cells were lysed with ice-cold 1X Complete Hypotonic Buffer, supplemented with NP-40, and then centrifuged at 12,000 g at 4?C for 10 minutes. The pellet was solubilized with ice-cold 1x Complete Nuclear Extraction Buffer, supplemented with protease and phosphatase inhibitors, and then centrifuged at 12,000 g for 15 minutes at 4?C; the supernatant was examined to analyze p53 activity in relation to protein quantification by the BCA assay (Thermo Fisher).

ELISA assay for BAX

BAX activity was determined using an ELISA kit

which analyzes the presence of BAX in cell lysates, according to the manufacturer's instructions. Briefly, 100 μL of sample or standard was added to each well and incubated at 37 μC for 90 minutes. Then, after washing, 100 μL of biotinylated antibody was added to each well and incubated for 60 minutes at 37 μC. Next, each well was washed and 100 μL of SABC working solution was added for 30 minutes at 37 μC. Then, 90 μL of TMB substrate was added and incubated for 15-30 minutes at 37 μC. Samples were analyzed using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration was expressed as pg/mL against a standard curve (range 0 to 2000 pg/mL) and results are expressed as a percentage (%) compared to the control (line 0).

SOD test

SOD level was measured according to the manufacturer's instructions (Cayman Superoxide Dismutase Assay Kit) as previously described. Briefly, in a 96-well plate, the SOD level present in cell lysates was measured by comparing the data with a standard curve (0.05-0.005 U/mL). The absorbance of all samples was measured using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 480 nm and the results are expressed as mean (%) compared to the control.

ELIS Kit for iNOS

iNOS activity was determined using an ELISA kit

which determines the presence of iNOS in cell lysates, according to the manufacturer's instructions. Samples were analyzed using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. Concentration is expressed as ng/mL relative to a standard curve (range 0.4 to 100 ng/mL) and results are expressed as a percentage (%) relative to the control (line 0).

Amyloid Precursor Protein (APP) ELISA Kit

Quantification of amyloid precursor protein (APP) was measured with the Amyloid Beta A4 Protein ELISA kit (Merck, Milan, Italy) on cell supernatants, as reported in the literature. Briefly, at the end of the treatments, cell supernatants were collected and each sample was analyzed with the ELISA kit. Biotinylated detection antibody specific for the target protein was added to each well and the plate was incubated for 1 h at room temperature. Then, after 45 min of incubation with HRP-conjugated streptavidin, TMB substrate solution was added for 30 min and, subsequently, the reaction was stopped by adding the stop solution. APP concentration was determined by measuring the absorbance using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm and the concentration was calculated using the ELISA kit. was calculated by comparing the results with the APP standard curve.

APOE 9 ELISA Kit

APOE activity was determined using an ELISA kit

which determines the presence of APOE in cell lysates, according to the manufacturer's instructions. Samples were analyzed using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. Concentration is expressed as ng/mL relative to a standard curve (range 1.6 to 400 ng/mL) and results are expressed as a percentage (%) relative to the control (line 0).

Thiobarbituric acid reactive substances (TBARS) test

Lipid peroxidation in cells was estimated using the thiobarbituric acid reactive substances (TBARS) assay. Briefly, 100?L of sample or standard was added to a 5mL vial and then 100?L of SDS solution was added. 4mL of Staining Reagent was added to each vial and the vials were boiled for 1 hour; after incubation, the vials were incubated for 10 minutes on ice. After 10 minutes, the vials were centrifuged for 10 minutes at 1600 x g at 4?C and then 150?L was added to the wells of the 96-well plate and the absorbance was read at 530-540 nm. Quantification of human pTAU

Quantification of TAU protein was measured with the Human Tau [pS199] ELISA kit on cell lysate. Briefly, 100 μL of standard or sample was added to each well and the plate was incubated at room temperature for 2 hours. At the end of the incubation, the wells were washed with 1X wash buffer and then 100 μL of rabbit anti-IgG HRP antibody solution was added to each well and incubated for 30 minutes at room temperature. The wells were washed again with 1X wash buffer and 100 μL of Stabilized Chromogen was added to each well. The plate was incubated at room temperature for 30 minutes and, finally, 100 μL of stop solution was added to each well. The absorbance was was detected using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm and the concentration is expressed in pg/mL relative to a standard curve (range 15.6 to 1000 pg/mL) and the results are expressed as a percentage (%) relative to the control (line 0).

