WO2024095199A1

WO2024095199A1 - Bacterial strains to support the mood and cognitive functionality, compositions and uses thereof

Info

Publication number: WO2024095199A1
Application number: PCT/IB2023/061058
Authority: WO
Inventors: Marco BOCCARUSSO; Giorgio Stefano CERANA; Peter Franck
Original assignee: Probionova Sa
Current assignee: Probionova Sa
Priority date: 2022-11-02
Filing date: 2023-11-02
Publication date: 2024-05-10
Anticipated expiration: 2025-05-02
Also published as: EP4611781A1; IT202200022464A1

Abstract

The present invention relates to bacterial strains belonging to the genus Bifidobacterium and Lactobacillus, their combination, and their use in therapy, particularly for mood regulation and support of cognitive function. The invention also relates to pharmaceutical, nutraceutical and dietary supplement compositions containing at least one of these strains belonging to the genus Bifidobacterium and Lactobacillus. In addition, the present invention relates to selected bacterial strains belonging to the species Bifidobacterium and Lactobacillus, and to mixtures and compositions thereof, for use in a method of treatment, preferably preventive or curative, of mood regulation and support of cognitive function, 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.

Description

Description of the invention having title:

“Bacterial strains to support the mood and cognitive functionality, compositions and uses thereof’

Mood disorders and cognitive decline are severe pathological conditions which affect a large part of the population.

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

Notwithstanding advances in drug development, most individuals being treated for a mood disorder do not achieve complete symptomatic remission and functional recovery, and in overall, the prevalence of mood disorders, especially treatment-resistant mood disorders, is increasing globally. Moreover, the pharmacological treatments indicated by the guidelines have not been developed based on the pathophysiology of the disease, and their impact on the neurobiology of these conditions remains incomplete. Another factor to consider is that a substantial percentage 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 indicated during mood episodes and euthymia in both MDD and BD. Furthermore, about 60% of patients with anxiety and depression have bowel function disorders, as in the case of irritable bowel syndrome Genes of the brain and intestinal tract are quite similar, especially related to the formation of the neuronal synapse, thus some gene mutations can lead to abnormalities in both the brain and gut. The gut microbiome is considered a key component of the gut-brain axis as there is ample evidence showing that it can influence symptoms of depression and anxiety. Specifically, the intestinal tract is controlled by intrinsic and extrinsic factors: the intrinsic factor is the intestinal neural system, which in turn is also controlled by extrinsic factors, including vagus and sacral parasympathetic nerve fibers and visceral sympathetic nerve fibers. The humoral connection includes the hypothalamic-pituitary-adrenal axis, which is responsible for regulating the stress response, and gut endocrine cells, which secrete neuropeptides and gut peptides that act locally and through the vagus nerve and spinal cord afferents or the blood-brain barrier to act on the brain. Analysis of the microbiota-gut-brain axis and the effects that bacteria in the microbiota may have on the functioning of the central nervous system has shown that, with regard to psychiatric disorders, an exciting approach is probiotic supplementation; probiotics are live bacteria that colonize the gastrointestinal tract and exert a beneficial effect on host health.

In particular, probiotics can regulate the constitution of the gut microbiota: some authors showed that feeding probiotic-containing foods to a patient with anxiety resulted in an increase in total sleep time in the third week, which is useful for relieving chronic stress, and a constitutional change in 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 symptoms of depression and anxiety: other authors found that rats given probiotics had better results than those given placebo and similar to those given diazepam, the standard reference substance, in performing a test on the degree of anxiety. One study indicates that behavioural and neurological changes induced by chronic stress can be alleviated through intervention with Bifidobacterium longum and other authors found that consumption of probiotics containing Lactobacillus acidophilus, Lactobacillus easel and Bifidobacterium bifidum over an eight-week period led to a significant reduction in depressive symptoms. Moreover, one study suggests that daily administration of L plantarum may lead to a decrease in depressive symptoms, cortical arousal and fatigue level, and an improvement in deep sleep quality.

Cognitive functions are the most complex properties of the nervous system since they are responsible for rational perception, cognition, and interaction with the outside world. They are important for the implementation of some highly intelligent complex tasks and even 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, resulting 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 proportion of older people considered to be at high risk for 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.

Considering this background, it is evident that there is an urgent need to find treatments that support cognitive function. The gut microbiome has emerged in recent decades as a critical factor influencing neurophysiological and psychophysiological functions, including cognition, emotional neurotransmission and neurodevelopment. Specifically, the gut microbiota undergoes a significant transition in its composition and function during aging and these alterations can affect health and age-related diseases. Recently, the emerging concept of gut-brain axis, referring to a bidirectional relationship between gut and brain, has linked gut microbiota to age-related neurodegenerative diseases, such as Alzheimer’s disease, and related cognitive disfunctions. The interplay between gut and brain involves a complex network of endocrinological, immunological, and neural mediators, which has been considered as 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 affect different pathways, could be used to repopulate the intestinal flora, and re-establish the physiology of the body. For example, probiotic consumption has been shown to increase 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 the treatment with Lactobacillus plantarum showed anti-Alzheimer's properties against D-Galactose-induced Alzheimer's disease, and again there are also scientific papers about the effectiveness of Lactobacillus paracasei in improving memory function through nerve growth factor-mediated neurogenesis.

In view of the above, it is evident that it would be useful to identify new probiotic-based treatments for improving mood disorders as well as the cerebral functions. the urological and gynecological disorders described above as an alternative to conventional phytotherapeutic and pharmacological treatments.

Following an extensive research and development activity, the Applicant surprisingly found that specific bacterial strains of the genus Bifidobacterium and Lactobacillus are able to exert an important beneficial activity in brain disorders, especially in mood disorders and also are effective in improving cerebral functions.

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

Lactobacillus paracasei “TJB8”, deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33129 by Probionova SA on May 17, 2019;

Bifidobacterium bifidum “novaBBFZ”, deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34336 by Probionova SA, on July 26, 2022; and

Bifidobacterium longum “novaBLG2'', deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34339 by Probionova SA, on July 26, 2022; and mixture thereof, are able to carry out an important beneficial activity in brain disorders, especially in mood disorders and also are effective in improving cognitive functionality.

All the bacterial strains mentioned in the present invention were deposited according to the provisions pursuant to the Budapest Treaty. The Depositing party of the bacterial strains described and/or claimed in the present patent application and the proprietor thereof express, from the outset, their consent to make available all the above strains for the whole duration of the patent.

The bacterial strains belonging to the species Lactobacillus paracasei are reclassified under the nomenclature Lacticaseibacillus paracasei.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the dose-response study on SHSY-5Y cells for assessment 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 the cell viability, TEER value and Butyric acid quantification. In panel A, cell viability assessed by MTT evaluated. In B, TEER analysis and in C Butyric acid quantification assessed by ELISA kit is reported. 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 the cell viability, analysis of JC-1 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 L-Glu; #p<0.05 vs single agents.

