The use of postbiotics in the treatment and / or prevention of anxiety disorders

Abstract

The present invention relates to a postbiotic composition comprising non-viable Bifidobacterium longum and Lactobacillus rhamnosus strains and related uses thereof in the treatment and / or prevention of anxiety disorders.

Description

Detailed Description of the Invention

[0001] The present invention relates to a postbiotic composition comprising non-viable Bifidobacterium longum and Lactobacillus rhamnosus strains and related uses thereof in the treatment and / or prevention of anxiety disorders.

[0002] [Background technology] There is growing evidence that bidirectional communication between the brain and gut, termed the "gut-brain axis," plays a potential role in maintaining gut homeostasis as well as brain function. Some studies suggest that distinct types of microbes, such as orally administered probiotics, can modify the gut-brain axis and control stress responses, suggesting that probiotics may play a therapeutic role in managing mood disorders such as depression or anxiety by modulating the "microbiota-gut-brain axis."

[0003] Many live probiotic strains and mixtures have been reported to affect anxiety and other behavioral indicators. However, there is very limited evidence of positive outcomes on anxiety for the use of non-living, inanimate, inactivated or heat-treated forms of microorganisms, including but not limited to inanimate forms of probiotics, all of which are known as postbiotics and are also described as ghost probiotics or paraprobiotics.

[0004] The use of these non-viable microbial strains allows the use of products in harsh environments, matrices or processes that may damage cell viability, making them highly suitable for industrial applications.

[0005] Therefore, there is a need to develop postbiotic compositions for the treatment of anxiety disorders.

[0006] Description of the Invention The present authors found that long-term feeding of a mixture of non-viable Lactobacillus rhamnosus and Bifidobacterium longum strains produced favorable results in a Danio rerio (zebrafish)-based model of behavior and anxiety. Surprisingly, the best results were obtained with a heat-treated postbiotic blend, rather than with either the viable or non-viable strains alone, or with the viable probiotic blend itself.

[0007] Furthermore, comparative assays with different combinations of heat-treated Lactobacillus rhamnosus (L. rhamnosus) and Bifidobacterium longum (B. longum) strains were performed in a Caenorhabditis elegans (C. elegans) anxiety model and showed a significant improvement and reduction in anxiety behavior associated with treatment with a blend of heat-treated B. longum strain CECT 7347 (also named HT-ES1 or HT-IATA-ES1) and L. rhamnosus strain CECT 8361 (also named HT-BPL15 or HT-BPL0015) when compared to other combinations of heat-treated strains belonging to the B. longum and L. rhamnosus species (Example 3).

[0008] Thus, in one aspect, the present invention relates to a postbiotic composition comprising the non-viable Bifidobacterium longum strain CECT 7347 and the non-viable Lactobacillus rhamnosus strain CECT 8361 ("the postbiotic composition of the invention" or "the composition of the invention").

[0009] Lactobacillus rhamnosus CECT 8361 (BPL15 or BPL0015) was isolated from the faeces of a healthy, lactating child under 3 months of age. The strain was deposited on 27 May 2013 at the Spanish Culture Collection (Building 3 CUE, Parc Cientificat Universitat de Valencia, C / Catedratico Agustin Escardino, 9, 46980 Paterna (Valencia) SPAIN) as an international depositary under the Budapest Treaty. The deposit number was CECT 8361.

[0010] B. longum CECT 7347 (ES1 or IATA-ES1) was isolated from the faeces of a healthy, lactating child under 3 months of age and deposited under the Budapest Treaty on 20 December 2007 at the Spanish Culture Collection as an international depository (Building 3 CUE, Parc Cientificitat Universitat de Valencia, C / Catedratico Agustin Escardino, 9, 46980 Paterna (Valencia) SPAIN). The deposit number was CECT 7347.

[0011] As used herein, the term "postbiotic composition" refers to any composition containing at least one postbiotic, including non-living bacteria or components thereof. According to the definition provided by the International Society for the Study of Probiotics and Prebiotics (ISAPP), postbiotics are "preparations of non-living microorganisms and / or their components that confer a health benefit to the host" (Sallines et al. 2021 Nature Rev Gastroenterol Hepatol. 18(9):649-667). In contrast, according to the World Health Organization (WHO), probiotics are "live microorganisms that, when administered in adequate amounts, confer a health benefit to the host."