ELISA kit for human SIRT1

Quantification of SIRT1 protein was measured with the Human SIRT1 ELISA kit on cell lysate. Briefly, 100 μL of standard or sample was added to each well and the plate was incubated at room temperature for 2.5 hours. At the end of the incubation, the wells were washed with 1X wash buffer and 100 μL of prepared biotin conjugate was added to each well and incubated for 1 hour at room temperature. The wells were washed again with 1X wash buffer and 100 μL of Streptavidin-HRP was added to each well. The plate was incubated at room temperature for 45 minutes and finally, 100 μL of TMB substrate was added to each well and the plate was incubated for 30 minutes at room temperature in the dark. Subsequently, 50 ?L of stop solution was added to each well and the absorbance was read with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as ng/mL against a standard curve (range 1.23 to 300 ng/mL) and the results are expressed as a percentage (%) compared to the control (line 0).

ELISA kit for BDNF

Quantification of brain-derived neurotrophic factor (BDNF) was measured with the Rat BDNF Elisa Kit, following the manufacturer's instructions. Briefly, biotinylated detection antibody was added to each well and the plate was incubated for 1 h at room temperature. Then, after 45 min of incubation with HRP-conjugated streptavidin, TMB substrate solution was added for 30 min and then the reaction was stopped by adding stop solution. BDNF concentration was determined by measuring absorbance using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm and calculated by comparing the results with the BDNF standard curve.

ELISA kit for NRF1

NRF1 activity was determined using an ELISA kit

which determines the presence of Caspase 9 in cell lysates, according to the manufacturer's instructions. Samples were analyzed using a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed in pg/mL relative to a standard curve (range 82.3-20000 pg/mL) and the results are expressed as a percentage (%) compared to the control (line 0).

Statistical analysis

Results are expressed as mean ? SD of at least 5 biological replicates for each experimental protocol and each replicate was repeated 3 times for each experimental protocol. Statistical comparisons between groups were performed using one-way ANOVA with Bonferroni post hoc test or Mann-Whitney U test, as appropriate, using GraphPad Prism 5. p<0.05 was considered statistically significant. All densitometric analysis data were normalized to control values (defined as 1). All other data from each experimental protocol were normalized to control values in percent (defined as 0%).

Results

In recent years, probiotics have received increasing attention for their wide clinical applications and beneficial health effects in various clinical disorders. Several studies have indicated that gut flora plays a more important role in regulating cognitive activities; furthermore, the present study investigates the role of different probiotics in improving cognitive functions. For this purpose, firstly, a 24-hour dose-response study was performed to select the best probiotics to be introduced in the new formulation, focusing on the possible toxic effect they could have on neuronal cells. As shown in Figure 8, B. Bifidum, B. Longum and L. Paracasei were found to be the best probiotics inducing the greatest effect on cell viability compared to the control and other tested probiotics (p<0.05); in particular B. Bifidum 10 mg/ml, B. Longum 5 mg/ml and L. Paracasei 10 mg/ml exerted the greatest effects on cell viability. cellular (p<0.05), suggesting that this concentration could be used for all subsequent experiments (Figure 8).

Analysis of the potential activity of different probiotics across the intestinal barrier

Before going into the details of the gut-brain axis, further investigations were performed to gain more insight into physiological absorption by running an intestinal barrier model, validated by EMA and FDA.

