Bifidobacterium animalis subsp lactis strain cect 8145 and lta thereof health uses

Abstract

The invention relates to health uses of a composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145, in the form of viable and non-viable cells. and / or a lipoteichoic acid (LT A) thereof, more particularly, for its use in anxiety, pathogen protection, gut barrier or age-related conditions in a subject. Given the associated effects, the invention is thus useful for exploitation in pharmaceutical and veterinary areas, food and beverages developments, animal feed, or medical foods among others.

Description

[0001] Bifidobacterium animalis subsp lactis strain CECT 8145 and LTA thereof health uses

[0002] The present invention falls within the food, feed and pharmaceutical industries. In particular, the invention relates to health uses of a composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145, in the form of viable and non-viable cells, and / or a lipoteichoic acid (LTA) thereof, more particularly, for its use in anxiety, pathogen protection, gut barrier or age-related conditions in a subject.

[0003] BACKGROUND ART

[0004] Life expectancy has increased globally by more than 6 years in the period from 2000 to 2019 while healthy life expectancy without disabilities has not improved at the same level (following WHO guidelines). In recent years, the role of human gut microbiota has been highlighted as key to maintain healthy aging and gut dysbiosis can contribute to the initiation and / or progress of many diseases (Coman, V. & Vodnar, D. C. Gut microbiota and old age: Modulating factors and interventions for healthy longevity. Exp Gerontol 141 , 111095, doi:10.1016 / j.exger.2020.111095 (2020)). In this context, discovering and understanding the mechanisms of action of nutraceuticals as probiotics and postbiotics that promote healthy aging is key to delay age-related pathologies.

[0005] It has been previously shown the effectiveness of the Bifidobacterium animalis subsp. lactis strain CECT 8145 (BPL1), its heat-treated form (HT-BPL1) and a novel postbiotic, the lipoteichoic acid (LTA), on fat reduction using the animal model Caenorhabditis elegans (C. elegans), as disclosed in Martorell, P. et al. Probiotic Strain Bifidobacterium animalis subsp. lactis CECT 8145 Reduces Fat Content and Modulates Lipid Metabolism and Antioxidant Response in Caenorhabditis elegans. J Agric Food Chem 64, 3462-3472, doi:10.1021 / acs.jafc.5b05934 (2016). LTA was one of the first key probiotic effector molecules reported in modulation of obesity (Balaguer, F. etal. Lipoteichoic acid from Bifidobacterium animalis subsp. lactis BPL1 : a novel postbiotic that reduces fat deposition via IGF-1 pathway. Microb Biotechnol, doi:10.1111 / 1751-7915.13769 (2021). On the other hand, some other strains belonging to Bifidobacterium animalis subsp. lactis are known to exert health beneficial effects, as well as in the improvement of particular conditions. For instance, in Dong W, et al., Front Psychol. 2021 Jan 13; 11 :570298, it is disclosed a study related to the effects produced by the intake of Bifidobacterium animalis subsp. lactis Bb-12 in athletes anxiety state and sports performance under stress.

[0006] Given the ever-increasing ageing of the population, there is a need in the field for finding alternative strains and compositions with positive health effects, as well as for the improvement of particular conditions, for instance in anxiety disorders, pathogen protection, intestinal integrity or age-related disorders.

[0007] DESCRIPTION OF THE INVENTION

[0008] The authors of the present invention have demonstrated new functionalities and beneficial health effects exerted by Bifidobacterium animalis subsp. lactis strain CECT 8145 (also known in the Example section as BPL1), including heat-treated cells (HT- BPL1), and its lipoteichoic acid (LT A).

[0009] In particular, inventors of the present invention have shown that viable and heat- treated cells of Bifidobacterium animalis subsp. lactis strain CECT 8145, as well as its LTA, (i) improve anxiety and stress related behaviors (as shown in Examples, in particular in section 2.2), (ii) exert ability to protect against pathogen (as shown in Examples, section 2.5), (iii) promote gut barrier integrity (as shown in Examples, section 2.6), and (iv) promote longevity effects having a positive impact in lifespan and age-related conditions, as shown, for instance in the lifespan assays (Examples, section 2.7), C. elegans Alzheimer’s model (section 2.2. in Examples) or in further examples of BPL1 , HT-BPL1 and LTA-BPL1 effects in aging and longevity (Examples, section 4).

[0010] Furthermore, comparative assays were performed in C.elegans models, showing that Bifidobacterium animalis subsp. lactis strain CECT 8145 has a better effect on anxiety / stress-related behaviors than other strains belonging to Bifidobacterium animalis or Bifidobacterium longum (Examples, sections 2.3 and 2.4, and section 5). Likewise, comparative assays in nematodes’ longevity were also carried out, showing that supplementation with Bifidobacterium animalis subsp. lactis strain CECT 8145 exerts a higher effect on C. elegans lifespan than other strains belonging to Bifidobacterium animalis (Examples, section 2.8) or Bifidobacterium longum (Examples, section 4.4).

[0011] Similarly, protection of Bifidobacterium animalis subsp. lactis strain CECT 8145 on pathogen infection in C. elegans was compared with other alternative Bifidobacterium animalis strains, showing that nematodes fed with Bifidobacterium animalis subsp. lactis strain CECT 8145 exhibited significant higher worms’ survival than the alternative B. animalis strains

[0012] Therefore, in the present invention, a composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145, and / or a lipoteichoic acid (LTA) thereof, hereinafter the “composition of the invention”, is disclosed for several uses.

[0013] In a first aspect, the invention relates to the composition of the invention, for use in the treatment and / or prevention of anxiety disorders in a subject, hereinafter the “anxiety therapeutic use of the invention”.

[0014] Another aspect relates to the the composition of the invention for use in the treatment and / or prevention of a pathogen infection in a subject, hereinafter the “antipathogenic therapeutic use of the invention”.

[0015] Another aspect of the invention relates to the the composition of the invention for use in restoring or maintaining the integrity of gut barrier in a subject, hereinafter the “gut barrier therapeutic use of the invention”.

[0016] Another aspect of the invention relates to the the composition of the invention for use in the improvement of age-related conditions and / or increasing the lifespan and / or increasing or promoting longevity in a subject, hereinafter the “age-related therapeutic use of the invention”. Bifidobacterium animalis subsp. lactis strain CECT 8145 was deposited on May 14th2012 under the Budapest Treaty in the Spanish Type Culture Collection as the International Depository Authority (based in Building 3 CUE, Parc Cientific Universitat de Valencia, 0 / Catedratico Agustin Escardino, 9, 46980 Paterna (Valencia) SPAIN). The deposit number assigned was CECT 8145. The terms “strain of the invention”, “B. animalis subsp. lactis strain 8145”, “8. anima / is CECT 8145”, “BPL0001”, “BPL1TM” or “BPL1” are interchangeably used in the present document to refer Bifidobacterium animalis subsp. lactis strain CECT 8145.

[0017] Members of the genus Bifidobacterium are among the first microbes to colonize the human gastrointestinal tract (GIT) and are believed to exert positive health benefits on their host. Bifidobacteria have been commercially exploited as probiotic agents due to their associated health benefits and to being generally recognised as safe. Due to their purported health-promoting properties, these bacteria have been incorporated into many functional foods as active ingredients.

[0018] Bifidobacteria naturally occur in a range of ecological niches that are either directly or indirectly connected to the animal GIT, such as the human oral cavity, the insect gut and sewage. To be able to survive in these particular ecological niches, Bifidobacterial must possess specific adaptations to be competitive.

[0019] Bifidobacterium are gram-positive, non-motile, and often branched anaerobic bacteria. They are one of the major genera of bacteria that make up the GIT microbiota in mammals, and comprise over 30 different species. Among them, Bifidobacterium animalis subsp. lactis is a Gram-positive lactic acid bacterium belonging to the Actinobacteria phylum. Originally isolated from fermented milk, Bifidobacterium animalis subsp. lactis inhabits the gut of healthy adults and infants.

[0020] The present invention contemplates the use of both, viable and non-viable, cells of the strain of the invention. As illustrated in the Examples section, viable and non- viable cells of BPL1 have been shown to have beneficial effects in a model organism.

[0021] Thus, in a preferred embodiment of the invention, alone or in combination with other preferred embodiments, the strain of the invention is in the form of viable cells. In another preferred embodiment of the invention, alone or in combination with other preferred embodiments, the strain of the invention is in the form of non-viable cells.

[0022] As used in the present document, the term “non-viable”, “inanimate”, “inactive” or “inactivated” relates to any microorganism metabolically or physiologically inactive i.e., not retaining its metabolic activity and the ability to elongate after the administration of nutrients. Viability is independent from the capacity of the microorganism to form colonies on solid media i.e., its culturability.

[0023] Some techniques known in the state of the art to render microorganisms in the form of non-viable cells are, without limitation to, thermal processing / heat, freezing or radiation, such as ultraviolet radiation.

[0024] Thermal processing is likely to be used in many instances to inactivate microorganisms, as there is a long history of thermal processing in the food industry. A common procedure to inactivate microorganisms is heat treatment. Heat is lethal to microorganisms, but each species has its own particular heat tolerance. During a thermal destruction process, such as pasteurization, tyndallisation and autoclaving, the rate of destruction is logarithmic, as is their rate of growth. Thus, bacteria subjected to heat are killed at a rate that is proportional to the number of organisms present. The process is dependent both on the temperature of exposure and the time required at this temperature to accomplish to desired rate of destruction.

[0025] In a more preferred embodiment of the invention, alone or in combination with other preferred embodiments, the non-viable cells are heat-treated. In some parts of the document, heat-treated strain of the invention is referred to as HT-BPL1 or BPL1 HT.

[0026] The present invention also contemplates those bacterial strains derived from B. animalis subsp. lactis strain CECT 8145 (parental strain) that may be part of the composition of the invention retaining any of the effects disclosed in the present document for the strain of the invention (i.e. anxiety / stress reduction; protection against pathogens; promoting gut barrier integrity; longevity effects promoting having a positive impact in lifespan and age-related conditions). Examples of bacterial strains derived from the parental strain comprised within the composition of the invention include genetically modified organisms which show variations in their genome compared to the genome of the strain of the invention, wherein the mutations do not affect the ability of strain and / or their components to exert the effects disclosed along the description and in the Examples section.

[0027] Strains derived from B. animalis subsp. lactis strain CECT 8145 may be naturally or intentionally produced by mutagenesis methods known in the art, such as, but not limited to, growing the parent strain in the presence of mutagenic agents or stressors or genetic engineering directed to the modification, deletion and / or insertion of specific genes.

[0028] In some specific embodiments, the present invention also contemplates bacterial components, metabolites and molecules secreted by B. animalis subsp. lactis strain CECT 8145 and / or compositions comprising said components.

[0029] Bacterial components include, without limitation to, components of the cell wall (by way of non-limiting example, peptidoglycan); nucleic acids; membrane components; and proteins, lipids, carbohydrates, and combinations thereof (such as lipoproteins, glycolipids or glycoproteins). Metabolites may include any molecule produced or modified by the bacterium as a result of its metabolic activity during growth, its use in technological processes or during its storage. Examples of these metabolites include, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, minerals or nucleic acids. Secreted molecules include any molecule secreted or released to the outside by the bacterium during growth, its use in technological processes (for example, food processing or drugs) or during its storage. Examples of these molecules include, but are not limited to, organic and inorganic acids, proteins, peptides, amino acids, enzymes, lipids, carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins, salts, minerals or nucleic acids.

[0030] As previously mentioned, the composition of the invention may include an LTA derived from B. animalis subsp. lactis strain CECT 8145 (“LTA thereof’), alone or in combination with the strain of the invention. Inventors of the present invention have demonstrated that an LTA obtained from B. animalis subsp. lactis strain CECT 8145, (also called as LTA thereof, LTA-BPL1 ™, or LTA of the invention) exerts different health beneficial effects, such as anxiety / stress reduction; protection against pathogens; promoting gut barrier integrity; longevity effects promoting having a positive impact in lifespan and age-related conditions.

[0031] LTA are macroamphiphilic molecules found as major constituents of bacterial cell walls in Gram-positive bacteria, anchored to cell membranes by its lipidic moieties. The conventional LTA polymer structure is formed by a poly-glycerol phosphate (GroP) backbone, bound to a glycolipid structure or anchor. The polyol phosphate backbone can be substituted with monosaccharides such as glucose or galactose, and amines, such as alanine or glucosamine. Classical LTAs have been classified in major classes (I, II, III, IV) that can be distinguished on the basis of their chemical structure. It is known in the state of the art that LTA I structures are found naturally in different species from Firmicutes phylum, including but not limited to Staphylococcus aureus, Bacillus subtilis and Streptococcus sp. Beneficial species such as Lactobacilli, Lactococci or Leuconostoc also show type I LTA structures, with single GroP repeating units. [Shiraishi et al., 2016, Bioscience of Microbiota, Food and Health, vol 35 (4) 147-161 ; Scheewind&Missiakas 2014, J. Bacteriology, 196 (6) 1133-1142],

[0032] Some microorganisms are known in the state of the art to produce atypical lipoteichoic acids that are generally referred to as lipoglycans, or cell-surface glycolipids. These unconventional LTAs have been grouped as type V LTAs and represent polysaccharides attached to lipids, instead of repeating phosphodiester-linked units. Type V LTAs include macroamphophiles such as lipoglycans, Gro-P- lipoglucogalactofuranan, and succinyl lipomannan. (Schneewind O. and Missiakas D., J Bacteriol, 196 (6), 1133-42 Mar 2014).

[0033] In the context of the present invention, the term “lipoteichoic acid thereof” or “LTA thereof’ refers to a lipoteichoic acid isolated from a B. animalis subsp. lactis strain CECT 8145. LTA isolation and purification from B. animalis subsp. lactis strain CECT 8145 (BPL1) is described below in Examples section ( 1.3. Preparation of LTA from BPL 1) and in Balaguer, F. et al. Lipoteichoic acid from Bifidobacterium animalis subsp. lactis BPL1 : a novel postbiotic that reduces fat deposition via IGF-1 pathway. Microb Biotechnol, doi:10.1111 / 1751-7915.13769 (2021). In a particular embodiment, the LTA of the invention is obtained by cultivating B. animalis subsp. lactis strain CECT 8145 in excess of sugars as carbon source, and by isolating the LTA from the cell wall of said strain.

[0034] The term “carbon source”, refers to the molecules used by an organism as the source of carbon for building its biomass. In the process for obtaining the LTA of the invention, the carbon source used is sugars. Sugars is a term referring to a broad category of all mono- and disaccharides: the simplest carbohydrates. Monosaccharides include glucose, galactose and fructose, and disaccharides include sucrose, lactose, maltose and trehalose. Preferably, the sugar is selected from the list consisting of glucose, galactose, fructose, sucrose, lactose, maltose and trehalose. More preferably, the sugar is glucose.

[0035] Conditions for carrying out the culture of Bifidobacterium and the isolation of the LTA from a Bifidobacerium as it is the strain of the invention are widely known in the state of the art. For instance, they are described in in Balaguer, F. et al. (2021).

[0036] In a preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to reduce and / or reverse stress and / or anxiety in a subject. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has this capacity are described in Examples section, in particular in section 1.5.1., section 1.5.2 and section 2.2.