Figure 4 shows BAX and Cyto-C activity. 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 5 shows IL6 and IL10 activity. 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 the bar p<0.05 vs single 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 assessment 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 the cell viability, TEER value and Butyric acid quantification. In panel A, cell viability assessed by MTT evaluated. In B, TEER analysis and in C Butyric acid quantification assessed by ELISA kit is reported. 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 the 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, 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 APP and APOE 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 14 shows Mitochondrial Metabolism and Lipid Peroxidation analysis. Data are expressed as mean±SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; y p<0.05 vs Fe³⁺; the 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 Fe³⁺; the 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 chosen from the group comprising or, alternatively, consisting of:

Lactobacillus paracasei “TJB8”, deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33129 by Probionova SA, on May 17, 2019;

Bifidobacterium bifidum “novaBBFZ”, deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34336 by Probionova SA, on July 26, 2022;

Bifidobacterium longum “novaBLG2” deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34339 by Probionova SA, on July 26, 2022; and mixture thereof.

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

- Lactobacillus paracasei “T JB8” DSM 33129, preferably in lyophilized form, or

- Bifidobacterium bifidum “novaBBF7” DSM 34336, preferably in lyophilized form, or

- Bifidobacterium longum “novaBLGZ DSM 34339, preferably in lyophilized 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 bacteria strains above, 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 , e.g., 1:3 to 3:1; or 1 :2 to 2: 1; or 1 :1, preferably all strains of bacteria are in lyophilized form. ii) Lactobacillus paracasei “TJB8" DSM 33129 and Bifidobacterium longum “novaBLGZ 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 , e.g., 1:3 to 3:1; or 1 :2 to 2: 1; or 1 :1, preferably all strains of bacteria are in lyophilized 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 , e.g., 1 :3 to 3:1 ; or 1 :2 to 2: 1 ; or 1 :1 , preferably all strains of bacteria are in lyophilized form.

Preferably, the present invention relates to a pharmaceutical or nutraceutical or food supplement association or combination comprising or, alternatively, consisting of three of the bacteria strains above, 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 bacteria strains are in lyophilized form.

Preferably, the present invention relates to a pharmaceutical or nutraceutical or food supplement composition 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 physiologically and/or pharmaceutically acceptable vehicle.

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

According to another of its aspects, the present invention relates to the use of at least one of said bacterial strains and/or their mixtures i), or ii), or iii), or iv), as set out above, in therapy and, more specifically, in brain disorders, including for treating and/or preventing mood disorders, such as anxiety and depression, and for improving cognitive functionality and treating and preventing 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 or food supplement association or combination or composition in therapy and, more specifically, in brain disorders, including the treatment for treating and/or preventing mood disorders, such as anxiety and depression, and for improving cognitive functionality and treating and preventing cognitive decline, such as in Alzheimer’s disease and senile dementia.

Preferably, the present invention relates to a composition comprising (w/w):

- from 1 mg to 100 mg of Lactobacillus paracasei T JB8, preferably from 5 mg to 50 mg, more preferably from 20 mg to 30 mg, preferably in lyophilized form, and/or

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

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

Preferably, the present invention relates to a composition comprising from 5 to 20 mg of Lactobacillus paracasei TJB8, from 5 to 20 mg of Bifidobacterium bifidum novaBBF7 and from 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 bacteria strain Lactobacillus paracasei TJB8 is administered at a daily dose comprised from 1 ,0x10⁶CFU/day to 1 ,0x10¹²CFU/day; preferably, from 1 ,0x10⁸CFU/day to 1 ,0x10¹°CFU/day, such as for example 3,0x10⁹ CFU/die.

Preferably, the bacteria strain Bifidobacterium bifidum novaBBF7 is administered at a daily dose comprised from 1,0x10⁶CFU/day to 1 ,0x10¹²CFU/day; preferably, from 1,0x10⁸CFU/day to 1 ,0x10¹⁰CFU/day, such as for example 1 ,0x10⁹ CFU/die. Preferably, the bacteria strain Bifidobacterium longum novaBLG2 is administered at a daily dose comprised from 1 ,0x10⁶CFU/day to 1 ,0x10¹²CFU/day; preferably, from 1,0x10⁸CFU/day to 1 ,0x10¹⁰CFU/day, such as for example 0,5x10⁹ CFU/die (CFU = Colony Forming Units).

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

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

According to one embodiment, the at least one physiologically and/or pharmacologically acceptable excipient may include prebiotics. According to an embodim ent, the at least one physiologically and/or pharmacologically acceptable excipient may include a monosaccharide, a disaccharide, a polysaccharide, soluble fibers and/or insoluble fibers, 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 could be selected from the group consisting of: inulin, a fructooligosaccharide (FOS), maltodextrin and their mixtures. The compositions according of the invention are preferably compositions for oral use, advantageously in the form of capsules. However, other pharmaceutical forms can be used.

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

In the Figures, the combination of the invention is referred to as MIX.

The combination of the invention showed to exert an unexpected synergistic activity, as it will be evident from the experimental tests below.

Mood disorders

First, assays to investigate the role of the microbiome in the development of mood disorders, especially the mechanisms that lead to anxiety and depression were carried out.

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 follow:

• Bifidobacterium Bifidum novaBBFT (herein after also referred to as B. Bifidum) ranging from 5mg/ml to 30mg/ml (corresponding to 0.5x10⁹-3x10⁹ CFU)

• Bifidobacterium Longum novaBLG2 (herein after also referred to as B. Longum) ranging from 1mg/ml to 30mg/ml (corresponding to 1 x10⁸-3x10⁹ CFU)

• Lactobacillus Paracasei TJB8 (herein after also referred to as L. Paracasei) ranging from 3pig/ml to 20mg/ml (corresponding to 1x10⁶-6x10⁹ CFU)

• one strain of L. Helveticus TJM deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33128 by Probionova SA, on May 17, 2019 ranging from 1.1 pig/ml to 17mg/ml (corresponding to 1.1x10⁵-1 ,7x10⁹ CFU) • one strain of L Plantarum TJA7 deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33132 by Probionova SA, on May 17, 2019 ranging from 100pg/ml to 2.5mg/ml (corresponding to 2.5x10⁸-1x10⁹ CFU)

• one strain of B. Psychaerophilum Q5 deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33131 by Probionova SA, on July 2, 2019 ranging from 3pg/ml to 20mg/ml (corresponding to 3x10⁵-2x10⁹ 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.

On the basis of the data obtained, the best concentration of each substance was tested for the intestinal barrier or by modulating the mediators released by probiotics. In this context, a gut/brain co-culture model was performed 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 the brain epithelium (SHSY-5Y cells) placed in the basolateral compartment for 48h to analyze mitochondrial metabolism. Then further experiments were performed to evaluate the activity of muscarinic receptor.

