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WO2024095199A1 - Souches bactériennes pour soutenir l'humeur et la fonctionnalité cognitive, compositions et utilisations associées - Google Patents

Souches bactériennes pour soutenir l'humeur et la fonctionnalité cognitive, compositions et utilisations associées Download PDF

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Publication number
WO2024095199A1
WO2024095199A1 PCT/IB2023/061058 IB2023061058W WO2024095199A1 WO 2024095199 A1 WO2024095199 A1 WO 2024095199A1 IB 2023061058 W IB2023061058 W IB 2023061058W WO 2024095199 A1 WO2024095199 A1 WO 2024095199A1
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cfu
dsm
day
bifidobacterium
novablg2
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Marco BOCCARUSSO
Giorgio Stefano CERANA
Peter Franck
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Probionova Sa
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Probionova Sa
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • 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.
  • 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.
  • 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.
  • MDD major depressive disorder
  • BD bipolar disorder
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • BDNF brain-derived neurotrophic factor
  • 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:
  • TJB8 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;
  • novaBBFZ 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.
  • the bacterial strains belonging to the species Lactobacillus paracasei are reclassified under the nomenclature Lacticaseibacillus paracasei.
  • 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.
  • panel A cell viability assessed by MTT evaluated.
  • 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.
  • panel A cell viability assessed by MTT evaluated.
  • 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 Fe 3+ ; 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 Fe 3+ ; the bar p ⁇ 0.05 vs single agent. DESCRIPTION OF THE INVENTION
  • the present invention relates to a bacterial strain chosen from the group comprising or, alternatively, consisting of:
  • TJB8 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;
  • novaBBFZ 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;
  • novaBLG2 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.
  • the bacterial strains belonging to the species Lactobacillus paracasei are reclassified under the nomenclature Lacticaseibacillus paracasei.
  • 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:
  • 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.
  • 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.
  • the present invention relates to a pharmaceutical or nutraceutical or food supplement composition
  • 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.
  • the present invention relates to a pharmaceutical or nutraceutical or food supplement composition
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention relates to a composition comprising (w/w):
  • Lactobacillus paracasei T JB8 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
  • Bifidobacterium bifidum novaBBF7 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
  • Bifidobacterium longum novaBLG2 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.
  • the present invention relates to a composition
  • 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.
  • compositions of the invention may comprise one or more physiologically and/or pharmacologically acceptable excipients and/or carriers.
  • the bacteria strain Lactobacillus paracasei TJB8 is administered at a daily dose comprised from 1 ,0x10 6 CFU/day to 1 ,0x10 12 CFU/day; preferably, from 1 ,0x10 8 CFU/day to 1 ,0x10 1 °CFU/day, such as for example 3,0x10 9 CFU/die.
  • the bacteria strain Bifidobacterium bifidum novaBBF7 is administered at a daily dose comprised from 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 for example 1 ,0x10 9 CFU/die.
  • compositions can be administered once or more times a day, preferably once a day.
  • 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.
  • the at least one physiologically and/or pharmacologically acceptable excipient may include prebiotics.
  • 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.
  • the at least one physiologically and/or pharmacologically acceptable excipient may include partially hydrolysed guar gum (PHGG).
  • PHGG partially hydrolysed guar gum
  • 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.
  • strains of the invention have been the subject of several experimental tests, reported below, to investigate the interaction between the microbiome and the brain.
  • MIX the combination of the invention
  • B. Bifidum novaBBFT Bifidobacterium Bifidum novaBBFT (herein after also referred to as B. Bifidum) ranging from 5mg/ml to 30mg/ml (corresponding to 0.5x10 9 -3x10 9 CFU)
  • B. Longum Bifidobacterium Longum novaBLG2 (herein after also referred to as B. Longum) ranging from 1mg/ml to 30mg/ml (corresponding to 1 x10 8 -3x10 9 CFU)
  • Lactobacillus Paracasei TJB8 (herein after also referred to as L. Paracasei) ranging from 3pig/ml to 20mg/ml (corresponding to 1x10 6 -6x10 9 CFU)
  • 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.
  • 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.
  • Plantarum TJA7 from 2.5 mg/mL to 30 mg/mL
  • 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.
  • 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).
  • 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).
  • a spectrometer Infinite 200 Pro MPlex, Tecan
  • BDNF Brain-derived neurotrophic factor quantification was measured by Rat BDNF Elisa Kit (Thermo ScientificTM, 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.
  • 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.
  • 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.
  • 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 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.
  • 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
  • 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 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
  • 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.
  • 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.
  • 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).
  • an 8mM glutamate pre-treatment was employed to simulate a condition of depression or more generally mood disorders as reported in literature.
  • pre-treatment with 8mM glutamate alone induces a significant reduction in cell viability
  • 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.
  • the combination of the invention was able to modulate both BAX and Cyto-C confirming the active role to maintain the neuronal cell homeostasis.
  • 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.
  • the combination of probiotics reverts the L-Glu-induced inflammation
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • SCFAs for example butyric acid
  • 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.
  • proinflammatory cytokines 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.
  • Bifidobacterium Bifidum novaBBF7 (herein after also referred to as B. Bifidum ranging from 5 mg/ml to 30 mg/ml (corresponding to 0.5x10 9 -3x10 9 CFU)
  • B. Longum Bifidobacterium Longum novaBLG2 (herein after also referred to as B. Longum) ranging from 1 mg/ml to 30 mg/ml (corresponding to 1x10 8 -3x10 9 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 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.
  • 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.
  • 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% CO 2 incubator.
  • 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.
  • 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).
  • 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 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.
  • 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 (%).
  • 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 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 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 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).
  • 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) 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.
  • a spectrometer Infinite 200 Pro MPlex, Tecan
  • 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).
  • TBARS Thiobarbituric acid reactive substances
  • 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.
  • TBARS thiobarbituric acid-reactive substance
  • TAU protein quantification was measured by Human Tau [pS199] ELISA Kit (Thermo ScientificTM, 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 ScientificTM, 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 Brain-derived neurotrophic factor quantification was measured by Rat BDNF Elisa Kit (Thermo ScientificTM, 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.
  • 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).
  • 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%).
  • 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.
  • intestinal passage analysis demonstrated that all selected agents are able to cross the intestinal epithelium.
  • 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.
  • 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.
  • the combination of the invention amplified the reduction compared to H 2 O 2 (about 8 times more and 10 times more respectively, p ⁇ 0.05), resulting in survival-favored conditions and confirming the reduction in cell loss.
  • 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.
  • BDNF neurotrophic protein which contributes to neuronal progression activity
  • NRF-1/PGC-1o neurotrophic protein which contributes to neurogenesis SIRT1
  • pTAU neuroprotection against neurodegeneration progression
  • BDNF neurotrophic protein which contributes to neuronal progression activity
  • pTAU neurotrophic protein which contributes to neurogenesis SIRT1
  • pTAU neurotrophic protein which contributes to neurogenesis SIRT1
  • pTAU which is an important marker to aging and neurodegeneration during oxidative stress and age-related disorders
  • SCFAs for example butyric acid
  • 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.