Therefore, cell viability, TEER value and metabolite production were evaluated to demonstrate the ability of the new formulation to maintain proper intestinal physiology without inducing cell damage. As shown in Figure 9, the intestinal passage analysis demonstrated that all the selected agents are able to cross the intestinal epithelium. In particular, cell viability shows an increase for all 3 concentrations tested compared to the control, although the greatest effect (p<0.05) was observed with the combination of the invention (about 33% compared to Bifidum; 1-fold more than Longum and 2-fold more than Paracasei), suggesting that the combination of the invention is safe for the intestinal epithelium, also suggesting its absorption. This effect was confirmed by the analysis of TEER values which reached a value of about 510 ? 10 !*cm<2 >for intestinal cells, as reported in the literature, confirming that the cells form an intact monolayer after the treatments, maintaining a correct intestinal homeostasis. In fact, all the tested probiotics were able to cross the intestinal barrier reaching the plasma environment (p < 0.05), as observed by the analysis of butyric acid at the basolateral level. In particular, the production of metabolites of the combination of the invention seems to follow a similar trend of cell viability, showing an increase in the production of SCFA (about 33% compared to Bifidum; 3 times more than Longum and 7 times more than Paracasei, p<0.05), amplifying the effects exerted by the individual agents, supporting the hypothesis of a synergistic activity between B. Longum, B. Bifidum and L. Paracasei.

Based on these results, it is possible to hypothesize that the combination of the invention and all selected agents are able to positively influence intestinal permeability without side effects (product safety), suggesting that the probiotic formulation is able to directly influence the target organ (Figure 9).

Ability of probiotics to modulate mechanisms related to cognitive functions under oxidative conditions.

To investigate the potential action of probiotics in preventing cell damage under oxidative conditions, cell viability, ROS production and mitochondrial potential were assessed by pre-treating neuronal cells with 200 μM H2O2 as reported in the literature. As shown in Figure 10, exposure to H2O2 significantly reduced cell viability by about 36% compared to the control; on the contrary, following treatment with intestinally produced probiotic metabolites, cell viability was significantly increased, but the greatest effect was obtained with the combination of the invention, which reversed cell loss compared to probiotics alone and compared to H2O2 (about 65%, p<0.05). Furthermore, considering that ERK/MAPK and PI3K-Akt pathways play a crucial role in regulating neuronal and brain survival, further experiments on their activity were performed. All probiotics alone confirmed their ability to improve cell viability by activating ERK and Akt mediators, as reported in Figures 4. The combination of B. Bifidum 10 mg/ml B. Longum 5 mg/ml L. Paracasei 10 mg/ml (the combination of the invention) amplified the activation of kinases compared to control and single administration during exposure to 200 μM H2O2 (approximately 3-fold more, p<0.05), showing a greater effect in PI3/Akt and ERK/MAPK, supporting the hypothesis that all neuronal survival signaling was activated. These results indicate that the probiotic combination reverses H2O2-induced cell loss by activating survival pathways (Figure 10).

At the same time, since the main theory underlying brain degeneration is the oxidative condition, ROS production and SOD3 and iNOS expressions were analyzed. Again, all probiotics, alone and in combination, were able to maintain ROS production below the physiological level (p<0.05 compared to control), supporting the hypothesis of their safety during exposure to 200 μM H2O2; in particular, all combined probiotics significantly reduced intracellular ROS production by about 70% compared to H2O2 (p<0.05) confirming the active role of the invention combination in counteracting oxidative damage. As reported in Figures 11, SOD3 and iNOS expressions significantly increased in the presence of 200 μM H2O2 compared to control (p<0.05), supporting the hypothesis of the involvement of oxidative stress in neuronal death. In contrast, treatment with B. Bifidum 10mg/ml, B. Longum 5mg/ml and L. Paracasei 10mg/ml alone significantly reduced the expression of both SOD3 and iNOS compared to H2O2 200 μM alone (p<0. 05), but a greater reduction was obtained with the combined stimulation (approximately 1.5-fold and 2.5-fold more than H2O2 200 μM, respectively, p<0.05), indicating a beneficial effect in counteracting cognitive dysfunction. These results indicate that the probiotic combination reverses H2O2-induced cell loss by activating survival pathways (Figure 11).