[0012] In the last few years, research has focused on members of the gut microbiota that exhibit health-promoting actions, such as protecting the host against pathogens by competitive exclusion or modulating the immune system. The term "gut microbiota" refers to a diverse group of microorganisms that have evolved to adapt and survive in the human gastrointestinal tract. The gut microbiota interacts with its human host in various ways. Microorganisms can be harmless commensals, opportunistic pathogens, or health-promoting or probiotic microorganisms. Research on the latter activity has increased significantly in the last two decades, and probiotic agents have been investigated in many clinical and animal model studies.

[0013] In the present invention, the postbiotic composition comprises non-viable bacteria belonging to the genera B. longum and L. rhamnosus, the classification of both species being detailed below.

[0014] [Table 1]

[0015] Members of the genus Bifidobacterium are among the first microorganisms to colonize the human gastrointestinal tract (GIT) and are thought to confer health benefits to their host. Due to their associated health benefits and general perception of safety, bifidobacteria have been commercially exploited as probiotic agents. Due to their purported health-promoting properties, bifidobacteria are included as active ingredients in many functional foods.

[0016] Bifidobacteria naturally occur in a variety of ecological niches that are directly or indirectly associated with the animal GIT, such as the human oral cavity, insect gut, sewage, etc. To survive in these specialized ecological niches, bifidobacteria require specific adaptations to enhance their competitiveness.

[0017] Bifidobacterium is a Gram-positive, non-motile, frequently branching anaerobic bacterium. It is one of the major genera of bacteria that compose the mammalian GIT microbiota and is composed of more than 30 species. Among them, B. longum is a catalase-negative, branching, rod-shaped bacterium.

[0018] Lactobacillus (now known as Lacticaseibacillus) is the largest genus in the family Lactobacillaceae. Prior to its reclassification into 23 genera (Zheng et al. 2020 Int J Syst Evol Microbiol. 70(4):2782-2858), it consisted of 196 valid published species, which are commonly isolated from environments associated with fermented foods such as fruits, meat, sourdough, vegetables, and wine, as well as from the gastrointestinal tract and vagina of humans and animals. Currently, they are widely applied in fields related to food, feed, medicine, and biotechnology, for example, as dairy starters, probiotics, vaccine carriers, and silage inoculants.

[0019] Lactobacillus bacteria are Gram-positive, rod-shaped, facultatively anaerobic or microaerophilic, non-spore-forming, acid-resistant, catalase-negative bacteria with a DNA G+C content of 46–58 mol%. Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus are phylogenetically and phenotypically closely related and were previously considered to be part of the L. casei group (Zheng et al. 2020 Int J Syst Evol Microbiol. 70(4):2782-2858). Probiotic strains that are part of this group are used worldwide as probiotics in fermented dairy products or food supplements to promote host health.

[0020] The postbiotic composition of the present invention comprises the non-viable forms of the bacteria B. longum and L. rhamnosus. As used herein, the terms "non-living", "non-living", "inactive" or "inactivated" refer to metabolically or physiologically inactive microorganisms, i.e., microorganisms that do not retain metabolic activity and elongation capacity after feeding. Viability is independent of the ability of the microorganism to form colonies on solid media, i.e., culturability.

[0021] Thermal processing has a long history in the food industry and is often used to inactivate microorganisms. A common method for inactivating microorganisms is heat treatment. Heat is lethal to microorganisms, but each species has its own specific heat tolerance. In thermal destruction processes such as pasteurization, tindalization, and autoclaving, the rate of destruction is logarithmic, as is the rate of growth. Thus, bacteria exposed to heat are killed at a rate proportional to the number of bacteria present. This process depends on both the exposure temperature and the time required at this temperature to achieve the desired rate of destruction.

[0022] In some embodiments of the invention, the non-viable Bifidobacterium longum and Lactobacillus rhamnosus strains are heat treated. Thus, preferred embodiments provide compositions of the invention, wherein the non-viable B. longum strain CECT 7347 is heat treated and the non-viable Lactobacillus rhamnosus strain CECT 8361 is heat treated.