[0037] In another preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to reduce gut permeability in a subject. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has this capacity are described in Examples section, in particular in section 1.7. and 2.6.

[0038] In another preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to increase lifespan in a subject. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has this capacity are described in Examples section, in particular in section 1.8. and section 2.7.

[0039] In another preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to reduce body paralysis in a subject, preferably wherein the subject has Alzheimer’s disease and / or proteotoxicity. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has this capacity are described in Examples section, in particular in section 1.5.3.

[0040] In another preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to reduce mortality associated with S. enterica infection, preferably S. enterica subsp. enterica. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has this capacity are described in Examples section, in particular in section 1.6. and section 2.5.

[0041] In another preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to reduce oxidative stress in a subject. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has this capacity are described in Examples section, in particular in section 1.9.

[0042] In another preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to reduce fat content in a subject. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has this capacity are described in patent document W02021130219A1 , page 39, in particular, Fat reduction assays in C.elegans and Triglyceride (TG) quantification section.

[0043] In a more preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention has the capacity to reduce and / or reverse stress and / or anxiety, and / or reduce mortality associated with S. enterica infection, preferably S. enterica subsp. enterica, and / or reduce gut permeability, and / or increase lifespan, and / or reduce body paralysis, and / or reduce the oxidative stress, and / or reduce fat content, in a subject. Assays to determine whether an LTA obtained / derived from B. animalis subsp. lactis strain CECT 8145 has these capacities have been mentioned before.

[0044] The LTA of the invention may undergo different treatments, such as heat or lyophilization, in order to sterilize, preserve or extend the shelf life of the LTA. Thus, in a particular embodiment, the LTA of the invention is heat-treated or lyophilized.

[0045] The term “lyophilized” as used herein refers to the method of removing free water from the solution containing the LTA of the invention by sublimation, subjecting the solution to quick freezing and then removing the ice by light vacuum heating which transforms the ice into steam.

[0046] In each case, the presentation of the composition of the invention, which comprises the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and / or the LTA of the invention, will be adapted to the type of administration used. The composition may be presented in the form of solutions or any other form of clinically permissible administration and in a therapeutically effective amount in some embodiments. The composition of the invention can be formulated into solid, semisolid or liquid preparations, such as tablets, capsules, powders, granules, solutions, suppositories, gels or microspheres.

[0047] In a preferred embodiment, alone or in combination with other preferred embodiments, the composition of the invention is formulated for administration in liquid form or in solid form.

[0048] In a more preferred embodiment, the solid formulation is selected from the group consisting of tablets, lozenges, sweets, chewable tablets, chewing gum, capsules, sachets, powders, granules, coated particles or coated tablets, tablets and gastro- resistant tablets and capsules and dispersible strips and / or films.

[0049] In another more preferred embodiment, the liquid formulation is selected from the group consisting of oral solutions, suspensions, droplets, emulsions and syrups. Likewise, various systems are known that can be used for sustained-release administration of the composition of the invention, including, for example, the encapsulation in liposomes, microbubbles, microparticles or microcapsules and the like. The suitable sustained-release forms as well as materials and methods for their preparation are well known in the state of the art. Thus, the orally administrable form of the composition of the invention is in a sustained-release form further comprising at least one coating or matrix. The sustained release coating or matrix includes, without limitation, natural semisynthetic or synthetic polymers, water-insoluble or modified, waxes, fats, fatty alcohols, fatty acids, natural, semisynthetic or synthetic plasticizers or a combination of two or more of the same. Enteric coatings can be applied using conventional processes known to those skilled in the art.

[0050] In addition to what has been described above, the present invention also encompasses the possibility that the composition of the invention may be administered to a subject together with other components or compounds, although these are not part of the composition of the invention. Examples of such components or compounds are detailed along the description.

[0051] The composition of the invention may be formulated for pharmaceutical administration, i.e. , forming part of pharmaceutical products to be administered to the subject by any means of administration; and / or for food administration, i.e., forming part of the foods or nutritional supplements consumed in the subject’s diet. Thus, in some embodiments, the composition of the invention is a pharmaceutical composition (“pharmaceutical composition of the invention”) and / or a nutritional composition (“nutritional composition of the invention”).

[0052] Additionally, the composition of the invention may further comprises (i) one or more components or compounds having any biological, pharmacological and / or veterinary useful activity, for the purpose of the present invention (i.e. for any of the uses of the invention); and / or (ii) one or more components that, upon administration to a subject, may increase, enhance and / or promote the activity of the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and / or the LTA of the invention. As understood by those skilled in the art, the additional components or compounds must be compatible with the effects exerted by the strain of the invention and / or LTA thereof.

[0053] The term "pharmaceutical composition" also encompasses veterinary compositions.

[0054] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0055] The term "excipient" refers to a substance that helps the absorption of any components or compounds of the composition of the invention, namely, the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and / or the LTA of the invention, or stabilizes the components or compounds and / or assists the preparation of the pharmaceutical composition in the sense of giving it consistency or flavours to make it more pleasant. Thus, the excipients may have the function, by way of example but not limited thereto, of binding the components (for example, starches, sugars or cellulose), sweetening, colouring, protecting the active ingredient (for example, to insulate it from air and / or moisture), filling a pill, capsule or any other presentation or a disintegrating function to facilitate dissolution of the components, without excluding other excipients not listed in this paragraph. Therefore, the term "excipient" is defined as that material that included in the galenic forms, is added to the active ingredients or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physical and chemical properties of the pharmaceutical composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of components or compounds of the pharmaceutical composition, that is, be compatible with the effect exerted by the strain of the invention (and / or bacterial strains and / or bacterial components derived therefrom) and / or the LTA thereof.

[0056] The "galenic form" or "pharmaceutic form" is the configuration to which the active ingredients and excipients are adapted to provide a pharmaceutical composition or a drug. It is defined by the combination of the form in which the pharmaceutical composition is presented by the manufacturer and the form in which it is administered. The "vehicle" or "carrier" is preferably an inert substance. Carrier functions are to facilitate the incorporation of other components or compounds, allow better dosage and administration and / or give consistency and form to the pharmaceutical composition. Therefore, the carrier is a substance used in the drug to dilute any of the components or compounds of the pharmaceutical composition of the present invention to a given volume or weight; or that even without diluting these components or compounds, it is able to allow better dosage and administration and / or give consistency and form to the drug. When the presentation is liquid, the pharmaceutically acceptable carrier is the diluent. The carrier can be natural or unnatural. Examples of pharmaceutically acceptable carriers include, without being limited thereto, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatine, lactose, starch, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0057] Furthermore, the excipient and the carrier must be pharmacologically acceptable, i.e., the excipient and the carrier are permitted and evaluated so as not to cause damage to the subject to whom it is administered.

[0058] In the event that the composition of the invention is a nutritional composition (“the nutritional composition of the invention”), said nutritional composition may be a food or be incorporated into a food or food product intended for human and / or animal consumption.

[0059] Thus, in a preferred embodiment, alone or in combination with other preferred embodiments, the nutritional composition of the invention is a food or a nutritional supplement.

[0060] In some embodiments, the nutritional composition of the invention is a nutritional or dietary supplement.

[0061] In the present invention, the term "nutritional composition" refers to any food which, regardless of providing nutrients to the subject who consumes it, beneficially affects one or more functions of the body, so as to leverage and / or provide better health and wellness.

[0062] The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement", "food supplement", or "alimentary supplement" or “alimentary complement” refers to products or preparations whose purpose is to supplement the normal diet of a subject consisting of sources of concentrated nutrients or other substances with a nutritional or physiological effect. In the present invention, the B. animalis subsp. lactis strain CECT 8145, LTA thereof and / or any strains and / or cellular components derived from said strain are the "substances" with a nutritional and / or physiological effect on the subject.

[0063] The food supplement may be in single or combined form and be marketed in dosage form, i.e., in capsules, pills, tablets and other similar forms, sachets of powder, ampoules of liquids and drop dispensing bottles and other similar forms of liquids and powders designed to be taken in a single amount.

[0064] There is a wide range of nutrients and other elements that may be present in alimentary complements including, among others, vitamins, minerals, amino acids, essential fatty acids, fibre, enzymes, plants and plant extracts. Since their role is to complement the supply of nutrients in a diet, they should not be used as a substitute for a balanced diet and intake should not exceed the daily dose expressly recommended by the doctor or the nutritionist. The composition of the invention can also be part of the so-called "food for special groups", i.e., foods that meet specific nutritional needs.

[0065] Examples of foods that may comprise the composition of the invention include, but are not limited to, feed, dairy products, vegetable products, meat products, snacks, chocolates, beverages, baby food, cereals, fried foods, industrial bakery products and biscuits. Examples of dairy products include, but are not limited to, products derived from fermented milk (for example, but not limited to, yogurt or cheese) or nonfermented milk (for example, but not limited to, ice cream, butter, margarine or whey). The vegetable product is, for example, but not limited to, a cereal in any form of presentation, fermented (for example, soy yogurt, oat yogurt, etc.) or unfermented, and a snack. The beverages include, but are not limited to, non-fermented milk.

[0066] In a preferred embodiment, alone or in combination with other preferred embodiments, the food product or food is selected from the group consisting of fruit or vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, milk powder, cereals, baked goods, milk-based products (such as milkshakes or smoothies), meat products and beverages.

[0067] As understood by the person skilled in the art, B.animalis subsp. lactis strain CECT 8145 (and / or strains and / or cellular components derived therefrom) and / or the LTA thereof, have to be present in an effective amount, preferably in a therapeutically effective amount, in order to exert their effects, such as the effects disclosed in the Example section, or along the description wherein the several uses are specifically explained.

[0068] Within the context of the present invention, an “effective amount” is any amount of the component or compound of the composition of the invention which, when administered to a subject, is sufficient to produce the desired effect. Said component or compound of the composition of the invention, including the nutritional or pharmaceutical composition, refers to B.animalis subsp. lactis strain CECT 8145 (and / or strains and / or cellular components derived therefrom) and / or the LTA thereof.

[0069] The effective amount may vary depending on, for example, the age, body weight, general health, sex and diet of the subject, as well as on the mode and time of administration, the excretion rate or any potential co-treatment with other drugs.

[0070] In some embodiments, alone or in combination with other preferred embodiments, the composition of the invention is administered to a subject through the diet.

[0071] In some embodiments of the invention, alone or in combination with other preferred embodiments, the administration regime of the composition of the invention is at least once daily; twice daily; or three times a day, one with each main food intake (breakfast and / or lunch and / or dinner). In a preferred embodiment, alone or in combination with other preferred embodiments, the strain of the invention is present in an amount between 105cfu and 1012cfu per gram or milliliter of the composition. More preferably, the strain of the invention is present in an amount of 107and 1011per gram or milliliter of the composition.

[0072] In another preferred embodiment, alone or in combination with other preferred embodiments, the LTA of the invention is present in an amount between 4 pg and 20 mg per gram.

[0073] In some embodiments, the composition of the invention may comprise other microorganisms in addition to B. animalis subsp. lactis strain CECT 8145. In a preferred embodiment, alone or in combination with other preferred embodiments, the composition of the invention further comprises a microorganism, and / or their components, selected from the group consisting of Bacillus sp., Lactobacillus sp., Lacticaseibacillus, Lactiplantibacillus sp., Levilactobacillus sp., Levilactobacillus sp., Ligilactobacillus sp., Limosilactobacillus sp., Streptococcus sp., Bifidobacterium sp., Saccharomyces sp., Kluyveromyces sp., and combinations thereof. More preferably, the composition of the invention further comprises a microorganism, and / or their components, selected from the group consisting of L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. kefir, L. brevis, L. casei, L. plantarum, L. fermentum, L. paracasei, L. acidophilus, L. paraplantarum, L. reuteri, Streptococcus thermophilus, B. longum, B. breve, B. bifidum, B. catenulatum, B. adolescentis, B. pseudocatenulatum, Saccharomyces cerevisiae, Saccharomyces boulardii, Kluyveromyces lactis, Kluyveromyces marxianus, and any combination thereof.

[0074] In some specific embodiments, the nutritional composition of the invention is a food or a nutritional supplement for farm animals and / or pets.

[0075] In some specific embodiments, the nutritional composition of the invention is a food or a nutritional supplement for humans.

[0076] Uses in anxiety disorders and conditions As previously mentioned, in a first aspect, the invention relates to the composition of the invention, for use in the treatment and / or prevention of anxiety disorders in a subject, hereinafter the “anxiety therapeutic use of the invention”.

[0077] As used herein, the term "to treat" or "treatment" comprises: inhibiting the disease, disorder or pathological condition, i.e., stopping its development; relieving the disease, disorder or pathological condition, i.e., causing regression of the disease, disorder or pathological condition; stabilizing the disease, disorder or pathological condition in a subject; ameliorating the symptoms associated to the disease, disorder or pathological condition in a subject; and / or slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Thus, the term "to treat" or "treatment" does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders. The treatment of a disorder, pathological condition or disease may, for example, lead to a halt in the progression of the disease, disorder or pathological condition (e.g., no deterioration of symptoms) or a delay in the progression of the disease, disorder or pathological condition (in case the halt in progression is of a transient nature only). The "treatment" of a disorder, disease or pathological condition may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject / patient suffering from the disease, disorder or pathological condition. Accordingly, the "treatment" may also refer to an amelioration of the disorder, pathological condition, or disease, which may, e.g., lead to a halt in the progression of disease, disorder or pathological condition, or a delay in the progression of the disease, disorder or pathological condition.

[0078] As used herein, the term "prevention" refers to the avoidance of occurrence of the disease, disorder or pathological condition in a subject, particularly when the subject has predisposition for the pathological condition, but has not yet been diagnosed, or minimize or hinder the occurrence of the disease or pathological condition in a subject It may also comprises, reducing the risk of developing a disease, disorder or pathological condition in a subject.

[0079] Within the context of the present invention, the term "subject" relates to any animal from any species. Examples of subjects include, but are not limited to: Animals of commercial interest such as birds (hens, ostriches, chicks, geese, partridges, etc.), rabbits, hares, pets (dogs, cats, etc.), sheep, goat cattle (goats, etc.), swine (boars, pigs, etc.), equine livestock (horses, ponies, etc.), and cattle (bulls, cows, oxen, etc.); animals of hunting interest, such as stags, deer, reindeer, etc.; and humans.

[0080] In some embodiments, the subject is a mammal; in some preferred embodiments, the mammal is a human being of any race, sex or age.

[0081] In some other preferred embodiments, the mammal is a farm animal, including but not limited to, cows, horses, goats, llamas, sheep, pigs, chickens, or any animal raised or bred for commercial purposes. In some other preferred embodiments, the mammal is a pet, including but not limited to, dogs, cats or rabbits.

[0082] The use of composition of the invention, comprising the strain of the invention and / or the LTA of the invention, may have a number of benefits over traditional treatments of anxiety disorders, avoiding typical side effects of standard medication, such as Fluoxetine, and including but not limited to insomnia, muscle tension, nausea, and sexual dysfunction.

[0083] Anxiety is an emotion characterized by feelings of tension, worried thoughts and physical changes like increased blood pressure. Whilst anxiety is a normal reaction to stress and can be beneficial in some situations, anxiety disorders differ from normal feelings of nervousness or anxiousness and involve excessive fear or anxiety. Anxiety may be experienced not only by humans but also by other animals, in particular by other non-human mammals.