Materials and methods

Cell cultures

SH-SY5Y cells (ATCC, Manassas, VA, USA) were cultured in a mixture of Dulbecco's Modified Eagle Medium F12 (Sigma-Aldrich, Milan, Italy) and Dulbecco's Modified Eagle Medium (Merck, Milan, Italy) at a ratio of 1 :1 , supplemented with 10% fetal bovine serum (FBS, Merck, Milan, Italy), and 2 mM HEPES (Merck, Milan, Italy), 2 mM L-Glutamine (Merck, Milan, Italy) and 1% penicillin/streptomycin (Merck, Milan, Italy). Cells are maintained in a 37°C incubator at 5% CO2 and 95% humidity

The CaCo-2 cell line, provided by the American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in DMEM/F12 (Merck, Milan, Italy) containing 10% FBS (Merck, Milan, Italy), 2 mM I- glutamine (Merck, Milan, Italy) and 1% penicillin-streptomycin (Merck, Milan, Italy) at 37°C in a 5% CO2 incubator.

Cells were treated with different concentrations of six different probiotics in order to find the minimum effective dose to be used in the final formulation:

• B. Bifidum novaBBF7, from 5 mg/mL to 30 mg/mL

• B. Longum novaBLG2, from 1 mg/mL to 30 mg/mL

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

• B. Psychaerophilum Q5, from 3 pg/mL to 20 mg/mL

• L. Paracasei TJB8, from 3 pg/mL to 20 mg/mL

• L. Helveticus TJM, from 1 pg/mL to 17 mg/mL

Probiotics with the dosages chosen for the final formulation were also tested after pre-treatment with 100 pM H2O2, 75 pM Fe³⁺ and 8mM Glutamate for 30 minutes to understand its role in regeneration of brain damage. Gut-Brain axis

Transwell® co-culture system was used to test the interaction between CaCo-2 cells and SHSY-5Y cells. SHSY- 5Y cells were pretreated to induce hypoxia-reoxygenation injury as previously described. A semi permeable membrane with a pore size of 0.4 pm (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 tests, quantification of ROS production, and evaluation of mitochondrial metabolism during the brain degenerative process were performed on the cells.

MTT test

At the end of the experiment, the MTT test 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 2h at 37°C in an incubator, and then cell viability was determined by measuring the absorbance through 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).

Acetycholine ELISA Kit

Choline/Ach Quantification Kit (Merck, Milan, Italy) was used to determine Ach level, as reported in literature. Briefly, the blood was centrifuged at 4,000 rpm for 10 min at 4°C to obtain the serum fraction. Twenty microliter serum sample was mixed with 30 pL of choline assay buffer and centrifuged at 13,000 rpm for 10 min. 50 pL of each sample were added to 50 pL of Reaction Mix into 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 through a spectrometer (Infinite 200 Pro MPlex, Tecan) at 570 nm and calculated by comparing results to choline standards (0- 5 nmol).

Brain-derived neurotrophic factor (BDNF) quantification was measured by Rat BDNF Elisa Kit (Thermo Scientific™, Waltham, MA, United States), following the manufacturer’s instructions. Briefly, biotinylated detection antibody was added into 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 Stop Solution. BDNF concentration was determined by measuring the absorbance by spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm and calculated by comparing results to BDNF standard curve.

Human HTR1A (S-hydroxytryptamine receptor 1A) ELISA Kit

HTR1A concentration was determined by Human HTR1A (5-hydroxytryptamine receptor 1A) ELISA Kit (FineTest, Wuhan, Hubei, China) in according to the instructions. Briefly, 100 pL of each sample were added into each well and the plate was incubated at 37°C for 90 minutes. At the end of incubation time, the material in each well was removed and wells were washed twice with Wash Buffer. 100 pL of Biotin-labeled antibody working solution were added into above wells and the plate was incubated at 37°C for 60 minutes. At the end of incubation, the solution in each well was removed and wells were washed three times with Wash Buffer. Then, 100 piL of SABC Working Solution were added into each well and plate was incubated at 37°C for 30 minutes. At the end, the wells were washed five times and 90 piL of TMB substrate were put in each well. After 10-20 minutes, 50 piL of Stop Solution were put in each well and the plate was red immediately at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve is plotted relating the intensity of the color (O D ) to the concentration of standards (range 0.156 ng/mL to 10 ng/mL).

GDNF ELISA Kit

GDNF activity was determined using an ELISA Kit (MyBioSource, Cambridge, UK) according to the manufacturer's instructions. Briefly, 100 piL of each standard or sample were added to each well and the plate was incubated for 90 minutes at 37°C. Then the liquid was removed and 100 piL of Biotin conjugate anti-Human GDNF Ab were added. After 1 h at 37°C, the liquid was removed, and the plate was washed 3 times before adding 100 pl of ABC working solution for 30 minutes at 37°C. After 5 washes, 90 pl of Color Developing Reagent were added and the plate was incubated for 15 minutes at 37°C. Finally, were added 10OpL of Stop solution and the samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm, and the concentration is expressed as ng/mL against a standard curve (range 31.2 pg/mL to 2000 pg/mL), and results are expressed as percent (%) against control (line 0).

Oxygen Consumption and Mitochondrial Membrane Potential

The oxygen consumption and mitochondrial membrane potential were immediately measured simultaneously following the manufacturer's instructions using an Oxygen Consumption/Mitomembrane Potential Dual Assay Kit (Cayman Chemical Company; Ann Arbor, Ml, USA). Briefly, 100pl of JC-1 Staining Solution were added and the plate was incubated for 15 minutes at 37°C. Then the plate was centrifugated for 5 minutes at 400xg at room temperature. After the removal of the supernatant, 200p of Assay Buffer were added and the plate was centrifugates again as before. These two passages were repeated again. Finally, WOpI of Assay Buffer were added. Healthy cells with mainly JC-1 J-aggregates can be measured by excitation and emission wavelengths at 540 and 570 nm, respectively, while apoptotic or unhealthy cells with mainly JC-1 monomers can me detected at an excitation/emission of 485/535 nm in a fluorescence spectrometer (Infinite 200 Pro MPlex, Tecan). The results are expressed as means ± SD (%) compared to control cells.

Interleukin 6 ELISA Kit

Interleukin 6 concentration was determined by ELISA Kit (FineTest) in according to the instructions. Briefly, 100piL of each sample were added into each well and the plate was incubated at 37°C for 90 minutes. At the end of incubation time, the material in each well was removed and wells were washed twice with Wash Buffer. 100 piL of Biotin-labeled antibody working solution were added into above wells and the plate was incubated at 37°C for 60 minutes. At the end of incubation, the solution in each well was removed and the wells were washed three times with Wash Buffer. Then, 1 OOpi L of HRP-Streptavidin Conjugate were added into each well and plate was incubated at 37°C for 30 minutes. At the end, the wells were washed five times and 90 piL of TMB substrate were put in each well. After 10-20 minutes, 50 piL of Stop Solution were put in each well and the plate was red immediately at 450nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards (range 4.688- 300 pg/ml).

Interleukin 10 ELISA Kit

Interleukin 10 concentration was determined by ELISA Kit (FineTest) in according to the instructions. Briefly, 10OpiL of each sample were added into each well and the plate was incubated at 37°C for 90 minutes. At the end of incubation time, the material in each well was removed and wells were washed twice with Wash Buffer. 100 piL of Biotin-labeled antibody working solution were added into above wells and the plate was incubated at 37°C for 60 minutes. At the end of incubation, the solution in each well was removed and the wells were washed three times with Wash Buffer. Then, 100 |ul_ of HRP-Streptavidin Conjugate were added into each well and plate was incubated at 37°C for 30 minutes. At the end, the wells were washed five times and 90 piL of TMB substrate were put in each well. After 10-20 minutes, 50 L of Stop Solution were put in each well and the plate was red immediately at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve is plotted relating the intensity of the colour (O.D.) to the concentration of standards (range 7.813- 500pg/ml).