Since the alteration of the formation of a proton gradient across the inner mitochondrial membrane is considered one of the key indicators of cell viability, mitochondrial potential was analyzed and, as expected, treatments with all probiotics, alone and in combination, induced a significant increase in JC-1 fluorescence, supporting the active role of probiotics and their combination on mitochondrial activity (p<0.05) even during oxidative stress induced by 200 μM H2O2. Furthermore, cells treated with H2O2 showed changes in the fluorescence signal leading to a decrease in the red fluorescence signal and an increase in the green fluorescence signal, indicating a significant dissipation of mitochondrial potential and cell loss compared to the control (p<0. 05); Conversely, the inventive combination reversed the dissipation of mitochondrial potential compared to H2O2200 μM alone (approximately 3.5-fold more, p<0.05), shifting the fluorescence signal from green to red. These results indicate that the inventive combination attenuates H2O2-induced apoptosis through the mitochondria-mediated pathway. Considering that the dissipation of mitochondrial potential under oxidative conditions is known to initiate a cascade of events leading to the activation of caspases, which in turn trigger apoptosis, and knowing that p53 is a key factor involved in aging, oxidative stress and neurodegeneration, and that BAX is a key regulator of both cellular energy metabolism and apoptosis, both were studied in neuronal cells under oxidative conditions. The data reported in Figure 12 show an induction of mitochondrial activity. of p53 and BAX in cells treated with H2O2 (p<0.05) compared to the control, resulting in apoptosis; on the contrary, probiotic stimulation alone reduced the activation of apoptosis, reversing the damage process. Furthermore, the combination of the invention amplified the reduction compared to H2O2 (approximately 8 and 10 times more, respectively, p<0.05), determining conditions favorable to survival and confirming the reduction of cell loss. These results indicate that the combination of probiotics exerts a beneficial effect by counteracting the mechanisms that lead to cognitive dysfunction caused by the oxidative condition (Figure 12).

Finally, since it is known that a natural consequence of apoptosis is cell loss, analysis of apolipoprotein E (APOE) and β-amyloid (APP) demonstrated the alteration in brain tissue. Indeed, H2O2 caused a significant increase in APOE and APP levels, supporting previous data on cell death and suggesting deterioration of cognitive functions. However, treatment with probiotics was able to reduce the damage by decreasing the level of APOE and APP compared to control and compared to H2O2 (p<0.05). Thus, the greatest effect was obtained when neuronal cells were treated with the combination of the invention (about 2.5 times more and 3.5 times more, respectively, compared to H2O2 200 μM, p<0.05), indicating the efficacy of the combination during cognitive deterioration. These results demonstrated that the combination of the invention is effective in preventing cognitive decline. able to reverse the damage induced in oxidative conditions, confirming the active role of probiotics in improving intestinal dysregulation that can modulate cell loss and cognitive dysfunction (Figure 13).

Evaluation of the activities of probiotics in modulating mechanisms related to cognitive functions in conditions of iron accumulation.

Iron can be progressively accumulated in the brain during normal aging and in neurodegenerative disorders it can be stored in abnormal accumulations. For this reason, cell viability and lipid peroxidation were assessed in neuronal cells, as reported in Figure 14. Exposure to 75 ?M Fe3+ significantly reduced cell viability by approximately 23% compared to control (p<0.05); on the contrary, following treatment with B. Bifidum 10mg/ml, B. Longum 5mg/ml and L. Paracasei 10mg/ml alone, cell viability was improved compared to control values, confirming their ability to repair damage (p<0.05). Furthermore, the combination of the invention was shown to be effective in reducing cell viability. was able to amplify the beneficial effect exerted by the single administration during the neurodegenerative process exerted by Fe3+ 75 ?M (about 46% compared to B. Bifidum 10 mg/ml; 35% compared to B. Longum and 40% compared to L. Paracasei, p<0.05), supporting the idea of its possible useful use during brain damage. The opposite trend was also observed during the analysis of lipid peroxidation, confirming the ability of the combination to counteract the oxidative condition caused by iron accumulation. These results indicate the ability of the combination to counteract iron-dependent damage, preventing its accumulation (Figure 14).