[0084] Anxiety disorders are the most common of mental disorders in humans and affect nearly 30% of adults at some point in their lives. Anxiety disorders include, but without limitations, generalized anxiety disorder, panic disorder, specific phobia, agoraphobia, social anxiety disorder (previously called social phobia), noise aversion, and separation anxiety disorder.

[0085] Generalized anxiety disorder involves persistent and excessive worry that interferes with daily activities. It may be accompanied by physical symptoms, such as restlessness, feeling on edge or easily fatigued, difficulty concentrating, muscle tension or problems sleeping.

[0086] Panic disorder is usually characterized by recurrent panic attacks, an overwhelming combination of physical and psychological distress with rather sever symptoms. The attacks may be expected, such as a response to a feared object, or unexpected, apparently occurring for no reason. They may occur with other mental disorders such as depression or post-traumatic stress disorder (PTSD).

[0087] A specific phobia is excessive and persistent fear of a specific object, situation or activity that is generally not harmful.

[0088] Agoraphobia is the fear of being in situations where escape may be difficult or embarrassing, or help might not be available in the event of panic symptoms. The fear is out of proportion to the actual situation and lasts generally six months or more and causes problems in functioning.

[0089] Social anxiety disorder is characterized by a significant anxiety and discomfort about being embarrassed, humiliated, rejected or looked down on in social interactions. The fear or anxiety causes problems with daily functioning and lasts at least six months.

[0090] The causes of anxiety disorders are currently unknown but likely involve a combination of factors including genetic, environmental, psychological and developmental.

[0091] Although each anxiety disorder has unique characteristics, most respond well to psychotherapy and medications. Whilst medications do not cure anxiety disorders, they provide significant relief from symptoms. The most commonly used medications are anti-anxiety medications (generally prescribed only for a short period of time) and antidepressants. Beta- blockers, used for heart conditions, are sometimes used to control physical symptoms of anxiety.

[0092] Within the present invention, the term “anxiety disorders” includes not only those anxiety disorders described above, but also related conditions including but not limited to stress-related disorders, such as, but without limitation to, post-traumatic stress disorder (PTSD) or acute stress disorder; obsessive-compulsive disorder; and adjustment disorders.

[0093] PTSD is a psychiatric disorder that may occur in people who have experienced or witnessed a traumatic event such as a natural disaster, a serious accident, a terrorist act, etc. or who have been threatened with death, sexual violence or serious injury. It can occur in all people, of any ethnicity, nationality or culture, and at any age. People with PTSD have intense, disturbing thoughts and feelings related to their experience that last long after the traumatic event has ended. They also experience sadness, fear or anger, and they may feel detached or estranged from other people.

[0094] Acute stress disorder occurs in reaction to a traumatic event, just as PTSD, and the symptoms are similar. However, the symptoms occur between three days and one month after the event, causing cause major distress and problems in the subject's daily lives. About half of people with acute stress disorder go on to have PTSD. Psychotherapy and medication can both help control symptoms and prevent development of the acute stress disorder into PTSD.

[0095] Obsessive-compulsive disorder (OCD) is a disorder in which people have recurring, unwanted thoughts, ideas or sensations (obsessions) that make them feel driven to do something repetitively (compulsions). These compulsions can significantly interfere with a person’s daily activities and social interactions, cause significant distress, and impair work or social functioning.

[0096] Adjustment disorder also occurs in response to a stressful life event (or events). The emotional or behavioural symptoms a person experiences in response to the stressor are more severe or more intense than what would be reasonably expected for the type of event that occurred. Symptoms may include sadness, hopelessness, and physical manifestations like tremors, palpitations, and headaches. These symptoms cause significant distress or problems functioning in important areas of the subject's life, and begin usually within three months of the stressful event; they last no longer than six months after the stressor or its consequences have ended. The disorder is typically treated with psychotherapy. As stated above, animals, in particular mammals, may also develop anxiety. For example, separation anxiety is commonly seen in pets. Separation anxiety is triggered when dogs or cats are separated from their guardians; escape attempts by the animals are often extreme and can result in self-injury. There is evidence that social stress may also play a critical role in pets’ welfare. Furthermore, there is evidence that social stress, transportation and high level of density in farms, may also play a critical role in farm animal disease prevention and animal farm’s welfare, and may be a mediator by which common management practices can increase disease risk. Social factors, including deprivation of social contact (‘social isolation’), reducing space allowance (‘crowding’) and disturbing social order (‘social instability’) trigger physiological and behavioural indicators of stress in livestock.

[0097] In other aspect, the present invention encompasses a method of treating or preventing anxiety disorders in a subject (“method of anxiety treatment or prevention of the invention”) comprising administering the composition of the invention to a subject.

[0098] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament for the treatment or prevention of anxiety disorders in a subject.

[0099] Other aspect of the invention relates to the use of the composition of the invention for the treatment or prevention of anxiety disorders in a subject.

[0100] All particular terms, definitions and preferred embodiments explained above in the “anxiety therapeutic use of the invention” are applicable herein and in the “method of anxiety treatment or prevention of the invention”.

[0101] In addition to the uses in the treatment and / or prevention of anxiety disorders, the composition of the invention can be also used in the reduction of an anxiety-related condition in a subject. As stated above in the present document, anxiety is a normal reaction to stress without being associated to a pathological state in some situations. In fact, anxiety can be beneficial, therefore, not involving a pathological condition. Anxiety disorders therefore differ from normal feelings of nervousness, fear or anxiousness, which are conditions related to anxiety as a normal and non-pathological reaction to a punctual stressful situation (i.e. not chronic).

[0102] Thus, other aspect relates to the non-therapeutic use of the composition of the invention in the reduction of anxiety and / or an anxiety- related condition in a subject.

[0103] As a way of example, anxiety can be a typical reaction to stress and new situations for an individual, encompassing feelings of, without being limited to, worry, unease, or nervousness in a subject. Therefore, the composition of the invention can provide relief from any or all these feelings to the subject. In a preferred embodiment, alone or in combination with other preferred embodiments, the anxiety related condition is selected from the list consisting of anxiousness, stress, worry, nervousness, unease and any combination thereof.

[0104] Moreover, based on the Examples included in the present application (Examples section) the composition of the invention can be used in reducing the perception of anxiety. The perception of anxiety encompasses feelings and conditions, such as feelings of nervousness, anxiousness or unease, present in healthy subjects, which may provoke some discomfort to said subject. Thus, other aspect of the invention relates to the non-therapeutic use of the composition of the invention in reducing the perception of anxiety and / or related conditions, including, but without limitation to, anxiousness, stress, worry, nervousness, unease, and any combination thereof.

[0105] In a preferred embodiment, the subject does not suffer from an anxiety disorder.

[0106] Antipathoqenic uses

[0107] Furthermore, the strain of the invention, both viable and non-viable cells such as HT- BPL1 , and the LTA of the invention, have pathogen protection effects. Thus, as previously mentioned in the present document, another aspect relates to the composition of the invention for use in the treatment and / or prevention of a pathogen infection in a subject, hereinafter the "antipathogenic therapeutic use of the invention".

[0108] The term “subject” has been previously described in a previous aspect, and its definition, as well as its preferred embodiments, apply equally to the present aspect of the invention.

[0109] Thus, the present use of the invention is directed to the treatment and / or prevention of infections caused by pathogen / s in a subject. The antipathogenic therapeutic use of the invention therefore encompasses the treatment and / or prevention of diseases, conditions and / or disorders caused by pathogen / s infection / s.

[0110] The term “pathogen”, as used in the present document, refers to a microorganism or infectious agent, which causes or can cause a disease or infection, including viruses, bacteria, protozoans, prions, viroids or any other infectious agent. It also encompasses foodborn pathogens and spoilage microorganisms.

[0111] Preferably, the pathogen is a bacterium. Pathogenic bacteria may be Gram-negative and / or Gram-positive bacteria. More preferably, the bacterium belongs to Salmonella, Staphylococcus, Escherichia, Clostridioides, Cronobacter, Porphyromonas, or Streptococcus genus.

[0112] Examples of pathogenic bacteria include, without limitation to, Salmonella enterica sp., Staphylococcus aureus sp., Escherichia coli (E. coli) sp., preferably E. coli O157:H7 strain, Clostridium difficile sp., Cronobacter sakazakii sp., Porphyromonas gingivalis sp. or Streptococcus mutans sp. Thus, in a more preferred embodiment, alone or in combination with other preferred embodiments, the bacterium belongs to Salmonella enterica sp., Staphylococcus aureus sp., Escherichia coli (E. coli) sp., preferably, E. coli 0157: H7 strain, Clostridium difficile sp., Cronobacter sakazakii sp., Porphyromonas gingivalis sp., Streptococcus mutans sp. or any combination thereof. More preferably, the bacterium belongs to Salmonella enterica sp. or Staphylococcus aureus sp. In an even more preferred embodiment, the bacterium is Salmonella enterica subsp. enterica serovar Typhimurium, and still more preferably, the bacterium is Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 14028.

[0113] In another even more preferred embodiment, the bacterium is Staphylococcus aureus sp. ATCC 25923 strain.

[0114] In other aspect, the present invention encompasses a method for the treatment and / or prevention of a pathogen infection (“antipathogenic method of the invention”), comprising administering the composition of the invention to a subject.

[0115] Thus, the antipathogenic method of the invention is directed to the treatment and / or prevention of infections caused by pathogen / s in a subject, and, therefore it encompasses the treatment and / or prevention of diseases, conditions and / or disorders caused by pathogen / s infection / s.

[0116] The terms "treatment", "prevention", “pathogen” and "subject" have been previously described, and its definitions, as well as their preferred embodiments, apply to the following aspects of the invention.

[0117] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament for the treatment and / or prevention of a pathogen infection in a subject.

[0118] Other aspect of the invention relates to the use of the composition of the invention for the treatment and / or prevention of a pathogen infection in a subject.

[0119] All particular terms, definitions and preferred embodiments explained above in the “antipathogenic therapeutic use of the invention” are applicable herein and in the “antipathogenic method of the invention”.

[0120] In addition to the above-mentioned uses, the composition of the invention can also have applications as non-therapeutic antipathogenic agent. Thus, other aspect of the invention relates to the non-therapeutic use of the composition of the invention in pathogen control or in alleviating unpleasant effects and / or discomfort associated to the presence of a pathogen in a subject (which can be also referred to as the "non-therapeutic antipathogenic use of the invention”).

[0121] The term “pathogen control” as used herein refers to controlling, reducing and / or eliminating a pathogen, or for preventing and / or reducing / inhibiting the growth, colonization and / or spreading of the pathogen.

[0122] As used herein, the term “controlling”, “reducing” or “eliminating” refers to any measurable decrease in the amount of pathogen with respect to the initial amount of pathogen, in particularly, it may mean a decrease by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% and / or 99% of the original pathogen count.

[0123] Preferably the pathogen is a bacterium (which can also be referred to as the “non- therapeutic antibacterial use of the invention”), more preferably wherein the bacterium belongs to Salmonella, Staphylococcus, Escherichia, Clostridioides, Cronobacter, Porphyromonas, or Streptococcus genus.

[0124] In a more preferred embodiment, the bacterium belongs to Salmonella enterica sp., Staphylococcus aureus sp., Escherichia coli (E. coli) sp., preferably E. coli O157:H7 strain, Clostridium difficile sp., Cronobacter sakazakii sp., Porphyromonas gingival is sp., Streptococcus mutans sp. or any combination thereof. Even more preferably, wherein the bacterium belongs to Salmonella enterica sp. or Staphylococcus aureus sp.

[0125] The term “pathogen” has been previously described, applying equally herein.

[0126] In an even more preferred embodiment, the bacterium is Salmonella enterica subsp. enterica serovar Typhimurium, and still more preferably, the bacterium is Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 14028.

[0127] In another even more preferred embodiment, the bacterium is Staphylococcus aureus sp. ATCC 25923 strain. In addition, since the strain of the invention, both viable and non-viable cells such as HT-BPL1 , and the LTA of the invention have demonstrated antipathogenic effects, they can also be used as an antipathogenic agent in in vitro / ex vivo pathogen control.

[0128] Thus, other aspect of the invention relates to the use of the composition of the invention in in vitro or ex vivo pathogen control.

[0129] The term “ex vivo”, as used herein, refers to pathogen control use of the composition of the invention outside a subject’s body, for instance, but without limitation to, in food / feed products, pharmaceutical, veterinary or biotechnological products, or in any non-food surfaces.

[0130] Terms, definitions, and preferred embodiments explained above in the “antipathogenic therapeutic use of the invention” are also applicable herein to the present aspect of the invention.

[0131] Gut barrier related uses

[0132] Inventors have shown that BPL1 , HT-BPL1 and LTA from BPL1 maintain, restore and improve the gut barrier integrity in C. elegans, which is a good model to study gut barrier integrity (Zhao, Y. et al. Lactic Acid Bacteria Protects Caenorhabditis elegans from Toxicity of Graphene Oxide by Maintaining Normal Intestinal Permeability under different Genetic Backgrounds. Scientific Reports 5, 17233, doi:10.1038 / srep17233 (2015)). Therefore, BPL1 , HT-BPL1 and LTA from BPL1 contribute to supporting and promoting the health of gut barrier

[0133] Thus, as previously mentioned in the present document, another aspect of the invention relates to the composition of the invention for use in restoring, maintaining, or improving the integrity of gut barrier in a subject, hereinafter the “gut barrier therapeutic use of the invention”. Likewise, due to the above-mentioned effects in gut barrier integrity, the composition of the invention can be used in preventing and or treating gut barrier damage or dysfunction. The phrase “restoring the integrity of gut barrier” refers to the gut barrier reparation, which may include the strengthening of its structural components and the recovery of its adequate function.

[0134] The phrase “maintaining the integrity of gut barrier” refers to the preservation and upholding the structural components and / or functional activity of the gut barrier. Maintaining the integrity of gut barrier helps to retain physiologically healthy levels of digestion and absorption of nutrients across the gut barrier without concomitant translocation of the gut barrier of harmful substances that will eventually cause, e.g., low-grade and / or chronic inflammation either systemically, in the gut, or in one or more extra-intestinal organs of the body of an individual, including a human being.

[0135] The gut carries out important functions including digestion, absorption, and endocrine regulation. The gut barrier lines the gastro-intestinal tract and separates the luminal, external part of the body from the systemic, internal part of the body. It protects the body from entering of luminal aggressors, such as antigens, toxins and pathogens. The gut barrier comprises the intestinal epithelium, a continuous monolayer of columnar epithelial cells, the enterocytes, sealed together by protein complexes, such as the tight junctions.

[0136] Various factors can compromise the integrity of the gut barrier, leading to increased permeability or "leaky gut." These factors may include chronic inflammation, infections, dietary factors (such as a high-sugar or high-fat diet), stress, certain medications, and imbalances in the gut microbiota.