BAX ELISA assay

BAX activity was determined using ELISA kit (MyBiosource, San Diego, CA, USA) analysing the BAX in cell lysates, according to the manufacturer’s instruction. Briefly, 100 pL of sample or standard were added in each well and incubated at 37°C for 90 minutes. Then, after washes, 100 pL of biotinylated antibody were added in each well and incubated for 60 minutes at 37°C. After, each well was washed and 100 pL of SABC working solution were added for 30 minutes at 37°C. Then, 90 pL of TMB substrate were added and incubated for 15-30 minutes at 37°C. The samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as pg/mL compared to a standard curve (range from 0 to 2000 pg/mL) and the results are expressed as percentage (%) versus control (0 line).

M1 muscarinic receptor ELISA Kit

M1 activity was determined using an ELISA Kit (Novus Biologicals, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, WOpil of each standard or sample were added to each well and the plate was incubated for 90 minutes at 37°C. Then the liquid was removed and 100|ul of Biotinylated Detection Ab were added After 1h at 37°C, the liquid was removed, and the plate was washed 3 times before adding 100 l of HRP Conjugate for 30 minutes at 37°C. After 5 washes, 90pil of Substrate Reagent were added and the plate was incubated for 15 minutes at 37°C. Finally, were added 50pil of Stop solution and the samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm, and the concentration is expressed as ng/mL against a standard curve (range 0.31 ng/mL to 20 ng/mL), and results are expressed as percent (%) against control (line 0).

M3 muscarinic receptor ELISA Kit

M3 activity was determined using an ELISA Kit (MyBioSource, Cambridge, UK) according to the manufacturer's instructions. Briefly, WOpil of each standard or sample were added to each well and the plate was incubated for 90 minutes at 37°C. Then the liquid was removed and WOpil of Biotinylated Detection Ab were added. After 1h at 37°C, the liquid was removed, and the plate was washed 3 times before adding 1 OOpil of HRP Conjugate for 30 minutes at 37°C. After 5 washes, 90 I of Substrate Reagent were added and the plate was incubated for 15 minutes at 37°C. Finally, were added 50pil of Stop solution and the samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm, and the concentration is expressed as ng/mL against a standard curve (range 0.188 ng/mL to 20 ng/mL), and results are expressed as percent (%) against control (line 0).

M5 muscarinic receptor ELISA Kit

M3 activity was determined using an ELISA Kit (Life Science, Singapore) according to the manufacturer's instructions. Briefly, WOpil of each standard or sample were added to each well and the plate was incubated for 90 minutes at 37°C. Then the liquid was removed and WOpil of Biotinylated Detection Ab were added. After 1h at 37°C, the liquid was removed, and the plate was washed 3 times before adding 1 OOpil of HRP Conjugate for 30 minutes at 37°C. After 5 washes, 90pil of Substrate Reagent were added and the plate was incubated for 15 minutes at 37°C. Finally, were added 50pil of Stop solution and the samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm, and the concentration is expressed as ng/mL against a standard curve (range 0.156 ng/mL to 10ng/mL), and results are expressed as percent (%) against control (line 0).

Results

Over the last few years, there are growing evidence of the use of probiotics for the benefit of psychiatric disorders evidenced by the increase in the number of clinical trials; considering the evidence about the interaction between the microbiome and the brain and about the role of the microbiome in the development of mood disorders, the present aimed to investigate the role of different probiotics able to improve cellular mechanisms related to cognitive functions. For this purpose, the first step consisted of a 24-hours dose-response study to determine which were the best concentrations of probiotics to use to create a new formulation. As shown in Figure 1 , B. Bifidum, B. Longum and L. Paracasei have been shown to have the most beneficial effect on cell viability compared to the control and other Probiotics tested (p<0.05); specifically, the concentrations with the greatest effect proved 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 from this first step, these concentrations were used for the following experiments (Figure 1).

Before conducting further analysis on cerebral epithelia, further investigations were performed to gain more information regarding the physiological absorption in an intestinal barrier model validated by EMA and FDA. To demonstrate the ability of the hypothesized new formulation to maintain proper cell physiology and fail to induce cell damage or irritability, cell viability, TEER values, and metabolite production were evaluated.

As shown in Figure 2, cell viability shows an increment in mitochondrial metabolism for all agents tested compared with the control, the largest effect (p<0.05) was observed with the combination of all probiotics. Hence, TEER analyses were performed to confirm the integrity of the intestinal monolayer. Transepithelial electrical resistance (TEER) values of 510 ± 10 !*cm² for CaCo2 intestinal cells are in accordance with literature results and suggest that the cells form an intact monolayer after treatments, maintaining a correct barrier integrity. Confirming this, all probiotics tested were able to cross the intestinal barrier reaching the plasma environment (p < 0.05), particularly this was observed by analysis of the presence of butyric acid at the basolateral level. Specifically, it was noted that the combination of the invention was able to amplify the effect of individual agents, resulting in increased absorption across 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 hypothesized that the combination of the invention and all 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 well-known involvement of glutamate in modulating mood tones, an 8mM glutamate pre-treatment was employed to simulate a condition of depression or more generally mood disorders as reported in literature. As is shown in figure 3, pre-treatment with 8mM glutamate alone induces a significant reduction in cell viability, in contrast, treatment with probiotics is able to reverse this trend and, as previously reported, the best results are shown by the combination of the invention with an increase in viability of 1.9% compared to L-Glu alone, of 12% compared to B. Bifidum, of 60% compared to B. Longum, and of 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 the mitochondrial potential compared with 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 was shown that the combination of the invention was able to reduce lipid peroxidation better than single Probiotics, specifically the combination of the invention reduced the peroxidation of 3 times compared to L-Glu alone, of 33% compared to B. Bifidum, of 13% compared to B. Longum, and of 60% compared to L. Paracasei. These results indicate that the combination of probiotics attenuates L-Glu-induced cellular damage by reducing oxidative stress and modulating mitochondrial well-being (Figure 3).