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Abstract

La présente invention concerne des souches bactériennes appartenant au genre Bifidobacterium et Lactobacillus, leur combinaison, et leur utilisation en thérapie, en particulier pour la régulation de l'humeur et le soutien de la fonction cognitive. L'invention concerne également des compositions pharmaceutiques, nutraceutiques et de compléments alimentaires contenant au moins une de ces souches appartenant au genre Bifidobacterium et Lactobacillus. De plus, la présente invention concerne des souches bactériennes sélectionnées appartenant aux espèces Bifidobacterium et Lactobacillus, et des mélanges et des compositions de celles-ci, destinées à être utilisées dans une méthode de traitement, de préférence préventif ou curatif, de régulation de l'humeur et de soutien de la fonction cognitive, de préférence pour une utilisation dans une méthode de traitement des troubles cérébraux, incluant le traitement et/ou la prévention des troubles de l'humeur, tels que l'anxiété et la dépression, et pour l'amélioration de la fonction cognitive et le traitement et la prévention du déclin cognitif, comme dans la maladie d'Alzheimer et la démence.
PCT/IB2023/061058 2022-11-02 2023-11-02 Souches bactériennes pour soutenir l'humeur et la fonctionnalité cognitive, compositions et utilisations associées Ceased WO2024095199A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120366154A (zh) * 2025-05-13 2025-07-25 河北源民生物科技有限公司 一株促进大脑发育、提升专注力、营养神经的瑞士乳杆菌ym1922及代谢产物、复合菌群、后生元和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016065419A1 (fr) * 2014-10-28 2016-05-06 Medlab Ip Pty Ltd Traitement de la dépression et des troubles dépressifs
US20180355445A1 (en) * 2015-09-15 2018-12-13 University-Industry Cooperation Group Of Kyung Hee University Novel Lactobacillus and Composition for Preventing, Improving, or Treating Degenerative Brain Diseases or Cognitive Function Disorders

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016065419A1 (fr) * 2014-10-28 2016-05-06 Medlab Ip Pty Ltd Traitement de la dépression et des troubles dépressifs
US20180355445A1 (en) * 2015-09-15 2018-12-13 University-Industry Cooperation Group Of Kyung Hee University Novel Lactobacillus and Composition for Preventing, Improving, or Treating Degenerative Brain Diseases or Cognitive Function Disorders

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Perelle 30 Compresse Tristrato: Utilizzo, effetti collaterali e prezzo | 1000farmacie", 1 February 2001 (2001-02-01), XP093129096, Retrieved from the Internet <URL:https://www.1000farmacie.it/perelle-30cpr-tristrato.html> [retrieved on 20240208] *
L. HELVETICUSPROBIONOVA SA, TJA4 DEPOSITED WITH THE DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GMBH (DSMZ) WITH FILING NUMBER DSM 33128, 17 May 2019 (2019-05-17)
PROBIONOVA SA: "deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33129", LACTOBACILLUS PARACASEI ''TJB8, 17 May 2019 (2019-05-17)
PROBIONOVA SA: "deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34336", BIFIDOBACTERIUM BIFIDUM ''NOVABBF7, 26 July 2022 (2022-07-26)
PROBIONOVA SA: "deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34339", BIFIDOBACTERIUM LONGUM ''NOVABLG2, 26 July 2022 (2022-07-26)
STEFAN SIEBRECHT ET AL: "THE INFLUENCE OF PARTIALLY HYDROLYZED GUAR GUM (PHGG) FIBER ON THE INTESTINAL MICROBIOTA", 1 January 2021 (2021-01-01), pages 42 - 45, XP093032642, Retrieved from the Internet <URL:https://www.teknoscienze.com/tks_article/the-influence-of-partially-hydrolyzed-guar-gum-phgg-fiber-on-the-intestinal-microbiota/> [retrieved on 20230317] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120366154A (zh) * 2025-05-13 2025-07-25 河北源民生物科技有限公司 一株促进大脑发育、提升专注力、营养神经的瑞士乳杆菌ym1922及代谢产物、复合菌群、后生元和应用

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