[0137] Disruption of the gut barrier has been associated with many gastrointestinal diseases, but also with extra-intestinal pathological condition. Examples of diseases or conditions associated with gut barrier disruption and / or with a reduction of the gut barrier integrity are, without limitation to, inflammatory bowel disease, irritable bowel syndrome, celiac disease, obesity, Diabetes type 2, Alzheimer’s disease, Parkinson disease, Major depression disorder, autism spectrum disorders, non-alcoholic fatty liver disease, colon carcinoma, type 1 diabetes, food allergies or microbial infections. Thus, a preferred embodiment provides the composition of the invention for use in restoring, improving or maintaining the integrity of gut barrier in the prevention and / or treatment of a disorder selected from the list consisting of inflammatory bowel disease, irritable bowel syndrome, celiac disease, obesity, Diabetes type 2, Alzheimer’s disease, Parkinson disease, major depression disorder, an autism spectrum disorder, non-alcoholic fatty liver disease, colon carcinoma, type 1 diabetes, food allergies, microbial infections, and any combination thereof.

[0138] Gut barrier integrity can be associated with a lack of gut or intestinal permeability, wherein a high level of gut permeability is indicative of low gut barrier integrity.

[0139] Methods to assess where restoration, maintenance, improvement of the gut barrier integrity in a subject are known in the state in the art, including, without limitation to measurement of altered expression and distribution of tight junction proteins; ex vivo measurement of increased passage of paracellular probe, mucosal passage of bacteria, internalization of bacteria; transcellular uptake of gliadin, urinary secretion of probe, levels of zonulin in blood, among others.

[0140] Approaches and techniques to assess barrier integrity are disclosed, for instance, in Grootjans J, et al., Non-invasive assessment of barrier integrity and function of the human gut. World J Gastrointest Surg. 2010 Mar 27;2(3):61-9. doi: 10.4240 / wjgs.v2.i3.61 , or in Schoultz I, Keita AV. The Intestinal Barrier and Current Techniques for the Assessment of Gut Permeability. Cells. 2020 Aug 17;9(8):1909. doi: 10.3390 / cells9081909.

[0141] In other aspect, the present invention encompasses a method for restoring, improving or maintaining the integrity of gut barrier in a subject, (“method of restoring or maintaining the gut barrier of the invention”) comprising administering the composition of the invention to a subject.

[0142] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament for restoring, improving or maintaining the integrity of gut barrier in a subject. Other aspect of the invention relates to the use of the composition of the invention for restoring, improving or maintaining the integrity of gut barrier in a subject.

[0143] All particular terms, definitions and preferred embodiments explained above in the “gut barrier therapeutic use of the invention” are applicable herein and in the “method of restoring or maintaining the gut barrier of the invention”.

[0144] As in the “gut barrier therapeutic use of the invention”, in some preferred embodiments, restoring or maintaining the integrity of gut barrier is for the treatment of a disorder selected from the list consisting of inflammatory bowel disease, irritable bowel syndrome, celiac disease, obesity, Diabetes type 2, Alzheimer’s disease, Parkinson disease, major depression disorder, an autism spectrum disorder, nonalcoholic fatty liver disease, colon carcinoma, type 1 diabetes, food allergies, microbial infections, and any combination thereof.

[0145] In addition to the above mentioned uses in the treatment and / or prevention of gut damage or dysfunctions, the composition of the invention can also have applications as non-therapeutic use in a subject gut barrier function improvement.

[0146] Thus, another aspect relates to the non-therapeutic use of the composition of the invention for improving the gut barrier function or maintaining the integrity of gut barrier of a subject.

[0147] In a preferred embodiment of the non-therapeutic use, the subject does not suffer from a gut barrier damage or dysfunction.

[0148] Age-related conditions uses

[0149] In addition, the composition of the invention has pro-longevity effects, improving age- related conditions and increasing lifespan, as shown in the Examples section, in particular, in 2.7. BPL 1, heat-treated BPL1 and LTA from BPL1 prolong lifespan via IGF-1 and protect against age-related phenotypes in C. elegans, 2.8. BPL 1 strain improves C. elegans lifespan in comparison to the alternative strains, and in section 4. Further Examples of BPL1 ™, BPL 1 ™ HT and LTA-BPL 1 ™ effects in aging and longevity, wherein several assays in C.elegans, which is a widely used experimental system for biomedical research having many convenient features that make it particularly suitable for aging research (Hildegard I.D. Mack, et al.', The nematode Caenorhabditis elegans as a model for aging research, Drug Discovery Today: Disease Models, Volume 27, 2018, Pages 3-13, ISSN 1740-6757), were carried out.

[0150] Thus, as previously mentioned in the present document, another aspect of the invention relates to the the composition of the invention for use in the improvement of age-related conditions and / or increasing the lifespan and / or promoting or increasing longevity in a subject, hereinafter the “age-related therapeutic use of the invention”.

[0151] As used herein, “age-related conditions” refers to conditions that appear often in aged and ageing adult population, and / or conditions, which usually worsen with age or which has ageing as an associated risk factor. It shall also mean any disease or disorder whose incidence in a population or severity in an individual correlates with the progression of age. Examples of age-related conditions include, without limitation to, gut barrier integrity, mobility disability, proteotoxicity, sarcopenia or related diseases, disorders, conditions or symptoms associated to a loss of muscle mass and function, or a neurodegenerative condition or disorder, preferably, wherein the neurodegenerative disorder is Alzheimer’s disease or wherein the neurogenerative condition is associated to Alzheimer’s disease. The improvement of age-related conditions may encompass the amelioration of age-related conditions with age, reduction of the age-related conditions severity, and / or the delay in the onset of age- related conditions. Therefore, the composition of the invention can be used in the prevention and / or delay of aging or associated symptoms.

[0152] In a particular embodiment, the age-related condition is a disease, condition or disorder whose incidence is at least 1 .5 fold higher among individuals greater than 60 years of age relative to human individuals between the ages of 30-40 and in a selected population of greater than 100,000 individuals.

[0153] At the biological level, ageing results from the impact of the accumulation of a wide variety of molecular and cellular damage over time. This leads to a gradual decrease in physical and mental capacity, a growing risk of disease and ultimately death. These changes are frequently neither linear nor consistent, and they may be only loosely associated with a person’s age in years.

[0154] As indicated before, the inventors of the present invention have demonstrated that viable and heat-treated cells of Bifidobacterium animalis subsp. lactis strain CECT 8145 (BPL1), as well as its LTA, have beneficial effects in conditions and symptoms associated with aging. In particular, examples in the present application carried out in C.elegans, which is a widely used experimental system particularly suitable for aging research, show a reduction of cell senescence, protection of muscle integrity, an increase in the frequency on pharyngeal pumping (frequency on pharyngeal pumping tends to decrease over the years) compared to control condition, an increase in the mean lifespan, a reduction in the body paralysis / proteotoxicity on an Alzheimer's Disease model, as well as antioxidant effects associated to the administration of viable or heat-treated cells of Bifidobacterium animalis subsp. lactis strain CECT 8145, as well as to the administration of an LTA thereof.

[0155] Thus, the composition of the invention described in the present document possess anti-aging effects, and therefore is beneficial in promoting / increasing longevity. In a particular embodiment, the composition of the invention is for use as a longevity extending agent. In another particular embodiment, the composition of the invention is for use in delaying the onset of one or more age-related conditions.

[0156] As mentioned before, BPL1 , heat-treated BPL1 and LTA from BPL1 prolong lifespan. As used herein, the term “lifespan” encompasses both the maximum lifespan (i.e. the maximum age that any member of a particular species has attained) and the average lifespan. The average lifespan may be the mean lifespan, or the median lifespan.

[0157] Examples and methodology for assessing lifespan is included in section 1 .8. Lifespan Assays, wherein nematodes of N2 or the corresponding mutant strains were cultured until L4 stage; Then, worms were transferred to NGM plates already seeded with E. coli OP50, or to NGM plates with a lawn of BPL1 / HT-BPL1. The LTA from BPL1 was also assessed with the wild-type strain N2. The plates were maintained at 20°C. The number of live nematodes were scored until the 100 % of population was dead. Age- synchronized nematodes (N2) were cultured in Nematode Growth Medium (NGM) as control condition or in the same medium supplemented with the different probiotics. Survival curves were obtained. Survival of the worms was monitored until 100% of the mortality was reached. Thus, by measuring the %survival rate in a subject, for instance, in N2 nematodes, it can be assessed whether an increasing of lifespan (which can be also referred in the present application as extending lifespan) occurs. As stated above, C.elegans is a widely used experimental system for biomedical research having many convenient features that make it particularly suitable for aging research (Hildegard I.D. Mack, et al.; The nematode Caenorhabditis elegans as a model for aging research, Drug Discovery Today: Disease Models, Volume 27, 2018, Pages 3-13, ISSN 1740-6757).

[0158] Lifespan may be extended by 5%, 10%, 15%, 20% or more, with reference to the expected lifespan for that species, including humans. Extended lifespan may be an increase in the maximum lifespan possible for any particular species of subject. Extended lifespan may be an increase in the average lifespan of an individual of that species who reaches adulthood. Thus, extended lifespan may be a 5%, 10%, 15%, 20% or more increase in maximum lifespan and / or a 5%, 10%, 15%, 20% or more increase in average lifespan.

[0159] For the human, the maximum lifespan is thought to be about 120 years and the average lifespan for an individual reaching adulthood in a developed nation is about 70-80 years depending on a number of factors including sex of the individual, nutrition and locality. In terms of years, lifespan may be extended by 1 , 2, 5, 10, 15, or even 20 or more years.

[0160] The age-related therapeutic use of the invention also encompasses the increasement or extending the healthspan in a subject. Thus, the invention also extends to healthspan.

[0161] As used herein, the “healthspan” is the period of time for which an individual is generally healthy and free of chronic illness. In particular, an individual will be substantially free from age-related disorders during his or her healthspan. By extending the healthspan, the present invention leads to a rise in the average age of onset of age-related disorders as compared to an untreated subject. The composition of the invention extends the period of time for which an individual is generally healthy and free of chronic illness and / or ameliorates disorders / conditions that appear often in aged and ageing adult population, including reduced gut barrier integrity or fitness, mobility disabilities, oxidative stress enhancement and proteotoxicity. These are well established indicators of ageing progression and are considered to be markers of healthspan.

[0162] In a preferred embodiment, alone or in combination with other preferred embodiments, the improved age-related condition in the subject is selected from the list consisting of gut barrier integrity, mobility disability, sarcopenia or related conditions or symptoms associated to a loss of muscle mass and function, macular degeneration, proteotoxicity, a neurodegenerative condition or disorder, preferably wherein the neurodegenerative condition or disorder is selected from the list consisting of Alzheimer’s disease, dementia, Parkinson’s disease, more preferably, wherein the neurodegenerative disorder is Alzheimer’s disease or wherein the neurogenerative condition is associated to Alzheimer’s disease, and any combination thereof.

[0163] The term “subject” has been previously described in a previous aspect, and its definition, as well as its preferred embodiments, apply equally to the present aspects of the invention. In a particular embodiment, alone or in combination with other preferred embodiments, the subject is a patient suffering from Alzheimer’s disease.

[0164] In other aspect, the present invention encompasses a method of improving age- related conditions and / or increasing lifespan and / or promoting longevity in a subject (“age-related condition method of the invention”) comprising administering the composition of the invention to a subject.

[0165] Other aspect of the invention relates to the use of the composition of the invention in the manufacture of a medicament for improving age-related conditions and / or increasing lifespan and / or promoting longevity in a subject. Other aspect of the invention relates to the use of the composition of the invention for improving age-related conditions and / or increasing lifespan and / or promoting longevity in a subject.

[0166] Other aspect of the invention relates to the non-therapeutic use of the composition of the invention for improving age-related conditions and / or increasing lifespan and / or promoting longevity in a subject.

[0167] All particular terms, definitions and preferred embodiments explained above in the “age-related condition therapeutic use of the invention” are applicable herein and in the “age-related condition method of the invention”.

[0168] In addition to the above mentioned uses in age-related conditions, the composition of the invention can also have non-therapeutic uses in the amelioration of age-related- conditions in a subject.

[0169] HT-BPL1 and LTA antioxidant use

[0170] Furthermore, inventors have demonstrated that HT-BPL1 and LTA, as well as BPL1 alive cells, exert a significant antioxidant activity. Thus, other aspect of the present invention relates to the strain of the invention in the form of non-viable cells, preferably heat-treated non-viable cells, and / or the LTA of the invention, or a composition comprising any or both of them, for use in the treatment and / or prevention of oxidative stress in a subject.

[0171] In other aspect, the present invention encompasses a method of treatment and / or prevention of oxidative stress in a subject, comprising administering the strain of the invention in the form of non-viable cells, preferably heat-treated non-viable cells, and / or the LTA of the invention, or a composition comprising any or both of them to a subject.

[0172] Other aspect of the invention relates to the use of the strain of the invention in the form of non-viable cells, preferably heat-treated non-viable cells, and / or the LTA of the invention, or a composition comprising any or both of them in the manufacture of a medicament for the treatment and / or prevention of oxidative stress in a subject.

[0173] Other aspect of the invention relates to the use of the strain of the invention in the form of non-viable cells, preferably heat-treated non-viable cells, and / or the LTA of the invention, or a composition comprising any or both of them, for the treatment and / or prevention of oxidative stress in a subject.

[0174] “Oxidative stress” refers to a condition characterized by an excess of oxidants and / or a decrease in antioxidant levels. Cellular oxidants may include for instance reactive oxygen species (ROS). Particular cellular oxidants agents include, without limitation to, one or more of: radicals of oxygen (superoxide anion, hydroxyl radical, and / or peroxy radicals); reactive non-radical oxygen species such as, for example, hydrogen peroxide and singlet oxygen; carbon radicals; nitrogen radicals; and sulfur radicals. The condition of oxidative stress may result in, for example, cellular damage, impaired performance of cells and / or cell death.

[0175] Oxidative stress and the evaluation of substances or probiotics can be assessed by methodologies known in the state of the art. For instance, an assay for assessing oxidative stress in a model organism is disclosed in Martorell, P. et al. Use of Saccharomyces cerevisiae and Caenorhabditis elegans as model organisms to study the effect of cocoa polyphenols in the resistance to oxidative stress. J Agric Food Chem 59, 2077-2085 (2011)).

[0176] In a preferred embodiment, alone or in combination with other preferred embodiments, the oxidative stress is associated with increased levels of ROS in the subject, wherein said increased levels has been determined by the comparison with reference values of ROS.

[0177] In the present invention “increased levels” shall mean that the ROS levels of the subject, with respect to a reference values, are higher by at least, 1 ,5-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more than the reference values. As used herein, “reference values” refers to ROS levels in a subject nonsuffering from an oxidative stress condition or the mean of ROS levels in a cohort of subjects non suffering from an oxidative stress.

[0178] Methods and techniques to evaluate levels of ROS, both in cells and in vivo, are known in the state of the art. For instance, in the review Murphy, M.P., Bayir, H., Belousov, V. et al. Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat Metab 4, 651-662 (2022), different approaches to do so are described.

[0179] Other aspect of the invention relates to the non-therapeutic use of the strain of the invention in the form of non-viable cells, preferably heat-treated non-viable cells, and / or the LTA of the invention, or a composition comprising any or both of them, as an antioxidant. In a preferred embodiment, the strain of the invention in the form of non-viable cells and / or the LTA of the invention, or the composition comprising any or both of them, is formulated in a cosmetic preparation, more preferably, for topical administration to a subject.

[0180] Other aspect relates to the non-therapeutic use of the strain of the invention in the form of non-viable cells, preferably heat-treated non-viable cells, and / or the LTA of the invention, or a composition comprising any or both of them for the manufacture of an antioxidant cosmetic preparation.