On the basis of the results obtained on the ability of Probiotics and the combination of the invention in particular to counteract cell damage, the effect of Probiotics against apoptosis was also analyzed since anxiety and depression are conditions known for high level of cell death. Therefore, the activity of BAX, a pro-apoptotic protein, and cytochrome C, an activator marker of the apoptotic process with no possibility of switch off. L-Glu enhanced both BAX and Cyto-C activities compared to control (p<0.05); in contrast, probiotics treatment inhibited the increased of these two apoptotic markers inducing a reduction of glutamate-exposure damage. As expected, the combination of the invention was able to modulate both BAX and Cyto-C confirming the active role to maintain the neuronal cell homeostasis. Specifically, Mix was able to reduce BAX activity of 28% compared to B. Bifidum, of 60% compared to B. Longum, and of 56% compared to L. Paracasei and to reduce Cyto-c activity of 60% compared to B. Bifidum, of 72% compared to B. Longum, and of 55% compared to L. Paracasei. These data indicate that the combination of the invention provides neuroprotection by dramatically improving BAX and Cyto-C overexpression (Figure 4) Furthermore, it is known that the condition of major depression is accompanied by significant activation in immune- inflammatory markers, such as proinflammatory cytokines that can lead to the disruption of the brain's regulatory and signalling mechanisms involving behavioral and emotional aspects. In this context, IL6 and IL10 have been found to be significantly elevated in patients with mood disorders. As shown in figure 5, the exposure to L-Glu improved the cytokines productions enhancing the inflammatory process compared to control (p<0.05). On the contrary, probiotics are able to ameliorate this condition reducing the production of inflammatory cytokines suggesting that this new formulation should be able to maintain its anti- inflammatory ability also during depression. The results obtained indicate that the combination of probiotics reverts the L-Glu-induced inflammation, in particular the combination of the invention is able to reduce the production of IL6 of 88%, 78% and 80% compared to B. Bifidum, L. Longum and L. Paracasei respectively. In addition, the combination of the invention was able to reduce I L10 activity of 84% compared to B. Bifidum, of 40% compared to B. Longum, and of 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. Thus, reduced expression of these receptors might increase Ach release and, indeed, increased central choline levels have been found in depressed patients. As reported in figure 6, M2-AChRs activity and Ach production were increased after treatment with L-Glu, confirming its effects in inducing neuronal damages (p<0.05); in contrast, probiotics treatment was able to revert this condition by decreasing both muscarinic receptors activity and Ach production compared to L-Glu (p<0.05); however, the maximum effect was observed when the probiotics are combined, confirming the cooperative role of this probiotics to modulate mood tones acting on cholinergic system. Specifically, particular the combination of the invention was able to reduce the M2 activity of 64%, 32% and 25% compared to B. Bifidum, L. Longum and L. Paracasei respectively. In addition, the combination of the invention was also able to reduce acetylcholine production of 72% compared to B. Bifidum, of 48% compared to B. Longum, and of 40% compared to L Paracasei. These results indicate that the probiotic combination acts on the cholinergic system by reversing the mechanisms underlying L-Glu-induced mood disorders due to their cooperative effects (Figure 6).

Ultimately, analysis on BDNF and GDNF activity and on Serotonin production were carried out due to the implication that nervous factors in the pathophysiology of depression may be of intestinal or cerebral origin and play essential roles in systemic homeostasis and in the regulation of the development and plasticity of neural circuits. Precisely, they co-modulate each other in mediating their physiological roles in regulating the development and plasticity of neural circuits. Serotonin stimulates the expression of BDNF/GDNF while BDNF/GDNF promotes neurogenesis and neuronal survival of serotonin. Impairment of the serotonin-BDNF signalling mechanism has been implicated in the pathophysiology of depression. This mechanism influences the development, survival, and differentiation of neurons, acting synergistically on the survival of damaged motor neurons in depressive episodes. As shown in figure 7, a significantly higher activity of GDNF/BDNF was observed in the L-Glu-treated samples compared to the control (p <0.05). However, the combination of the invention was able to restore damage by activating the survival pathway and maintaining the balance between BDNF/GDNF activity better than single agents during L-Glu exposure (p <0.05) Specifically, the combination of the invention was able to increase the BDNF activity of 37% compared to B. Bifidum, of 20% compared to B. Longum, and of 48% compared to L Paracasei. Moreover, the combination of the invention was able to reduce GDNF activity of 52%, 41 % and 43% compared to B. Bifidum, L. Longum and L. Paracasei respectively. Finally, the combination of the invention was able to increase Serotonin production of 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), whose production plays a key role in the pathophysiology of stress by triggering a deterioration of synaptic plasticity, neuronal dysfunction and neuroinflammation.

The above assays showed that the supplementation of the specific strains of the invention can restore proper intestinal metabolism, which is related to the maintenance mood.

The presence of the secretion of SCFAs, for example butyric acid, capable of functioning as second messengers and activating various mechanisms that are impaired in conditions of cognitive disfunction.

It can be assumed that a maintained intestinal homeostasis can 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 modulating apoptosis-related protein expression, by downregulating the synthesis of proapoptotic BAX and upregulating antiapoptotic bcl-2 expression. Analysis on the neuroprotection effects of the combination of the invention in neuronal cell injury model, mediated via serotonergic-cholinergic interactions, underling the importance of said strains in neuroprotective mechanism leading to mood disorders.

The inhibition of proinflammatory cytokines is the basis for decrease neuroinflammation in depression, capable of disrupting the brain’s regulatory and signalling mechanisms involving behavioural and emotional aspects.

Cognitive functionality

Assays to investigate the role of the microbiome in connection with cognitive impairments were also carried out. Firstly, a dose-response study was conducted on an in vitro model using SHSY-5Y cells to evaluate the safety of different probiotics and substances, as follow:

• Bifidobacterium Bifidum novaBBF7 (herein after also referred to as B. Bifidum ranging from 5 mg/ml to 30 mg/ml (corresponding to 0.5x10⁹-3x10⁹ CFU)

• Bifidobacterium Longum novaBLG2 (herein after also referred to as B. Longum) ranging from 1 mg/ml to 30 mg/ml (corresponding to 1x10⁸-3x10⁹ CFU)

Lactobacillus Paracasei T JB8 (herein after also referred to as L. Paracasei) ranging from 3 pig/ml to 20 mg/ml (corresponding to 1x106-6x109 CFU).

• a strain of L Helveticus JM ranging from 1 .1 pig/ml to 17mg/ml (corresponding to 1.1x105-1.7x109 CFU)

• a strain of L. Plantarum TJA7 ranging from 100pig/ml to 2.5mg/ml (corresponding to 2.5x108-1x109 CFU)

• a strain of B. Psychaerophilum Q5 ranging from 3pig/ml to 20mg/ml (corresponding to 3x105-2x109 CFU)

All substances were prepared directly in the stimulation medium.