Since iron accumulation is considered a key factor in cognitive dysfunction, the role of BDNF, a neurotrophic protein that contributes to neuronal progression activity, NRF-1/PGC-1?, which is implicated in neurogenesis, SIRT1, which is widely recognized as playing a role in neuroprotection against the progression of neurodegeneration, and pTAU, an important marker of aging and neurodegeneration during oxidative stress and age-related disorders, was evaluated.

As expected, stimulation with Fe<3+ > increased oxidative stress and inflammation, but this condition was reversed when probiotics were used (p<0.05). In fact, all probiotics tested alone were able to restore the damage compared to Fe<3+ > (p<0.05) and this reduction was amplified when all probiotics were combined, confirming the positive role of the invention combination in counteracting brain damage (p<0.05). All these beneficial effects were induced by the invention combination as it is able to enhance BDNF production (about 3 times more than H2O2, p<0.05) and, consequently, activates several biological pathways aiming to restore the damage caused by iron accumulation.

These data suggest that the invention combination is able to enhance BDNF production even during injury induction, supporting the efficacy of the invention combination in maintaining neuronal activity and also indicate the ability of the combination to prevent iron-dependent cell loss and damage, activating a rescue mechanism able to restore and maintain neuronal homeostasis (Figure 15).

From the tests described above, it can be deduced that specific concentrations of the strains of the invention are able to stimulate the gut/brain axis and restore proper intestinal metabolism, which is related to the maintenance of cognitive functions.

The presence of SCFA secretion, for example butyric acid, is able to function as second messengers and activate various mechanisms that are compromised in conditions of cognitive dysfunction.

Maintaining gut homeostasis may improve brain function by decreasing cell loss.

The tested substances did not induce any damage in the intestinal epithelia, confirming their safety properties by enhancing antioxidant mechanisms and antineuroinflammation activity during brain damage, also acting on several molecular pathways involved in neuronal well-being, such as ERK/MAPK and PI3K/AKT.

The combination of B. Bifidum 10 mg/ml B. Longum 5 mg/ml L. Paracasei 10 mg/ml was able to restore the negative effect by improving the production of BDNF which modulates several brain proteins involved in cognitive functions, such as SIRT1, pTAU and APP linked to cognitive dysfunction by acting directly on specific biomarkers.

Claims (10)