[0181] Other aspect relates to a non-therapeutic method for improving the antioxidant function in a subject.

[0182] DESCRIPTION OF THE DRAWINGS

[0183] Fig. 1. (A) Percentage of nematodes with the different cellular localization of DAF-16 after feeding with BPL1 (108cfu / plate), BPL1 HT (108cells / plate) and LTA from BPL1 (10 pg / mL); (B) Representative images of the DAF-16 nucleus translocation of nematodes fed with BPL1 , BPL1 HT and LTA from BPL1. Image taken with a Nikon- SMZ18 Fluorescence Stereomicroscope (Scale bar 250 pm); (C) Percentage of Nile red fluorescence of N2 wild type worms of pmk-1 (km25) and jnk-1 (gk7) mutant strains fed with BPL1 (108cfu / plate), BPL1 HT (108cells / plate) or LTA from BPL1 (10 pg / mL). Nile red staining fluorescence was quantified at young adult stage. Orlistat (6 pg / mL) was used as a positive control. Data are mean ± SD and were calculated from two independent experiments (n=120 / condition for fat deposition, n=50 / condition for DAF-16 nuclear translocation). *** P < 0.001 , ** P < 0.01. One-Way ANOVA was applied.

[0184] Fig. 2. (A) Quantification of the time to respond to the Octanol after applying starvation during 20 min in N2 wild type worms fed with BPL1 (108cfu / plate), BPL1 HT (108cells / plate) or LTA from BPL1 (10 pg / mL) Data are the average from two independent assays (n= 80 / condition) “* P < 0.001 , ** P < 0.01. Student’s t-test was applied; (B) Quantification of the number of N2 wild type worms distributed in each zone after with the addition of Denubil, and fed with BPL1 (108cfu / plate), BPL1 HT (108cells / plate) and LTA from BPL1 (10 pg / mL) (*** P < 0.001 , ““ P < 0.0001). (### P < 0.001). Data are the average of two independent assays (n= 60 / condition). Two way ANOVA was applied.

[0185] Fig. 3. Percentage of CL4176 non-paralyzed worms fed with BPL1 overnight lawn, BPL1 HT (108cells / plate) and LTA from BPL1 (10 pg / mL) after induction by temperature raising. Ginkgo biloba extract (EGb 761) at 100 pg / mL was included as a positive control. Nematodes without temperature induction were also included as a negative control. BPL1 (*** P < 0.001), BPL1 HT and LTA from BPL1 (**** P < 0.0001). Data correspond to two independent assays (n=100 / condition). Log Rank T-test was applied.

[0186] Fig. 4. Effect of BPL1 and the alternative Bifidobacterium animalis strains on C. elegans avoidance behavior (anxiety-like behavior). One-way ANOVA was applied. Data are the average of two independent experiments. ****p<0,0001 **p<0,01 , * p<0,05.

[0187] Fig. 5. Quantification of N2 wild type worms distributed in each zone after the addition of Denubil, and fed with BPL1 or the alternative Bifidobacterium animalis strains (***p< 0.001 , ### p< 0.001 , # p< 0.05, NS not significant). Data are the average of two independent assays. Two way ANOVA was applied.

[0188] Fig. 6. (A) Survival rate of N2 wild type nematodes fed with E. coli OP50, S. enterica subsp. enterica serovar Typhimurium and BPL1 (108cfu / plate), BPL1 HT (108cells / plate) or LTA from BPL1 (10 pg / mL) in the presence of the pathogen. (P<0.0001) Data are the average of two independent experiments (n= 100 / condition). Log Rank T-test was applied; (B) Survival rate of N2 wild type nematodes fed with E. coli OP50, S. aureus and BPL1 (108cfu / plate), BPL1 HT (108cells / plate) or LTA from BPL1 in the presence of the pathogen. (P<0.0001). Data are the average of two independent experiments (n= 100 / condition). Log Rank T-test was applied.

[0189] Fig. 7. (A) Percentage of fluorescence of nematodes without intestinal damage (Control) or worms with damage provoked with 0.8 mM of H2O2 and the different conditions tested. Two independent experiments were performed (n=60 / condition); (B) Percentage of fluorescence of nematodes damaged with 0.8 mM of H2O2, fed with BPL1 (108cfu / plate), BPL1 HT (108cells / plate) or LTA from BPL1 (10 pg / mL). ** P < 0.01. Values are the average of two independent assays (n=60 / condition). One-way ANOVA was applied; (C) Representative images of fluorescence nematodes treated with 0.8 mM of H2O2 and fed with BPL1 , BPL1 HT or LTA from BPL1. Image taken with a Nikon-SMZ18 Fluorescence Stereomicroscope (Scale bar 250 pm).

[0190] Fig. 8. Survival rate of N2 wild type worms fed with (A) BPL1 alive cells, (B) heat- treated BPL1 and (C) LTA from BPL1. Curve comparison vs E. coli OP50 are indicated (p-values). Two independent experiments were performed (n=100-200 / condition). Log Rank T-test was applied.

[0191] Fig. 9. Survival rate of C. elegans N2 wild type strain fed with BPL1 HT (108cells / plate) and LTA from BPL1 (5 pg / mL) after acute oxidative stress. NGM was used as a control feeding condition. ** P < 0.01 , * P < 0.05. Data correspond to two independent experiments (n= 100 / condition) One way ANOVA was applied.

[0192] Fig. 10. (A) Effect of BPL1 and the alternative Bifidobacterium animalis strain BPL27 on C. elegans N2 lifespan. Log rank survival test was applied. Data from two independent assays. Both strains ****p<0.0001 vs NGM; BPL1 was significantly higher than BPL27 (*p-value<0.05). (B) Effect of BPL1 and the alternative Bifidobacterium animalis strain BPL30 on C. elegans N2 lifespan. Log rank survival test was applied. Data from two independent assays. Both strains ****p<0.0001 vs NGM. BPL1 was significantly higher than BPL30 (*p-value<0.05).

[0193] Fig. 11. % Median fluorescence intensity of Normal Human Dermal Fibroblasts (NHDF) after treatment with 0.4 mM H2O2 in the presence or absence of BPL1 ™ HT (108cells / mL) and LTA-BPL1 ™ (100 pg / mL). Epigallocatechin-3-gallate (EPCG) was included as positive control. Cellular senescence was estimated by the measurement of p-galactosidase activity in NHDF cells using the CellEvent™ Senescence Green Probe. For each condition, at least 5000 cells were analyzed. Data correspond to the average of two independent assays±sd. One-way Anova test was applied. “** significant at p-va / ue<0.0001 ; *“ significant at p-value< 0.001.

[0194] Fig. 12. Frequency of pharyngeal pumping (Hz) of nematodes C. elegans N2 fed in presence or absence of BPL1 ™ (overnight lawn), BPL1 ™ HT (108cells) and LTA- BPL1 ™ (10 pg / mL) at day 1 and day 7 of adulthood. At least, 35 worms per condition were measured. A t-test was applied. . ““ significant at p-value<0.000 ; “ significant at p-va / ue<0.01 ; * significant at p-va / ue<0.05; ns: not significant.

[0195] Fig. 13. Percentage of worms C. elegans DM8005 included in each sarcomere structure group (well organized, moderate disorganized or severe disorganized) fed with BPL1 ™ (overnight lawn), BPL1 ™ HT (108cells / plate) or LTA-BPL1 ™ (10 pg / mL) at day 1 , 7 and 11 of adulthood. 45 worms were analyzed per condition and time point. Statistics Chi-square test was applied. *“ significant at p-v,a / ue<0.001 ; * significant at p-value<0.05; ns: not significant.

[0196] Fig. 14. Effect of BPL1 and Bifidobacterium longum strains BB536, 1714 and 35624 on C. elegans N2 lifespan. Log rank survival test was applied. Data from two independent assays. All strains were ****p<0.0001 vs NGM.

[0197] Fig. 15. Effect of BPL1 and Bifidobacterium longum strains BB536, 1714 and 35624 at dose of 108cells / plate on avoidance behavior (anxiety) in C. elegans. One-way ANOVA was applied. Data are the average of two independent experiments.*** significant at p-value< 0.001 ; **p-v,a / ue<0.01 ; * p-value< 0.05.

[0198] Fig. 16. Survival rate of N2 wild type nematodes fed with E. coli OP50, S. aureus or with BPL1 or with alternative Bifidobacterium animalis strains B420, BPL27 and BPL30 (dose of 108cells / plate) in the presence of the pathogen. Data are the average of two independent experiments (n= 50 worms / condition). Log Rank T-test was applied.

[0199] Fig. 17. Survival rate of N2 wild type nematodes fed with E. coli OP50, S. enterica subsp. enterica serovar Typhimurium or with BPL1 or with alternative Bifidobacterium animalis strains B420, BB12 and BPL30 (dose of 108cells / plate) in the presence of the pathogen. Data are the average of two independent experiments (n= 50 worms / condition). Log Rank T-test was applied.

[0200] Examples

[0201] 1. Materials and methods

[0202] 1.1. C. elegans strains and maintenance conditions

[0203] Caenorhabditis elegans N2, Bristol (wild-type) strain and the mutant strains: GR1307 daf-16(mgDf50) I; CB1370 daf-2(e1370) III; KU25 pmk-1(km25) IV; VC8 jnk-1(gk7) IV; TJ356 zls356[daf-16p::daf-16a / b::GFP + rol-6(su1006)] IV and DA650 npr-1 (g320) were provided by the Caenorhabditis Genetic Center (CGC), University of Minnesota (USA). The nematodes were maintained on Nematode Growth medium (NGM) plates with Escherichia coli OP50 as normal diet. Worms were synchronized by isolating eggs from gravid adult nematodes grown at 20°C on freshly NGM plates with OP50.

[0204] For paralysis assays the transgenic strain CL4176 (smg-1ts [pAF29(myo-3 / Ab1- 42 / let UTR)+pRF4(rol-6(su10069))]) was provided by Dr. Christopher D. Link. This strain was routinely propagated on OP50 plates at 16°C. 1.2. Bacterial strains and culture conditions

[0205] Bifidobacterium animalis subsp. lactis BPL1 (CECT 8145) was routinely grown in MRS medium (Peptone from casein, tryptic digest 10 g / L; meat extract 10 g / L; yeast extract 10 g / L; D-glucose 20 g / L; K2HPO42 g / L; di-ammonium hydrogen citrate 2 g / L; sodium acetate 5 g / L; MgSO4 0.2 g / L; MnSO4 0.05 g / L; Tween 80 1 g / L) supplemented with cysteine (Sigma, 0.05% wt / vol)), MRS-Cys, for 18 h at 37°C in an anaerobiosis atmosphere generated by GasPakTM EZ Anaerobe Container System (BD), as described elsewhere (Martorell, P. et al. Probiotic Strain Bifidobacterium animalis subsp. lactis CECT 8145 Reduces Fat Content and Modulates Lipid Metabolism and Antioxidant Response in Caenorhabditis elegans. J Agric Food Chem, 64, 3462-3472 (2016); Balaguer, F. et al. Lipoteichoic acid from Bifidobacterium animalis subsp. lactis BPL 1: a novel postbiotic that reduces fat deposition via IGF-1 pathway. Microb Biotechnol, doi:10.1111 / 1751-7915.13769 (2021)).

[0206] As previously stated in the description, the terms “strain of the invention”, "B.animalis subsp. lactis strain 8145”, "B.animalis CECT 8145”, “BPL0001” or“BPL1™” or“BPL1” are interchangeably used in the present document to refer Bifidobacterium animalis subsp. lactis strain 8145.

[0207] For BPL1 heat inactivation, the same protocol applied in previous studies was followed (Balaguer, F. et al., 2021).

[0208] Escherichia coli OP50 was routinely cultured in LB broth medium (Bacto-tryptone 10 g / L; Bacto-yeast 5 g / L; NaCI 5 g / L) at 37°C for 18 h and then was used for the seeding of NGM plates.

[0209] Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028) was grown on Luria-Bertani (LB) medium and Staphylococcus aureus (ATCC 25923) was cultured on tryptic soy broth (TSB) aerobically.

[0210] 1.3. Preparation of LTA from BPL1 Lipoteichoic acid from BPL1 was obtained by isolation and purification from BPL1 cultures as previously described in Balaguer, F. et al. Lipoteichoic acid from Bifidobacterium animalis subsp. lactis BPL 1: a novel postbiotic that reduces fat deposition via IGF-1 pathway. Microb Biotechnoi, doi:10.1111 / 1751-7915.13769 (2021). Cells were overnight cultured under anaerobic conditions at 37°C in MRS+Cys medium with 20 g / L of glucose (excess of glucose). Afterwards, briefly, cells were harvested by centrifugation at 12,000xg 15 minutes from overnight grown cultures, washed twice with sodium citrate 50 mM pH 4.7 and mechanically disrupted in a PANDA PLUS 2000 homogenizer (GEA) at 1 ,000 bar. Pellet cell membrane material was mixed with an equal volume of n-butanol and stirred for 45 min at 37°C. After centrifugation (12,000 xg 90 min at 4°C) lower aqueous phase was collected. Sample was freeze-dried and subjected to hydrophobic interaction chromatography (HIC) on an octyl-Sepharose column using a linear gradient from 15% to 65% n-propanol in 0.05 M citrate buffer at pH 4.7. Phosphate-containing fractions (molybdenum blue test detected) were pooled and lyophilized. The purified LTA from BPL1 was resuspended in MilliQ water to obtain different concentrations prior the different assays.

[0211] 1.4. Fat reduction assays

[0212] The C. elegans fat content under the different feeding conditions was measured using Nile Red (Sigma, St. Louis, MO, USA) staining and subsequent fluorescence quantification following the protocol previously described (Martorell, P. et al. Probiotic Strain Bifidobacterium animalis subsp. lactis CECT 8145 Reduces Fat Content and Modulates Lipid Metabolism and Antioxidant Response in Caenorhabditis elegans. J Agric Food Chem 64, 3462-3472, doi:10.1021 / acs.jafc.5b05934 (2016)). Nematodes were fed with BPL1 , BPL1 HT and LTA purified from BPL1. In the case of live and heat-treated BPL1 , the cells were added directly to the surface of NGM plates with OP50 at a dose of 108cells / plate. The purified LTA isolated from BPL1 was added to the surface of NGM plates at a final concentration of 10 pg / mL. As a positive control of the assay, the anti-obesity drug Orlistat was used at a final concentration of 6 pg / mL. Two independent experiments were carried out, analyzing 120 worms / per condition.

[0213] 1.5. Behavioural assays 1.5.1. Avoidance behaviour

[0214] Octanol avoidance was used as an anxiety-related behaviour assay and the experiments were carried out using the “smell-on-a-stick” assay described elsewhere (Chao, M. Y., et al.. Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit. Proc Natl Acad Sci U S A 101 , 15512- 15517, doi:10.1073 / pnas.0403369101 (2004)). For the assays, the blunt end of a hair of a paintbrush (Loew-Cornell (Teaneck, NJ) 9000 Kolinsky 7 paintbrush) taped to a Pasteur pipette, was immersed in freshly prepared 30% octanol in EtOH (vol / vol) and placed in front of a forward-moving nematode.