Based on the data obtained, the best concentration of each substance was tested for the intestinal barrier or by modulating the mediators released by probiotics. In this context, a gut/brain co-culture model was performed 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. Therefore, safety test and the analysis of barrier integrity were performed in order to avoid irritability, also evaluating the active role of their metabolites crossing intestine. Consequently, samples metabolized by intestinal cells were used to stimulate the brain epithelium (SHSY-5Y cells) placed in the basolateral compartment analysing the main biological activity exerted by probiotics during cognitive disfunctions. More in details, further experiments were performed to evaluate two different biological aspects involved in brain ageing and neurodegeneration: oxidative stress and irondependent damage. Indeed, the role of oxidative stress was investigated by pre-treatment for 30 min with 200 pM H2O2 on neuronal cells. Cell viability, mitochondrial membrane potential and ROS production were analyzed to evaluate the ability of all probiotics, alone or combined, to prevent or restore damage caused by oxidative stress, with H2O2. Finally, to induce neurodegeneration, cells were pre-treated with catalytic iron (Fe³*) 75 pM for 24 hours and then treated with all probiotics, alone and combined, for 24 h to investigate the protection exerted by the combination, analysing viability, lipid peroxidation and activated intracellular pathways during iron accumulation which led cognitive disfunction. Materials and methods

Cell cultures

SH-SY5Y cells (ATCC, Manassas, VA, USA) were cultured in a mixture of Dulbecco's Modified Eagle Medium F12 (Sigma-Aldrich, Milan, Italy) and Dulbecco's Modified Eagle Medium (Merck, Milan, Italy) at a ratio of 1 : 1 , supplemented with 10% fetal bovine serum (FBS, Merck, Milan, Italy), and 2 mM HEPES (Merck, Milan, Italy), 2mM L-Glutamine (Merck, Milan, Italy) and 1% penicillin/streptomycin (Merck, Milan, Italy). Cells are maintained in a 37°C incubator at 5% CO2 and 95% humidity.

The CaCo-2 cell line, provided by the American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in DMEM/F12 (Merck, Milan, Italy) containing 10% FBS (Merck, Milan, Italy), 2 mM I- glutamine (Merck, Milan, Italy) and 1% penicillin-streptomycin (Merck, Milan, Italy) at 37 °C in a 5% CO₂ incubator.

Gut-Brain axis

Transwell® co-culture system was used to test the interaction between CaCo-2 cells and SHSY-5Y cells. SHSY- 5Y cells were pre-treated to induce oxidative stress injury as previously described. A semipermeable membrane with a pore size of 0.4pm (BD Biosciences) was used to separate the two chambers filled with DM EM medium. CaCo-2 cells were seeded in the upper chamber at the density of 50000cell/well and reached at confluences for 28 days (cells maturation time in which intestinal cells produce microvilli); when intestinal cells lead to monolayer, SHSY-5Y cells were seeded in the lower chamber at density of 50000 cell/well and maintained in culture until they reached confluence.

MTT test

Intestinal integrity analysis

The TEER values of the inserts were measured on the alternate days continuously for 21 days using EVOM3 and the experiments were started when TERR reached >500 0 cm2. In literature it is reported that the TEER values >500 ± 52.9Q cm2 are recommended for the transport study.

Butyric Acid quantification

In order to quantify probiotics metabolites, butyric acid production was analyzed using ELISA kit (Cloud-Clone, Wuhan) according to manufacture instruction. The absorbance of each sample was measured after the addition of stop solution at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan) and the CD were inter-polarized with a standard curve (from 10.000 pg/mL to pg/ml), expressing the data as mean (pg/ml) compared to control.

ROS Production

The rate of superoxide anion release was used to examine ROS produced by astrocytes after stimulations. After treatment, in all samples (treated or not), 100 pl of cytochrome C was added, and in another sample, 100 pl superoxide dismutase was also added for 30 min in an incubator (all substances were from Sigma-Aldrich). The absorbance was measured by a spectrometer (Infinite 200 Pro MPlex, Tecan), at 550 nm, and O2 was expressed as of nanomoles per reduced cytochrome C per microgram of protein compared to the control on percentage (%). Mitochondrial Membrane Potential

The mitochondrial membrane potential was analyzed by the Oxygen Consumption/Mito membrane Potential Dual Assay Kit (Cayman Chemical Company) following manufacturer's instructions. The mitochondrial membrane potential was measured using JC-1 aggregates at an excitation/emission of 560/590 nm and monomers at an excitation/emission of 485/535 nm in a fluorescence spectrometer (Infinite 200 Pro MPlex, Tecan). The results are expressed as (%) compared to control cells.

AKT/ERK ELISA Kit

AKT/ERK activation was measured by the InstantOneTM ELISA (Thermo Fisher, Milan, Italy) on chondrocytes lysates as reported in literature. The strips were measured by a spectrometer at 450nm (Infinite 200 Pro MPlex, Tecan). The results were expressed as mean absorbance (%) compared to control.

P53 ELISA Kit

P53 activity was measured by 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. The nuclear extraction was obtained by the classical technique using a complete buffer present in the kit. Briefly, the 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 min. The pellet was solubilized with ice-cold Complete Nuclear Extraction Buffer 1x supplemented with protease and phosphatase inhibitors and then centrifuged at 12,000 g for 15 min at 4°C; the supernatant was examined to analyze the activity of p53 related to the protein quantification through the BCA assay (Thermo Fisher).

BAX ELISA assay

BAX activity was determined using ELISA kit (MyBiosource, San Diego, CA, USA) analyzing the presence of BAX in cell lysates, according to the manufacturer's instruction. Briefly, 100 pL of sample or standard were added in each well and incubated at 37°C for 90 minutes. Then, after washes, 100 pL of biotinylated antibody were added in each well and incubated for 60 minutes at 37°C. After, each well was washed and 100 pL of SABC working solution were added for 30 minutes at 37°C. Then, 90 pL of TMB substrate were added and incubated for 15-30 minutes at 37°C. The samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as pg/mL compared to a standard curve (range from 0 to 2000 pg/mL) and the results are expressed as percentage (%) versus control (0 line).

SOD assay

The level of SOD was measured following the manufacturer’s instructions (Cayman's Superoxide Dismutase Assay Kit) as previously described. Briefly, in a 96 well plate, the level of SOD present on cell lysates was measured by comparing data to a standard curve (0.05-0.005 U/mL). The absorbance of all samples was measured through a spectrometer (Infinite 200 Pro MPlex, Tecan) at 480 nm and the results are expressed as a means (%) compared to control. iNOS ELISA kit i NOS activities were determined using ELISA kit (Thermoscientific, Waltham, Massachusetts, USA) determining the iNOS presence in cell lysates, according to the manufacturer's instruction. The samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as ng/mL compared to a standard curve (range from 0.4 to 100 ng/mL) and the results are expressed as percentage (%) versus control (0 line).

Amyloid Precursor Protein (APP) ELISA Kit

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

APOE 9 ELISA kit

APOE activities were determined using ELISA kit (Thermoscientific, Waltham, Massachusetts, USA) determining the APOE presence in cell lysates, according to the manufacturer’s instruction. The samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as ng/mL compared to a standard curve (range from 1.6 to 400 ng/mL) and the results are expressed as percentage (%) versus control (0 line).

Thiobarbituric acid reactive substances (TBARS) assay

Lipid peroxidation in the cells was estimated using the thiobarbituric acid-reactive substance (TBARS) assay Briefly, 10OpL of sample or standard was added into 5mL vial and then 10OpL of SDS solution was added. 4mL of the Color Reagent was added in each vial and boiled the vials for 1 hour and, at the end of 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 150pL were added into wells of 96 plate and red the absorbance at 530-540 nm.