1. A pharmaceutical, nutraceutical or food supplement composition comprising or, alternatively, consisting of at least one strain of bacteria selected from the group comprising or, alternatively, consisting of: - Lactobacillus paracasei "TJB8" with DSM deposit number 33129; - Bifidobacterium bifidum "novaBBF7" with DSM deposit number 34336; - Bifidobacterium longum "novaBLG2" with DSM filing number 34339; or a mixture thereof and, optionally, together with at least one physiologically and/or pharmaceutically acceptable, or food grade, excipient and/or vehicle. 2. The composition according to claim 1, wherein said composition comprises or, alternatively, consists of at least two strains of bacteria: (i) Lactobacillus paracasei "TJB8" DSM 33129 and Bifidobacterium bifidum "novaBBF7" DSM 34336; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example, 1:3 to 3:1; or 1:2 to 2:1; or 1:1; or (ii) Lactobacillus paracasei "TJB8" DSM 33129 and Bifidobacterium longum "novaBLG2" DSM 34339; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example, 1:3 to 3:1; or 1:2 to 2:1; or 1:1; or (iii) Bifidobacterium bifidum "novaBBF7" DSM 34336 and Bifidobacterium longum "novaBLG2" DSM 34339; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example, 1:3 to 3:1; or 1:2 to 2:1; or 1:1. 3. The composition according to claim 1, wherein said composition comprises or, alternatively, consists of at least three strains of bacteria: (iv) Lactobacillus paracasei "TJB8" DSM 33129, Bifidobacterium bifidum "novaBBF7" DSM 34336 and Bifidobacterium longum "novaBLG2" DSM 34339; preferably in a weight ratio of 1:1:2, or 1:2:1, or 2:1:1, or 1:1:1. 4. The composition according to any of claims 1 to 3, wherein said composition comprises (wt/wt): - from 1 mg to 100 mg of Lactobacillus paracasei TJB8, preferably from 5 mg to 50 mg, more preferably from 20 mg to 30 mg, preferably in lyophilized form, and/or - 1 mg to 100 mg of Bifidobacterium bifidum novaBBF7, preferably 5 mg to 50 mg, more preferably 20 mg to 30 mg, preferably in lyophilized form, and/or - 1 mg to 100 mg of Bifidobacterium longum novaBLG2, preferably 5 mg to 50 mg, more preferably 10 mg to 20 mg, preferably in lyophilized form. 5. The composition according to any of claims 1 to 4, wherein said composition comprises (wt/wt): - from 5 to 20 mg of Lactobacillus paracasei TJB8, and - from 5 to 20 mg of Bifidobacterium bifidum novaBBF7, e - 1 to 10 mg of Bifidobacterium longum novaBLG2, preferably in freeze-dried form, more preferably 10 mg of Lactobacillus paracasei TJB8, 10 mg of Bifidobacterium bifidum novaBBF7 and 5 mg of Bifidobacterium longum novaBLG2. 6. The composition according to any of claims 1 to 5, wherein said bacterial strain Lactobacillus paracasei TJB8 is administered at a daily dose in the range of 1.0x10<6 >CFU/day to 1.0x10<12 >CFU/day; preferably, from 1.0x10<8 >CFU/day to 1.0x10<10 >CFU/day, for example 3.0x10<9 >CFU/day; said bacterial strain Bifidobacterium bifidum novaBBF7 is administered at a daily dose in the range of 1.0x10<6 >CFU/day to 1.0x10<12 >CFU/day; preferably, from 1.0x10<8 >CFU/day to 1.0x10<10 >CFU/day, for example 1.0x10<9 >CFU/day; said bacterial strain Bifidobacterium longum novaBLG2 is administered at a daily dose in the range of 1.0x10<6 >CFU/day to 1.0x10<12 >CFU/day; preferably, from 1.0x10<8 >CFU/day to 1.0x10<10 >CFU/day, e.g. 0.5x10<9 >CFU/day. 7. The composition according to any of claims 1 to 6, wherein said at least one bacterial strain is for use as a medicament for brain disorders. 8. The composition for use according to claim 7, wherein said bacterium is for use in brain disorders, including treatment to treat and/or prevent mood disorders, such as anxiety and depression, and to improve cognitive function and treat and prevent cognitive decline, such as in Alzheimer's disease and dementia. 9. A strain of bacteria selected from the group comprising or, alternatively, consisting of: - Lactobacillus paracasei "TJB8" with DSM deposit number 33129; - Bifidobacterium bifidum "novaBBF7" with DSM deposit number 34336; - Bifidobacterium longum "novaBLG2" with DSM filing number 34339; or a mixture of them. 10. The bacterial strain of claim 9 wherein said bacterium is for use as a medicament for brain disorders; preferably is for use for brain disorders, including treatment to treat and/or prevent mood disorders, such as anxiety and depression, and to improve cognitive function and treat and prevent cognitive decline, such as in Alzheimer's disease and dementia.