[0215] Control condition nematodes were maintained in NGM with OP50 E. coli. and were incubated 20 min in fresh plates before testing. For the anxiety condition, nematodes were incubated 20 min in NGM plates without OP50. This food deprivation induces anxiety-related behaviour due to a decreased levels of serotonin. To evaluate treatments, wild-type nematodes were age-synchronized in NGM plates supplemented with BPL1 (108cfu / plate), BPL1 HT (108cells / plate) or LTA from BPL1 (10 pg / mL). At young-adult stage, nematodes were transferred to NGM plates with each treatment but no food during 30 min. Afterwards, worms were placed in NGM plates without OP50 and with each treatment and tested 20 min later. The amount of time that worms took to move backward was measured. Two independent assays were carried out with 80 worms analyzed per condition.

[0216] In addition, a comparative assay of BPL1 strain (108cfu / plate) against other Bifidobacterium animalis strains (BPL21 , obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from faeces from breastmilk fed baby; BPL27, obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from breastmilk; BPL30, obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from infant faeces; BB-12 (®; Jungersen M., The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis BB-12(®). Microorganisms. 2014 Mar 28;2(2):92-110) (Benchmark) originates from Chr. Hansen’s collection of dairy cultures; B420 (Benchmark, Howaru; Uusitupa HM, et al., Bifidobacterium animalis subsp. lactis 420 for Metabolic Health: Review of the Research. Nutrients. 2020 Mar 25;12(4):892. ) isolated from fermented milk, with similar conditions were carried out. Each strain amount was also 108cfu / plate.

[0217] 1.5.2. Reduction of stress

[0218] In this experiment, the nematode’s exploratory behaviour was analyzed after the addition of the psychostimulant drug Denubil. The nematodes were incubated in NGM plates with BPL1 (108cfu / plate), heat-treated BPL1 (108cells / plate) or LTA from BPL1 (10 pg / mL) and transferred to NGM plates supplemented with the psychostimulatory drug until they reached young adult stage. These nematodes were picked and moved to the assay plates.

[0219] The assay plates (NGM seeded with E. coli OP50), were divided in three concentric areas measuring <0.9 cm, 0.9-1.8 cm and 1.8-2.8 cm to the center point. The nematodes were placed in the middle of the plate and then scored for its position after 2 min. These assays were carried out in duplicate with 60 worms per condition.

[0220] In addition, a comparative assay of BPL1 strain (108cfu / plate) against other Bifidobacterium animalis strains (BPL21 , BPL27, BPL30, BB12 (Benchmark), B420 (Benchmark); each strain amount was also 108cfu / plate) with similar conditions were carried out.

[0221] 1.5.3. Paralysis assays (C. elegans Alzheimer’s model)

[0222] Paralysis experiments were performed using the transgenic C. elegans strain CL4176 as previously described Martorell, P. et al. A nutritional supplement containing lactoferrin stimulates the immune system, extends lifespan, and reduces amyloid p peptide toxicity in Caenorhabditis elegans. Food Sci Nutr 5, 255-265, doi:10.1002 / fsn3.388 (2017). Briefly, nematodes were synchronized by isolating eggs from gravid worms at 16°C in NGM plates (control condition) and NGM plates with an overnight lawn of BPL1 , heat-treated BPL1 (1x108cells / plate), or different doses of LTA from BPL1 (0.05, 0.1 , 1 and 10 pg / mL). As a positive control, G. biloba extract EGb 761® (100 pg / mL) (Tanakene, Ipsen Pharma, S.A., Sant Feliu de Llobregat, Spain) was also added on NGM plates. The induction of the muscle-specific A 1-42 transgene expression was carried out by raising the temperature from 16°C to 25°C, starting 48 h after the synchronization and maintained for 24 h, when the paralysis was scored. Paralysis in induced worms was compared with non-induced worms (maintained at 16°C until the end of the paralysis assay). Experiments were carried out in duplicate, analyzing 100 worms per condition.

[0223] 1.6. Infection assays

[0224] Infection assays were performed as described previously with minor modifications in Kim, Y. & Mylonakis, E. Caenorhabditis elegans immune conditioning with the probiotic bacterium Lactobacillus acidophilus strain NCFM enhances gram-positive immune responses. Infect Immun 80, 2500-2508, doi: 10.1128 / iai.06350-11 (2012)). Wild-type synchronized eggs were seeded in NGM plates with OP50 E. coli or with different doses of purified LTA from BPL1 (1.5 and 10 pg / mL), BPL1 or BPL1 HT at 108cells / plate. Once the worms reached the young adult stage, they were transferred to the infection plates containing a lawn of the respective pathogen (S. aureus ATCC 25923 or S.enterica subsp. enterica serovar Typhimurium ATCC 14028). A negative control condition without infection (NGM medium with the strain OP50 E. coli) and a positive control condition of infection only with the corresponding pathogen were included. Worms were maintained at 25 °C in the different conditions and scored for survival during 5-10 days. Worms were counted as alive or dead by stimulation with the platinum stick. Two independent assays were carried out with a total of 100 worm analyzed per condition.

[0225] 1.7. Gut barrier integrity

[0226] Age-synchronized nematodes of the wild-type strain N2 were obtained and maintained in NGM plates or NGM plates supplemented with the strain BPL1 (107and 108cfu / plate), heat-treated BPL1 (107and 108cells / plate) or LTA from BPL1 (10 pg / mL and 1 pg / mL) at 20°C. To induce intestinal permeability L4-larvaes were exposed to H2O2 (0.8 mM) for 24 h (Forsyth, C. B. et al. Role for intestinal CYP2E1 in alcohol-induced circadian gene-mediated intestinal hyperpermeability. Am J Physiol Gastrointest Liver Physiol 305, G185-195, doi: 10.1152 / ajpgi.00354.2012 (2013)). A control condition without damage was also included. To evaluate the intestinal permeability in H2O2 exposed-nematodes, Nile Red staining (0.05 g / mL) was used. A total of 30 worms were randomly selected from each condition and observed in a fluorescence stereomicroscope Nikon SMZ18 (Tokyo, Japan) equipped with NIS- ELEMENT image software. Results are shown as the percentage of fluorescence in each treatment with respect to the H2O2-treated nematode population. Two independent assays were performed.

[0227] 1.8. Lifespan Assays

[0228] For lifespan assays, synchronized nematodes of N2 or the corresponding mutant strains were cultured until L4 stage at 20°C. Then, worms were transferred to NGM plates already seeded with E. coli OP50, or to NGM plates with a lawn of BPL1 . In the case of heat-treated BPL1 , cells were added to a final concentration of 109and 1010cells / plate. The LTA from BPL1 was assessed at different doses (0.1 , 0.5, 1 , 5, 10, 25 and 50 pg / mL) with the wild-type strain N2. The plates were maintained at 20°C. The number of live nematodes were scored until the 100 % of population was dead. Two independent experiments were conducted of each condition with 100 / 200 worms analyzed.

[0229] In addition, a lifespan comparative assay of BPL1 strain against other Bifidobacterium animalis strains (BPL1 , BPL21 , BPL27, BPL30, BB12 (Benchmark), B420 (Benchmark); Lawn of bacteria, as with BPL1) was conducted in the C. elegans w'M- type strain N2 in order to test the efficacy of the probiotic strains on the nematode’s longevity.

[0230] Age-synchronized nematodes (N2) were cultured in Nematode Growth Medium (NGM) as control condition or in the same medium supplemented with the different probiotics. Survival curves were obtained with data from an automatized system based on artificial vision, developed by Biopolis. Survival of the worms was monitored until 100% of the mortality was reached.

[0231] 1.9. Oxidative stress assay The antioxidant activity of LTA from BPL1 and BPL1 HT was assessed in C. elegans by means a previously described assay using wild-type N2 strain (Martorell, P. et a / . Use of Saccharomyces cerevisiae and Caenorhabditis elegans as model organisms to study the effect of cocoa polyphenols in the resistance to oxidative stress. J Agric Food Chem 59, 2077-2085 (2011)). Briefly, egg-synchronized worms were placed in NGM plates with four different doses of LTA from BPL1 (1 , 5, 10 and 25 pg / mL). An oxidative stress was applied with the addition of H2O2 (2 mM) during 5 h and the viability of the worms was measured after this time. Vitamin C (10 pg / mL) was used as positive control of the assay. The experiment was carried out in duplicate, analyzing 100 worms per condition.

[0232] 1.10. DAF-16 nuclear translocation

[0233] The intracellular localization of DAF-16 was assessed with the C. elegans TJ356 reporter strain, following the protocol previously described (Sugawara, T. & Sakamoto, K. Killed Bifidobacterium longum enhanced stress tolerance and prolonged life span of Caenorhabditis elegans via DAF-16. Br J Nutr 120, 872-880, doi: 10.1017 / s0007114518001563 (2018)). Briefly, worms were synchronized in NGM plates with BPL1 (108ufc / plate), heat-treated BPL1 (108cells / plate) or LTA from BPL1 (10 pg / mL) until they reached young adult stage. Then, worms were transferred to plates with 1 % agarose and 0.1 % sodium azide (Sigma, St. Louis, MO, USA) and nuclear translocation was measured using the Nikon SMZ18 (Tokyo, Japan) fluorescence stereomicroscope equipped with NIS-ELEMENT image software. Fifty worms per condition were assessed in two different experiments.

[0234] 1.11. Statistical analysis

[0235] Results are showed as mean ± standard deviation. For fat reduction, oxidative stress and gut permeability assays data was analyzed by one-way ANOVA, using Tukey’s multiple comparison test. Log Rank T-test was applied for the lifespan, infection and body paralysis assays. Octanol avoidance assay was analyzed using Student’s T-test and cholinergic stress assays were analyzed using two-way ANOVA. All the analysis were performed with GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) adjusting the level of significancy at 5%. 2. Results

[0236] 2.1. Fat reduction effect of BPL1, heat-treated BPL1 and LTA from BPL1 requires the insulin-like signaling pathway (IGF-1 ) and not the MAPK pathway in C. elegans

[0237] Previous studies with BPL1 alive cells have reported that its fat reducing effect is mediated by the IGF-1 pathway (Martorell, P. etal. 2016). Furthermore, BPL1 HT and purified LTA from BPL1 also require this signalling pathway to exert the activity (Balaguer, F. et al. 2021).

[0238] To further determine the mechanism of action, first we checked the activation of DAF- 16 by a nuclear translocation assay using the TJ356 reporter strain, in the presence of BPL1 , BPL1 HT or LTA from BPL1. By fluorescence microscopy we observed an increase of nuclear DAF-16 in nematodes fed with the three ingredients when compared with control nematodes with OP50 standard diet (Fig. 1A and 1 B). This increase revealed that BPL1 , its inactivated form, as well as LTA from BPL1 , promote the activation of DAF-16, required for their functionality.

[0239] Furthermore, we explored other pathways that may be involved in BPL1 functionality. For this purpose, fat reduction activity of BPL1 , BPL1 HT and LTA from BPL1 was evaluated in C. elegans mutant strains through alternative pathways. Besides acting in the IGF-1 pathway, DAF-16 is downstream of the mitogen-activated protein kinase pathway (MAPK). Two protein kinases were selected, PMK-1 / p38 and JNK-1 , as representative of MAPK family, who can be activated by environmental stress factors which ultimately activate the nuclear localization of DAF-16 (Kondo, M. et al. The p38 signal transduction pathway participates in the oxidative stress-mediated translocation of DAF-16 to Caenorhabditis elegans nuclei. Meeh Ageing Dev 126, 642-647, doi:10.1016 / j.mad.2004.11.012 (2005); Oh, S. W. et al. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor / DAF-16. Proc Natl Acad Sci U S A 102, 4494-4499, doi: 10.1073 / pnas.0500749102 (2005)). The fat reduction assays showed that BPL1 , heat-treated BPL1 and LTA from BPL1 maintain their activity in pmk-1 (km25) and jnk-1(gk7) C. elegans mutant strains (Fig. 1C). These results indicate that BPL1 does not require either PMK-1 / p38 or JNK-1 pathways to exert its fat reduction activity, acting through DAF-16 via the IGF-1 pathway.

[0240] 2.2. BPL1, heat-treated BPL1 and its LTA improve C. elegans anxiety, stress- related behaviors and had positive effect on C. elegans Alzheimer’s model.

[0241] Previous studies performed with the strain BPL1 have indicated an upregulation of tryptophan synthesis after supplementation in nematode’s diet (Martorell etal., 2016). Inventors of the present invention further assessed BPL1 , BPL1 HT and its LTA role in different C. elegans behaviour models.

[0242] Firstly, the avoidance behaviour was used as an anxiety-related conduct in C. elegans, which is mediated by serotonin pathway Chao, M. Y., Komatsu, H., Fukuto, H. S., Dionne, H. M. & Hart, A. C. Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit. Proc Natl Acad Sci U SA 101 , 15512-15517, doi:10.1073 / pnas.0403369101 (2004). Fig. 2A represents the time it took for the nematodes to start the backwards movement when they smelled octanol. Control anxious worms without food took longer to avoid octanol than control conditions nematodes with E. coli OP50. However, the nematodes fed with BPL1 , BPL1 HT or LTA from BPL1 significantly reduced the avoidance time under food deprivation (p-value <0.05), therefore counteracting the anxiety-related behaviour caused by the lack of food.

[0243] Then, it was also analyzed the nematode’s exploratory behavior after the addition of the psychostimulant drug Denubil. BPL1 , BPL1 HT and LTA from BPL1 were evaluated in this cholinergic-induced model for stress, developed in the laboratory, in order to test if they are able to counteract this behavior. Fig. 2B shows the distribution of worms in solid NGM plates at each feeding condition, including a non-stressed control and stressed conditions induced with Denubil. Under non-stressed condition, nematodes tended to accumulate in the center of the plates (Zone 1), while when stressed with the Denubil nematodes acquired a pattern with a trend to move to the edge of the plates (Zone 3). Moreover, when stressed-induced nematodes were fed with BPL1 , BPL1 HT or LTA from BPL1 , a reduction of the number of nematodes in Zone 3 was determined (p-value <0.001), with a behaviour pattern similar to the control (non-stressed) nematodes. Thus, BPL1 , BPL1 HT and LTA from BPL1 reversed the induced stress and decreased the exploratory capacity of the nematodes.

[0244] Finally, the potential benefits of BPL1 , BPL1 HT or LTA from BPL1 were also tested on the Alzheimer's Disease model in C. elegans by scoring the body paralysis in the CL4176 nematode strain. BPL1 , BPL1 HT and LTA from BPL1 provoked a reduction on the nematodes body paralysis when compared with control conditions (Figure 4C). Particularly, LTA from BPL1 caused a high delay on body paralysis between 26 h to 32 h after the induction (p-value <0.0001), presenting a major positive effect than the alive and heat-treated cells (Fig. 3).

[0245] All these results showed the potential role of BPL1 , BPL1 HT and LTA from BPL1 in the modulation of anxiety-related behaviours and the effectiveness of reducing the body paralysis / proteotoxicity on the Alzheimer's Disease model in C. elegans.

[0246] 2.3. BPL1 has a better effect on anxiety-related behavior than other Bifidobacterium animalis strains

[0247] We used the avoidance behavior assay in response to octanol in C. elegans as an anxiety-related behavior to evaluate the five Bifidobacterium animalis alternative strains and BPL1 as a control. In this assay, anxious control worms take longer to avoid octanol than control conditions nematodes (NGM, control without anxiety). The results represent the relative percentages of each strain against the anxious control population (Fig. 4).