Human pTAU quantification

TAU protein quantification was measured by Human Tau [pS199] ELISA Kit (Thermo Scientific™, Waltham, MA, United States) on cell lysate. Briefly, 100 pL of standard or sample were added in each well and the plate was incubated at room temperature for 2 hours. At the end of incubation, the wells were washed with 1X Wash Buffer and then 100 pL of Anti-Rabbit IgG HRP solution were added into each well and incubate for 30 minutes at room temperature. The wells were washed again with 1X Wash Buffer and 100 pL of Stabilized Chromogen were added in each well. The plate was incubated at room temperature for 30 minutes and, at the end, 100 pL of Stop Solution were added in each well. The absorbance was red through a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm and the concentration is expressed as pg/mL compared to a standard curve (range from 15.6 to 1000 pg/mL) and the results are expressed as percentage (%) versus control (0 line).

Human SIRT1 ELISA Kit SIRT1 protein quantification was measured by Human SIRT1 ELISA Kit (Thermo Scientific™, Waltham, MA, United States) on cell lysate. Briefly, 100 pL of standard or sample were added in each well and the plate was incubated at room temperature for 2.5 hours. At the end of incubation, the wells were washed with 1X Wash Buffer and 10OuL of prepared biotin conjugate were added into each well and incubate for 1 hour at room temperature. The wells were washed again with 1X Wash Buffer and 100pL of Streptavidin-HRP were added in each well. The plate was incubated at room temperature for 45 minutes and, at the end, 100 pL of TMB Substrate were added in each well and the plate was incubated for 30 minutes at room temperature in the dark. Then, 50 pL of Stop Solution were added in each well and the absorbance was red through a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as ng/mL compared to a standard curve (range from 1 .23 to 300 ng/mL) and the results are expressed as percentage (%) versus control (0 line).

BDNF ELISA Kit

NRF1 ELISA kit

NRF1 activities were determined using ELISA kit (Thermoscientific, Waltham, Massachusetts, USA) determining the Caspase 9 presence in cell lysates, according to the manufacturer's instruction. The samples were analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration is expressed as pg/mL compared to a standard curve (range from 82.3-20000 pg/mL) and the results are expressed as percentage (%) versus control (0 line).

Statistical Analysis

Results are expressed as mean ± SD of at least 5 biological replicates for each experimental protocol, and each replicate was replicated 3 times for each experimental protocol. Statistical comparisons between groups were performed using one-way ANOVA with Bonferroni's post hoc test or the Mann-Whitney U test, as appropriate, using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). 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 the control values in percent (defined as 0%).

Results

In recent years, probiotics have received increasing attention for their extensive clinical applications and beneficial health effects on various clinical disorders. Several studies have indicated that the intestinal flora plays a more important role in regulating cognitive activities; moreover, the present study investigates the role of different probiotics able to improve cognitive functions. To do this, firstly, a 24-hour dose-response study was performed to screen 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 appeared to be the best probiotics able to induce the greatest effect on cell viability compared to the control and to other probiotics tested (p<0.05); in particular B. Bifidum 10mg/ml, B. Longum 5 mg/ml and L. Paracasei 10 mg/ml exerted the greatest effects on cell viability (p<0.05) suggesting that this concentration could be the use for all successive experiments (Figure 8).

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

Before going in detail about the gut-brain axis, further investigations were performed to gain more information on physiological absorption performing a gut barrier model, validated by EMA and FDA.

Therefore, cell viability, TEER value and the metabolite production were evaluated to demonstrate the ability of the new formulation hypothesized to maintain the correct intestinal physiology without inducing cell damages. As shown in Figure 9, intestinal passage analysis demonstrated that all selected agents are able to cross the intestinal epithelium. In particular, cell viability shows an increase for all 3 concentrations tested compared to control, although the greatest effect (p<0.05) was observed with the combination of the invention (about 33% vs. Bifidum; 1 time more vs Longum and 2 times more vs. Paracasei), suggesting that the combination of the invention is safe for the intestinal epithelium, also suggesting its absorption. This effect was confirmed by TEER values analysis which reached a value around 510 ± 10 !*cm² for intestinal cells, as reported in literature confirming that the cells form an intact monolayer after treatments, maintaining a correct intestinal homeostasis. Indeed, all the probiotics tested were able to cross the intestinal barrier reaching the plasma environment (p < 0.05), as observed by Butyric acid analysis at the basolateral level. In particular, the combination of the invention metabolite production appears to follow a similar cell viability trend, showing an increase in SCFA production (about about 33% vs. Bifidum; 3 times more vs Longum and 7 times more vs. Paracasei, p<0.05), amplifying the effects exerted by the single agents, supporting the hypothesis of a synergistic activity between B. Longum, B. Bifidum and L Paracasei.

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

Ability of probiotics to modulate the mechanisms connected to cognitive functions in oxidative conditions.

In order to investigate the potential action of probiotics to prevent cellular damages under oxidative condition, cell viability, ROS production, and mitochondrial potential were evaluated by pre-treating neuronal cells with 200 pM H2O2 as reported in literature. As shown in figure 10, exposure to H2O2 significantly reduced cell viability of about 36% compared to the control; conversely, following treatment with probiotics metabolites produced at intestinal level, the cell viability was significantly increased but the greatest effect was obtained with the combination of the invention, which reverted the cell loss compared to probiotics alone and compared to H2O2 (about 65%, p<0.05). Moreover, considering that the ERK/MAPK and PI3K-Akt pathways play a crucial role in the regulation of neuronal and brain survival, additional experiments on their activity were performed. All probiotics alone confirmed their ability to improve the viability of cells, 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 kinase activation compared to the control and to single administration during exposure to 200 pM H2O2 (about 3 times more, p<0.05) showing a greater effect in PI3/Akt and ERK/MAPK, supporting the hypothesis that all neuronal survival signalling was switched on. These results indicate that the combination of probiotics revert the H2O2- induced cell loss activating survival pathways (Figure 10).

At the same time, since the main theory at the basis of brain degeneration regards the oxidative condition, ROS production was investigated as well as SOD3 and iNOS expressions. Also in this case, all probiotics, alone and combined, were able to maintain the ROS production under the physiological level (p<0.05 vs. control), supporting the hypothesis of their safety during the exposure to 200 pM H₂O₂; in particular, all probiotics combined significantly reduced the intracellular ROS production of about 70% compared to H2O2 (p<0.05) confirming the active role of the combination of the invention to counteract oxidative damages. As reported in Figures 11, SOD3 and INOS expressions significantly increased in the presence of 200 pM H2O2 compared to control (p<0.05), supporting the hypothesis of the involvement of oxidative stress in neuronal death. On the contrary, the treatment with B. Bifidum 10mg/ml, B. Longum 5mg/ml and L Paracasei 10 mg/ml alone significantly reduced the expression of both SOD3 and iNOS compared to 200 pM H2O2 alone (p<0.05), but a greater reduction was obtained by the combined stimulation (about 1.5 times more and 2.5 times more respectively compared to 200 pM H2O2 p<0.05), indicating a beneficial effect in counteracting the cognitive disfunctions. These results indicate that the combination of probiotics revert the F^-induced cell loss activating survival pathways (Figure 11).

Since the alteration of the formation of a proton gradient across the inner mitochondrial membrane is considered to be one of the key indicators of cellular viability, the mitochondrial potential was analyzed and as expected, treatments with all probiotics, alone combined induced a significant increase of JC-1 fluorescence, supporting the active role of probiotics and their combination on mitochondrial activity (p<0.05) also during oxidative stress induced by 200 piM H2O2. In addition, FkC treated cells exhibited changes in the fluorescence signal which led to a decreased red fluorescence signal and increased green fluorescence signal, indicating a significant dissipation of mitochondrial potential and cell loss compared to the control (p<0 05); conversely, the combination of the invention reversed dissipation of mitochondrial potential compared to 200 pM H2O2 alone (about 3.5 times more, p<0.05), shifting the fluorescence signal from green to red. These results indicate that the combination of the invention attenuates the FhCk-induced apoptosis through the mitochondrial-mediated pathway. Considering that the dissipation of mitochondrial potential under oxidative condition 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 ageing, oxidative stress, and neurodegeneration, and BAX is a key regulator of both cellular energetic metabolism and apoptosis, they were both investigated in neuronal cells under oxidative conditions. Data reported in figure 12, showed and induction in p53 and BAX activity in H2O2-treated cells (p<0.05) vs control, leading to apoptosis process; conversely, probiotics stimulation alone reduced apoptosis activation reverting the damages process. Moreover, the combination of the invention amplified the reduction compared to H₂O₂ (about 8 times more and 10 times more respectively, p<0.05), resulting in survival-favored conditions and confirming the reduction in 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 a natural consequence of apoptosis is known to be cell loss, the apolipoprotein E (APOE) and IB- amyloid analysis (APP) demonstrated the alteration in brain tissue. Indeed, H2O2 caused a significant increase of the APOE and APP level, supporting previous data about cell death and suggesting the impairment in cognitive functions. However, the treatment with probiotics was able to reduce the damage by decreasing APOE and APP level compared to control and compared to H2O2 (p<0.05). Therefore, 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 200 pM H2O2, p<0.05), indicating the effectiveness of the combination during cognitive impairment. These results demonstrated that the combination of the invention is able to revert the damages induced under oxidative condition, confirming the active role of probiotics in ameliorating intestinal dysregulation which can modulate cell loss and cognitive disfunction (Figure 13).

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

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

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

As expected Fe³* stimulation increased oxidative stress and inflammation, but this condition was reverted when probiotics were used (p<0.05). Indeed, all probiotics tested alone were able to restore the damage compared to Fe³* (p<0.05) and this reduction was amplified when all probiotics were combined, confirming the positive role of the combination of the invention to counteract brain damage (p<0.05). All these beneficial effects were induced by the combination of the invention since it is able to improve BDNF production (about 3 times more compared to H2O2, p<0.05) and, consequently, activates different biological pathways with the aim to restore iron accumulation damages.

These data suggest that the combination of the invention is able to improve BDNF production even during injury induction, supporting the efficacy of the combination of the invention to maintain neuronal activity and they 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 above assays, it can be derived that specific concentration of the strains of the invention are able to stimulate the gut/brain axis and that they can restore proper intestinal metabolism, which is related to the maintenance of cognitive function.

The presence of the secretion of SCFAs, for example butyric acid, capable to function as second messengers and to activate various mechanisms that are impaired in conditions of cognitive disfunction.

A maintained intestinal homeostasis can improve brain activity by decreasing cell loss.

The substances tested did not induce any damage in the intestinal epithelia, confirming their safety properties enhancing antioxidant mechanisms and anti-neuroinflammation activity during brain damages by also acting on several molecular pathways involved in neuronal well-being, such as ERK/MAPK and PI3K/AKT. The combination between B. Bifidum 10 mg/ml + B. Longum 5 mg/ml + L Paracasei 10 mg/ml was able to restore the negative effect by improving BDNF production which modulate several brain proteins involved in cognitive functions, such as SIRT1 , pTAU and APP connected to cognitive disfunction acting directly on specific biomarkers.

Claims

1 . A pharmaceutical or nutraceutical or food supplement composition comprising or, alternatively, consisting of at least two bacteria strains selected from group comprising or, alternatively, consisting of:

with deposit number DSM 33129;

- Bifidobacterium bifidum ”novaBBF7” with deposit number DSM 34336;

- Bifidobacterium longum "novaBLG2" with deposit number DSM 34339; or a mixture thereof, and, optionally together with at least one excipient and/or physiologically and/or pharmaceutically acceptable vehicle, or food grade.

2. The composition according to claim 1, wherein said composition comprises or, alternatively, consists of at least two bacteria strains:

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 , e.g., 1:3 to 3:1; or 1 :2 to 2: 1; or 1 :1; or ii) Lactobacillus paracasei“~[ B8’ 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 , e.g., 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 , e g., 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 bacteria strains: 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 one of claims 1 to 3, wherein said composition comprises (w/w):

5. The composition according to any one of claims 1 to 4, wherein said composition comprises (w/w):

- from 5 to 20 mg of Lactobacillus paracasei TJB8, and - from 5 to 20 mg of Bifidobacterium bifidum novaBBF7, and

- from 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.

6. The composition according to any one of claims 1 to 5, wherein said bacteria strain Lactobacillus paracasei TJB8 is administered at a daily dose comprised from 1 ,0x10⁶CFU/day to 1,0x10¹²CFU/day; preferably, from 1 ,0x10⁸CFU/day to 1 ,0x10¹⁰CFU/day, for example 3,0x10⁹CFU/die; said bacteria strain Bifidobacterium bifidum novaBBF7 is administered at a daily dose comprised from 1 ,0x10⁶CFU/day to 1 ,0x10¹²CFU/day; preferably, from 1 ,0x10⁸CFU/day to 1 ,0x10¹⁰CFU/day, for example 1,0x10⁹ CFU/die; said bacteria strain Bifidobacterium longum novaBLG2 is administered at a daily dose comprised from 1,0x10⁵CFU/day to 1 ,0x10¹²CFU/day; preferably, from 1 ,0x10⁸CFU/day to 1 ,0x10¹⁰CFU/day, for example 0,5x10⁹CFU/die.

7. The composition according to any one of claims 1 to 6, wherein said at least one bacteria strain is for use as medicament for brain disorders.

8. The composition for use according to claim 7, wherein said bacteria is for use for brain disorders, including the treatment for treating and/or preventing mood disorders, such as anxiety and depression, and for improving cognitive functionality and treating and preventing cognitive decline, such as in Alzheimer's disease and senile dementia.

9 A bacteria strain selected from group comprising or, alternatively, consisting of:

- Lactobacillus paracasei “T JB8” with deposit number DSM 33129;

- Bifidobacterium bifidum "novaBBF7” with deposit number DSM 34336;

- Bifidobacterium longum "novaBLG2" with deposit number DSM 34339; or a mixture thereof.

10. The bacteria strain according to claim 9, wherein said bacteria is for use as a medicament for brain disorders; preferably it is for use for brain disorders, including the treatment for treating and/or preventing mood disorders, such as anxiety and depression, and for improving cognitive functionality and treating and preventing cognitive decline, such as in Alzheimer's disease and senile dementia.