[0248] Results indicate that BPL1 provided 38.19% of reduction of the time of response when the octanol was presented to the worms, showing a high positive effect reducing the anxious behavior while the alternative strains reduced between 12.21 and 18.41 % (Table 1). Therefore, these results indicate that BPL1 has a better effect on anxiety-related behavior than other Bifidobacterium animalis strains.

[0249] Table 1. Percentage of reduction of time that takes for the avoidance behavior (anxiety model) in C. elegans, obtained with BPL1 and the alternative Bifidobacterium animalis strains included in this study.

[0250] 2.4. BPL1 strain reverses the stressed behavior while the alternative strains have no effect

[0251] We analyzed the nematode’s exploratory behavior after the addition of the psychostimulant drug Denubil. BPL1 and the alternative Bifidobacterium animalis strains were evaluated in this cholinergic-induced model for stress, in order to test if they are able to counteract this behavior. Fig. 5 shows the distribution of worms in solid NGM plates at each feeding condition, including a non-stressed control (NGM) and stressed conditions induced with Denubil. Under non-stressed condition, nematodes tended to accumulate in the center of the plates (Zone 1), while when stressed with the Denubil nematodes acquired a pattern with a trend to move to the edge of the plates (Zone 3). When stressed-induced nematodes were fed with BPL1 a reduction of the percentage of nematodes in Zone 3 was obtained, with a behavior pattern similar to the control (non-stressed) nematodes (Table 2). These results show that BPL1 reversed the induced stress while the alternative Bifidobacterium animalis strains did not show any effect. Table 2. Percentage of N2 wild type worms in zone 3 after the addition of Denubil, and fed with BPL1 or the alternative Bifidobacterium animalis strains.

[0252] 2.5. BPL1, heat-treated BPL1 and LTA from BPL1 exert protective effect against pathogen infection.

[0253] Infection assays in C. eleganswere performed with a Gram (+) (S. aureus) and Gram (-) (S. enterica) pathogen bacteria. As showed in Fig. 6A, S. enterica subsp. enterica serovar Typhimurium (ATCC 14028) infection provoked an increase in mortality between days 6-9 of infection. However, the addition of BPL1 , heat-treated BPL1 or LTA from BPL1 to the nematodes’ diet counteracted this effect, and survival significantly increased during the infection period (p-value<0.0001). This effect was particularly higher in the case of LTA from BPL1 at a dose of 10 pg / mL. In the case of S. aureus infection, this pathogen provoked mortality up to 80% at day 4, and nearly 100% at day 5 (Fig. 6B). Conversely, nematodes fed with BPL1 or BPL1 HT significantly increased the survival having a 40% of survival at day 4 and 20% of survival at day 5 (p-value<0.0001) (Fig. 6B)). Thus, BPL1 and BPL1 HT showed protection against both Gram + and Gram - pathogens, and LTA from BPL1 exerted a high protective effect against S. enterica subsp. enterica serovar Typhimurium (ATCC 14028) infection.

[0254] 2.6. Gut barrier integrity assays During aging, intestinal permeability worsens causing age-associated decreases in cognition or physical functions (Nagpal, R. et al. Gut microbiome and aging: Physiological and mechanistic insights. Nutr Healthy Aging 4, 267-285, doi:10.3233 / nha-170030 (2018)). In this sense, C. elegans has emerged as a good model to study the gut barrier integrity using the Nile Red staining (Zhao, Y. et al. Lactic Acid Bacteria Protects Caenorhabditis elegans from Toxicity of Graphene Oxide by Maintaining Normal Intestinal Permeability under different Genetic Backgrounds. Scientific Reports 5, 17233, doi:10.1038 / srep17233 (2015)).

[0255] To study whether BPL1 , BPL1 HT and LTA from BPL1 could protect against intestinal permeability, leaky gut experiments were performed. Firstly, as shown in Fig. 7A, the addition of H2O2 (0.8 mM) to the nematodes damaged the intestine causing the passage of dye to the body increasing the fluorescence (30.06%). By contrast, the treatment with either BPL1 , BPL1 HT and LTA from BPL1 induced a significant fluorescence reduction (Figure 7B and 7C), suggesting therefore, that either BPL1 , HT-BPL1 or LTA from BPL1 maintain the integrity of the nematodes gut barrier.

[0256] 2.7. BPL1, heat-treated BPL1 and LTA from BPL1 prolong lifespan via IGF-1 and protect against age-related phenotypes in C. elegans

[0257] Inventors of the present invention have assessed whether BPL1 , heat-treated BPL1 and LTA from BPL1 could be effective on longevity.

[0258] As shown in Fig. 8A, BPL1 cells significantly increased the C. elegans lifespan compared with NGM control, with an increase in the mean lifespan of 1 day (8.33 %), and in the final lifespan of 4 days (19.05%) (p-value<0.0001). Likewise, heat-treated BPL1 cells also provoked a significant positive effect on nematode's lifespan (p- value<0.01), increasing the mean lifespan in 1 day (6.67%) and 3 days (13.04%) the final lifespan (Fig. 8B), while the purified LTA from BPL1 increased in 1 day (7.7%%) the mean lifespan and 2 days (8.7%) the final lifespan (p-value<0.01) (Fig. 8C).

[0259] In order to identify the underlaying mechanism of action of the pro-longevity effect of BPL1 , BPL1 HT and LTA from BPL1 , additional lifespan experiments were performed with different C. elegans mutant strains of the IGF-1 (DAF-2 / DAF-16) and MAPK pathways (PMK-1 / p38 and JNK-1).

[0260] The positive effect on lifespan observed for BPL1, BPL1 HT and LTA from BPL1 was completely lost in C. elegans daf-16 and daf-2 mutant strains, whereas this increase in C. elegans lifespan remained statistically significant in the other mutant strains analyzed pmk-1 and jnk-1 (Table 3). These evidences indicate that, likewise fat reduction activity, this positive effect on C. elegans lifespan was mainly regulated by the insulin-like signalling pathway IGF-1.

[0261] Table 3. Effect of BPL1 , HT-BPL1 and LTA on different C .elegans mutant strains lifespan.

[0262] Previous studies have shown an age-related increase of oxidative stress mediated by the accumulation of reactive oxygen species (ROS) that could provoke molecular damage (Sohal, R. S. & Weindruch, R. Oxidative stress, caloric restriction, and aging. Science 273, 59-63, doi:10.1126 / science.273.5271.59 (1996)., Back, P., Braeckman, B. P. & Matthijssens, F. ROS in aging Caenorhabditis elegans: damage or signaling? Oxid Med Cell Longev 2012, 608478, doi: 10.1155 / 2012 / 608478 (2012)). As we had previously reported that BPL1 alive cells had a significant antioxidant activity (Martorell, P. et al. 2016), now we wanted to determine if BPL1 HT and LTA from BPL1 had antioxidant effect. By oxidative stress assays on C. elegans model we observed that both heat-treated BPL1 and LTA from BPL1 showed a protective effect against oxidative stress in C. elegans, as a significant survival increase was determined in treated nematodes (p-value <0.05) (Fig. 9).

[0263] 2.8. BPL1 strain improves C. elegans lifespan in comparison to the alternative strains

[0264] We evaluated the effect of BPL1 and 2 alternative Bifidobacterium animalis strains on the nematodes' longevity (BPL27 and BPL30). Fig. 10A and 10B show the survival curves obtained in worms cultured in nematode growth media (NGM, control) or in NGM supplemented with the strains BPL1 , BPL27 or BPL30. The results showed that the 3 strains significantly increased the lifespan of the worms. However, the increase on worm’s survival provoked by BPL1 was significantly higher than survival provoked by strains BPL27 and BPL30 (p-value<0.05). Moreover, BPL1 increased the final lifespan in 6 and 2 days, when 100% of mortality has been reached, comparing with the strains BPL27 and BPL30, respectively (Table 4). Therefore, these results show that BPL1 exerts higher effect on C. elegans' lifespan than the alternative Bifidobacterium animal is strains.

[0265] Table 4. Summary of the effect of alternative Bifidobacterium animalis strains BPL27 and BPL30 in comparison to BPL1 on C. elegans' N2 lifespan.

[0266] Mean lifespan: 50% survival. Final lifespan: 100% mortality.

[0267] 3. Discussion

[0268] Previous studies have shown Bifidobacterium animalis subsp. lactis BPL1, as a probiotic strain with a high fat reduction effect. This anti-obesity function was firstly assessed in the Caenorhabditis elegans model (Martorell, P. et al. 2016), and then in pre-clinical studies using Zucker rats and Wistar rats under cafeteria diet (Caimari, A. et al. Heat-killed Bifidobacterium animalis subsp. Lactis CECT 8145 increases lean mass and ameliorates metabolic syndrome in cafeteria-fed obese rats. Journal of Functional Foods 38, 251-263, doi:https: / / doi.org / 10.1016 / j.jff.2017.09.029 (2017); Carreras, N. L. et al. Anti-obesity properties of the strain Bifidobacterium animalis subsp. lactis CECT 8145 in Zucker fatty rats. Benef Microbes 9, 629-641 , doi:10.3920 / bm2017.0141 (2018)). Moreover, the effect of both BPL1 and its heat treated form, was substantiated in a study with obese human volunteers. Further investigations with BPL1 conducted by our group, allowed the characterization of the molecule responsible for the functional activity of the strain, the Lipotheichoic Acid (LT A) (Balaguer, F. eta / . Lipoteichoic acid from Bifidobacterium animalis subsp. lactis BPL1: a novel postbiotic that reduces fat deposition via IGF-1 pathway. Microb Biotechnol, doi:10.1111 / 1751-7915.13769 (2021)). Mechanism of action analysed with the model organism C. elegans showed that the fat reduction activity of BPL1 required the FOXO transcription factor DAF-16. DAF-16 is a well-known regulator of the insulin-like signalling pathway IGF-1 in C. elegans. Interestingly, additional research with the novel LTA from BPL1 and the heat treated form of the strain, reveal that both act through IGF-1 to perform their fat reduction activity. Moreover, in the present study we corroborated that in the presence of BPL1 , BPL1 HT or LTA from BPL1 , DAF-16 is activated and translocated to the nucleus.

[0269] Besides, in this study we explored some alternative pathways that could act in parallel to the IGF-1 pathway. The JNK signaling pathway has been shown to act jointly with the IGF-1 pathway and PMK-1 / p38 could transmit environmental stress to DAF-16. Our results demonstrate that the IGF-1 pathway is the main metabolic target for BPL1 and LTA from BPL1 anti-obesity effect while the genes jnk-1 and pmk-1, belonging to the JNK-1 / DAF-16 pathway and p38 MAPK pathway respectively, are not required. These results suggest that BPL1 and its LTA fat reduction effect requires predominantly the IGF-1 pathway (Figure 5).

[0270] Our results demonstrate that BPL1 , BPL1 HT and the postbiotic LTA from BPL1 have positive effects on C. elegans lifespan. As seen previously with BPL1 fat reduction activity, the positive effect on C. elegans lifespan only required the IGF-1 signalling pathway.

[0271] Furthermore, the comparative assays carried out, highlights the high efficacy of BPL1 in anti-aging related C. elegans models. Furthermore, these anti-aging properties are more pronounced with BPL1 when compared with other strains of the same species. Therefore, the evidences provided herein expose a novel surprising functionality of the probiotic strain Bifidobacterium animalis subsp. lactis BPL1 on aging-related C. elegans models, and these beneficial effects are higher in BPL1 than in other Bifidobacterium animalis strains.

[0272] Preceding studies with BPL1 showed a protective effect against oxidative stress (Martorell, P. et al. 2016). In this study we validated that BPL1 HT and LTA from BPL1 can exert protective effect against oxidative stress in a similar way that previously described for the alive cells.

[0273] In our work we have also tested the protective effect of BPL1 , BPL1 HT and LTA from BPL1 against Gram (+) and Gram (-) pathogens. Both BPL1 and its heat treated form as well as LTA from BPL1 improved viability of the nematodes during the infection with S. enterica ATCC 14028 and S. aureus infection.

[0274] Moreover, a positive activity of BPL1 , BPL1 HT and LTA from BPL1 in anxiety- associated behaviour in C. elegans is shown in the present disclosure. Nematodes treated with our probiotic strain or LTA from BPL1 show behavioural differences between anxious nematodes under deprivation of food. Similarly, nematodes submitted to a psychostimulant stress through exposition to the drug Denubil, tended to move to the edge of the plates under control conditions, but nematodes fed with BPL1 , BPL1 HT or LTA from BPL1 presented different behavioural pattern with nematodes staying in the center of the plate.

[0275] Finally, BPL1 , BPL1 HT and LTA from BPL1 were evaluated on the Alzheimer’s Disease model in C. elegans. Alzheimer’s Disease (AD) is a neurodegenerative disease that usually affects elder people. AD pathogenesis consists on the accumulation of the beta amyloid (AfB) peptide outside the neurons. In this model, the CL4176 strain expresses a muscle-specific A 1-42 [SEQ ID NO: 1 : DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIA] that under a temperature shift induces the paralysis of the worms. With this strain, we demonstrate that BPL1 , BPL1 HT and LTA from BPL1 improve the nematodes' body paralysis. These results highlight the potential of BPL1 , heat-treated BPL1 and the LTA from BPL1 as anti-aging strain by exerting a positive effect on a specific neurodegenerative disease that affects mainly to elderly individuals. Overall, this study investigates the metabolic targets whereby BPL1 strain and its LTA exert their beneficial effect. Results highlight the pivotal role of the Insulin-like signalling pathway in the fat reduction activity as well as the newly confirmed beneficial effects in anxiety conditions, age-related conditions, anti-pathogenic effect, gut barrier integrity, against oxidative stress, and a potential improvement of cognitive function. 4. Further Examples of BPL1 ™, BPL1 ™ HT and LTA-BPL1 ™ effects in aging and longevity

[0276] Additional assays demonstrating beneficial effects of BPL1 ™, BPL1 ™ HT and LTA- BPL1 ™ to prevent aging and promoting longevity were carried out by the inventors.

[0277] 4.1. Oxidative Stress-Induced Cellular Senescence

[0278] Normal Human Dermal Fibroblasts (NHDF; Promocell) were cultured in Dulbecco’s Modified Eagle Medium (DM EM) supplemented with 10 % of fetal bovine serum (FBS), 2 mM L-Glutamine and penicillin (100 U / mL) and streptomycin (100 pg / mL) in a humidified 5% CO2 incubator at 37°C. All cell culture reagents were obtained from Thermo Fisher. Cells were seeded in 24-well plates at a density of 1.5 x 104cells / well and incubated for 24 h. Next day, cells were treated with a non-cytotoxic dose of 0.4 mM H2O2 in DM EM medium without FBS for 2h in the presence or absence of heat- treated BPL1 ™ (108cells / mL) and LTA-BPL1 ™ (100 pg / mL). Epigallocatechin-3- gallate was used as inhibition control of the oxidative senescence process. Afterwards, cells were washed once with PBS and further incubated in complete DMEM medium for 72 h at 37°C. After that time, cells were harvested by trypsinization, washed once with PBS and fixed upon 10 min incubation with a formaldehyde solution (4 % v / v). Cellular senescence was estimated by the measurement of [3-galactosidase activity in the cell population using the CellEventTM Senescence Green Probe (Thermo Fisher) following manufacturer’s instructions. To this purpose, fixed cells were stained with the probe (dilution 1 :1000) for 2h protected from light at 37°C without CO2. Cells were resuspended in dPBS containing 1% bovine serum albumin and analyzed on a flow cytometer using the FITC channel. For each condition, at least 5000 cells were analyzed. Results were expressed as Median Fluorescence Intensity (MFI). Data are the mean of two independent experiments.

[0279] P-galactosidase activity is a biomarker of senescence (Gerasymchuk et al., 2022. Modeling of the Senescence-associated phenotype in human skin fibroblasts. I nt J Mol Sci. 23:7124. doi: 10.3390 / ijms23137124). In this assay, the [3-galactosidase activity is proportional to the fluorescence obtained. Figure 11 indicate that worms fed with BPL1 ™ HT and LTA-BPL1 ™ showed a significantly lower fluorescence intensity values than cell population treated with H2O2 (p-v,a / ue<0.001). Moreover, this reduction was higher with LTA-BPL1 ™ condition (p-va / ue<0.0001). Therefore, BPL1 ™ HT and LTA-BPL1 ™ are able to reduce [3-galactosidase activity involved in the cellular senescence.

[0280] 4.2. Pharyngeal pumping measurement in C. elegans

[0281] C. elegans Wild-type synchronized eggs were seeded in NGM plates with the E. coli OP50 strain and different compounds: BPL1 ™ (overnight lawn), BPL1 ™ HT (108cells / plate) and LTA from BPL1 ™ (10 pg / mL). Once the worms reached the L4 stage they were transferred to fresh plates with the same compounds and FUdR (0.2 mM) to avoid progeny. Worms were maintained at 20°C. Previously to each measurement, worms were stimulated with serotonin hydrochloride 98% 10 mM (Fisher Scientific). The pharyngeal pumping rate was measured at day 1 and 7 using Nemametrix system. Two independent assays were carried out with at least 35 worms analyzed per condition.

[0282] Pharyngeal pumping (intensity and mean frequency) declines progressively with aging in C. elegans (Chow et al., 2006. Sarcopenia in the Caenorhabditis elegans pharynx correlates with muscle contraction rate over lifespan. Exp. Gerontol. 41 : 252- 260. doi: 10.1016 / j.exger.2005.12.004). Figure 12 indicate the mean frequency of the pharyngeal pumping of worms fed with BPL1 ™, BPL1 ™ HT or LTA-BPL1 ™ at day 1 and day 7 of adulthood. Worms fed with BPL1 ™ and BPL1 ™ HT significantly improved the pharyngeal pumping at day 1. However, BPL1 ™, BPL1 ™ HT or LTA- BPL1 ™ exerted a best positive effect at 7 days-old adults, significantly increasing the frequency of worms’ pharyngeal pumping (Figure 12). Therefore, BPL1 ™, BPL1 ™ HT or LTA-BPL1 ™ improved pharyngeal pumping with age.

[0283] 4.3. Sarcopenia in C. elegans (muscle sarcomere structure)

[0284] The effect of the ingredients on the muscle integrity of the nematode C. elegans was visualized in a transgenic strain expressing green fluorescent protein (GFP)-tagged sarcomeres (DM8005 strain: rals5 [myo-3p::GFP::myo-3 + rol-6(su1006)] using fluorescence microscopy. Worms were synchronized on NGM plates supplemented with E. coli OP50 as food and with BPL1 ™ (overnight lawn), BPL1 ™ HT (108cells / plate) or LTA-BPL1 ™ (10 pg / mL). Animals were transferred onto fresh plats every 2 days after young adult stage to remove progeny and prevent population starvation. To image sarcomere structure, 20-25 animals per condition / time point were picked onto a microscope slide containing 4% agarose and imaged immediately using a fluorescence microscope Zeiss Axio Imager M2. Images of the body wall were captured with the 20x objective from the central part of the animal using an Axiocam 705 mono camera. The worms were classified into 3 groups based on the integrity of their muscle cells: i) Well organized: the myofilaments are well organized in a tight and parallel way. More than 70% of the total cells that appear in the image are in perfect condition; ii) Moderately disorganized: gaps appear between the filaments, they are not completely straight and the ends seem to fray. There are some areas with well-aligned filaments. If from the image, between 50-70% of the cells are in good condition; iii) Severely disorganized: large gaps appear between the muscle myofilaments and areas with straight filament can barely be identified. The filaments have an irregular orientation, breaks and abnormal curvatures. If from the image, less than 50% of the cells are in good condition. Two independent assays were performed (a total of 45 worms per condition and day). The number of worms on each sarcomere structure group was determined at day 1 , day 7 and day 11 of adulthood.

[0285] Sarcopenia in C. elegans is a parameter also related with aging (Christian and Benian, 2020. Animal models of sarcopenia. Aging Cell. 19: e13223. DOI: 10.1111 / acel.13223). As the worm ages, sarcomere structure loses integrity (Slade etal., 2023. Bisphosphonates attenuate age-related muscle decline in Caenorhabditis elegans. J. Cachexia Sarcopenia Muscle. 14: 2613-2622. DOI: 10.1002 / jcsm.13335). Worms fed with BPL1 ™, BPL1 ™ HT and LTA-BPL1 ™ showed a protection of the muscle structure integrity, exhibiting a significant lower percentage of worms with severe and moderate disorganization than control fed-population at day 11 (p- value< 0.001) (Figure 13). Therefore, BPL1 ™, BPL1 ™ HT and LTA-BPL1 ™ preserve sarcomere structure with age.

[0286] 4.4. Additional lifespan comparative assays Additional lifespan comparative experiment was conducted in the C. elegans wild-type strain N2 in order to test the efficacy of the probiotic strains on the nematode’s longevity. Effect of BPL1 on lifespan in C. elegans in comparison to Bifidobacterium longum benchmark strains BB536® (commercially available;), 35624™ (commercially available), 1714™ (commercially available) was assessed.

[0287] Age-synchronized nematodes (N2) were cultured in Nematode Growth Medium (NGM) as control condition or in the same medium supplemented with BPL1 ™ or with B. longum strains BB536, 1714 and 35624 (overnight lawn). Survival curves were obtained with data from an automatized system based on artificial vision, developed by Biopolis. Survival of the worms was monitored until 100% of the mortality was reached.

[0288] We evaluated the effect of BPL1 and 3 Bifidobacterium longum strains on the nematodes' longevity (BB536, 35624 and 1714). Survival curves obtained with worms cultured with the strain BPL1 ™ exhibited a significant higher survival increase of the worms in comparison to B. longum strains BB536, 35624 and 1714 (Figure 14 and Table 5). Moreover, BPL1 increased the mean lifespan in 3 days and final lifespan in 2-3 days in comparison to B. longum strains. Therefore, these results show that BPL1 ™ exerts higher effect on C. elegans' lifespan than other Bifidobacterium longum strains (BB536, 35624 and 1714).

[0289] Table 5. Summary of the effect of Bifidobacterium longum strains BB536, 35624 and 1714 in comparison to BPL1 on C. elegans' N2 lifespan.

[0290] Mean lifespan: 50% survival. Final lifespan: 100% mortality. 5. Additional comparative assays in anxiety C.elegans model

[0291] Effect of BPL1 on anxiety behavior in C. elegans in comparison to Bifidobacterium longum benchmark strains BB536, 35624™, 1714™ was assessed.

[0292] Octanol avoidance was used as anxiety- related behavior assay in the wild-type strain of C. elegans (N2). The experiments were carried out using the “smell-on-a-stick” assay. For octanol avoidance assays, the blunt end of a hair (Loew-Cornell (T eaneck, NJ) 9000 Kolinsky 7 paintbrush) taped to a Pasteur pipette, was immersed in freshly prepared 30% octanol in EtOH (vol / vol) and placed in front of a forward-moving nematode. The amount of time that worms took to move backward was measured. Control condition nematodes were maintained in NGM with E. coli OP50. Animals were transferred to NGM plates seeded with freshly overnight E. coli OP50. Nematodes were incubated 20 min in these plates before testing. For the anxiety condition, nematodes were incubated during 20 min in NGM plates without OP50. This food deprivation induces anxiety-related behavior due to a decrease of levels of serotonin. To evaluate treatments, wild-type nematodes (N2) were age-synchronized in NGM plates supplemented with BPL1 ™ or with B. longum strains BB536, 1714 and 35624 (dose of 108cells / plate). At young-adult stage, nematodes were transferred to NGM plates with each treatment but no food during 30 min. Afterwards, worms were placed in NGM plates without OP50 and with each treatment and tested 20 min later. The percentage of reduction time to respond to octanol in comparison to control anxious is represented.

[0293] Results indicate the relative percentages of each strain against the anxious control population (Figure 15), which take longer to avoid octanol. BPL1 ™ provided 36.6% of reduction of the time of response when the octanol was presented to the worms, while B. longum strains BB536, 1714 and 35624 exhibited 17.7, 20.5 and 13.5%, respectively. These data showed a higher significant positive effect of BPL1 ™ reducing the anxious behavior in comparison to B. longum strains BB536, 1714 and 35624 (Figure 15).

[0294] 6. Pathogen protection infection comparative assays Protection of BPL1 on pathogen infection in C. elegans was compared with other alternative Bifidobacterium animalis strains:

[0295] -BPL27, obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from breastmilk;

[0296] -BPL30, obtained from the applicant’s collection, namely Biopolis, S.L. collection, isolated from infant faeces;

[0297] -BB12 (®; Jungersen M., The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis BB-12(®). Microorganisms. 2014 Mar 28;2(2):92-110) (Benchmark) originates from Chr. Hansen’s collection of dairy cultures: and

[0298] -B420. (Benchmark, Howaru; Uusitupa HM, et al., Bifidobacterium animalis subsp. lactis 420 for Metabolic Health: Review of the Research. Nutrients. 2020 Mar 25;12(4):892. ) isolated from fermented milk, with similar conditions were carried out)

[0299] Wild-type synchronized eggs were seeded in NGM plates with OP50 E. coli or with BPL1 ™ or other alternative B. animalis strains (BPL27, BPL30, BB12 and B420), all of them at dose of 108cells / plate. Once the worms reached the young adult stage, they were transferred to the infection plates containing a lawn of the respective pathogen (S. aureus ATCC 25923 or S. enterica subsp. enterica serovarTyphimurium ATCC 14028). A negative control condition without infection (NGM medium with the strain OP50 E. coli) and a positive control condition of infection only with the corresponding pathogen were included. Worms were maintained at 25 °C in the different conditions and scored for survival during 5-10 days. Worms were counted as alive or dead by stimulation with the platinum stick. Two independent assays were carried out with a total of 50 worms analyzed per condition.

[0300] As showed in Figure 16, nematodes fed with BPL1 ™ exhibited significant higher worms’ survival than the alternative B. animalis strains BPL27, BPL30 and B420 (p- value< 0.001). Table 6 indicates the p-value (Log rank test) of nematodes survival curve fed with BPL1 ™ in comparison to strains B. animalis strains BPL27, BPL30 and B420.

[0301] Table 6. p-value (Log rank test) of nematodes survival curve fed with BPL1 ™ and infected with S. aureus in comparison to strains B. animalis BPL27, BPL30 and B420.

[0302] Concerning data from S. enterica subsp. Enterica serovar Typhimurium (ATCC 14028) infection, worms fed with BPL1 ™ showed higher resistance to Salmonella infection compared with other B. animalis strains BPL30, BB12 and B420 (Figure 17).

[0303] Table 7 indicates the p-value (Log rank test) of nematodes survival curve fed with BPL1 and infected with Salmonella enterica in comparison to strains BPL30, BB12 and B420. Table 7. p-value (Log rank test) of nematodes survival curve fed with BPL1 and infected with S. aureus in comparison to strains BPL27, BPL30 and B420.

Claims

CLAIMS1. Composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145 and / or a lipoteichoic acid (LTA) thereof, for use in the treatment and / or prevention of anxiety disorders in a subject.

2. Composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145 and / or a lipoteichoic acid (LTA) thereof, for use in the treatment and / or prevention of a pathogen infection in a subject, preferably wherein the pathogen is a bacterium.

3. Composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145 and / or a lipoteichoic acid (LTA) thereof, for use in restoring the integrity of gut barrier in a subject.

4. Composition for use according to any one of claims 1 to 3, wherein the Bifidobacterium animalis subsp. lactis strain is in the form of viable cells.

5. Composition for use according to any one of claims 1 to 3, wherein the Bifidobacterium animalis subsp. lactis strain is in the form of non-viable cells, preferably wherein the non-viable cells are heat-treated.

6. Composition for use according to any one of claims 1 to 5, wherein the strain is present in an amount between 105cfu and 1012cfu per gram or milliliter of the composition.

7. Composition for use according to any one of claims 1 to 6, wherein the composition is formulated for administration in liquid form or in solid form.

8. Composition for use according to claim 7, wherein the solid formulation is selected from the group consisting of tablets, lozenges, sweets, chewable tablets, chewing gum, capsules, sachets, powders, granules, coated particles or coated tablets, tablets and gastro-resistant tablets and capsules and dispersible strips and / or films.

9. Composition for use according to claim 7, wherein the liquid formulation is selected from the group consisting of oral solutions, suspensions, droplets, emulsions and syrups.

10. Composition for use according to any one of claims 1 to 9, wherein the composition is a pharmaceutical composition or a nutritional composition.

11. Composition for use according to claim 10, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or an excipient.

12. Composition for use according to claim 10, wherein the nutritional composition is a food or a nutritional supplement.

13. Composition for use according to claim 12, wherein the food is selected from the group consisting of fruit or vegetable juices, ice cream, infant formula, milk, yogurt, cheese, fermented milk, milk powder, cereals, baked goods, milk-based products, meat products and beverages.

14. Non-therapeutic use of a composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145, and / or a lipoteichoic acid (LTA) thereof, in the reduction of anxiety and / or an anxiety-related condition in a subject.

15. Non-therapeutic use according to claim 14, wherein the anxiety related condition is selected from the list consisting of anxiousness, stress, worry, nervousness and unease.

16. Non-therapeutic use of a composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145, and / or a lipoteichoic acid (LTA) thereof, in alleviating unpleasant effects and / or discomfort associated to the presence of a pathogen in a subject.

17. Non-therapeutic use according to claim 16, wherein the pathogen is a bacterium belonging to Salmonella or Staphylococcus genus.

18. Non-therapeutic use of a composition comprising Bifidobacterium animalis subsp. lactis strain CECT 8145, and / or a lipoteichoic acid (LTA) thereof, for improving the gut barrier function or maintaining the gut barrier integrity in a subject.

19. Use of a composition comprising Bifidobacterium animalis subsp. lactis strainCECT 8145, and / or a lipoteichoic acid (LTA) thereof, for improving age-related conditions, and / or increasing lifespan and / or promoting longevity in a subject.

20. LTA obtained by cultivating Bifidobacterium animalis subsp. lactis strain CECT 8145 in excess of sugars as carbon source, wherein the LTA has the capacity to reduce and / or reverse stress and / or anxiety in a subject.

21. Use of the LTA according to claim 20, for the elaboration of a food or feed product, preferably a nutritional supplement.

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