[0023] The present invention also contemplates bacterial strains derived from B. longum CECT 7347 and L. rhamnosus CECT 8361 strains ("parent strains") that may be part of the postbiotic compositions of the present invention, as these bacterial strains retain the ability to reduce and / or ameliorate symptoms of anxiety disorders in a subject.

[0024] Examples of bacterial strains derived from parent strains that may be comprised within the compositions of the invention include genetically modified organisms that exhibit a mutation in their genome compared to the genome of the strain of the invention, where the mutation does not affect the ability of the strain and / or its components to reduce and / or ameliorate symptoms of an anxiety disorder in a subject.

[0025] Strains derived from B. longum CECT 7347 and L. rhamnosus CECT 8361 can be produced naturally or intentionally by mutagenesis methods known in the art, including but not limited to, growing the parent strain in the presence of a mutagenic agent or stress factor, or by genetic engineering directed modification, deletion and / or insertion of specific genes.

[0026] In some embodiments, the compositions of the present invention contemplate genetically modified organisms derived from B. longum CECT 7347 and L. rhamnosus CECT 8361 strains that retain the ability to prevent, reduce and / or ameliorate symptoms of anxiety disorders in a subject, and thus contemplate said compositions as useful for the treatment and / or prevention of said disorders.

[0027] In some specific embodiments, the present invention also contemplates bacterial components, metabolites and molecules secreted by B. longum CECT 7347 and / or L. rhamnosus CECT 8361 strains, and / or compositions comprising said components, and their use for the treatment and / or prevention of anxiety disorders.

[0028] Bacterial components include, but are not limited to, components of the cell wall (non-limiting examples include peptidoglycan); nucleic acids; membrane components; and proteins, lipids, carbohydrates and combinations thereof (such as lipoproteins, glycolipids or glycoproteins). Metabolic products include any molecule produced or modified by bacteria as a result of metabolic activity during growth, use of bacteria in technological processes, or storage of bacteria. Examples of these metabolic products 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 bacteria during growth, use of bacteria in technological processes (e.g., food processing or drugs), or storage of bacteria. 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.

[0029] The composition of the present invention can be formulated for pharmaceutical administration, i.e., to form part of a pharmaceutical product administered to a subject by any administration means; and / or for dietary administration, i.e., to form part of a food or nutritional supplement consumed in the diet of a subject.Thus, in some embodiments, the composition of the present invention is a pharmaceutical composition ("pharmaceutical composition of the present invention") and / or a nutritional composition ("nutritional composition of the present invention").

[0030] The compositions of the invention include non-viable B. longum CECT 7347 and L. rhamnosus CECT 8361 (and / or any strains and / or cellular components derived therefrom) in any suitable concentration.

[0031] Additionally, the compositions of the invention may include (i) one or more components or compounds having any biologically, pharmacologically and / or veterinarily useful activity, such as an activity useful for the prevention and / or treatment of anxiety disorders in a subject; and / or (ii) one or more components that, upon administration to a subject, may increase, enhance and / or promote the activity of the postbiotics contained in the compositions of the invention. As will be appreciated by those of skill in the art, the additional components or compounds should not affect the effects exerted by the non-viable strains of the compositions of the invention.

[0032] The term "pharmaceutical composition" also includes veterinary compositions.

[0033] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier and / or excipient.

[0034] The term "excipient" refers to a substance that aids in the absorption of any component or compound of the composition of the invention, i.e. the strain of the invention, or that stabilizes the component or compound, and / or that aids in the preparation of the pharmaceutical composition in the sense of giving it consistency or imparting a flavor to make it more pleasant. Thus, the excipient may have, but is not limited to, the function of binding the component (e.g., starch, sugar or cellulose), sweetening, coloring, protecting the active ingredient (e.g., shielding it from air and / or moisture), filling the tablet, capsule or any other presentation, or disintegrating to facilitate the dissolution of the ingredient, without excluding other excipients not mentioned in this paragraph. Thus, the term "excipient" is defined as a substance that is included in the galenic form and added to the active ingredient or its related, allowing their preparation and stability, modifying their organoleptic properties, or determining the physical and chemical properties of the pharmaceutical composition and its bioavailability. A "pharmaceutical acceptable" excipient must allow the activity of the components or compounds of the pharmaceutical composition, i.e., must not affect the effect exerted by the strain of the present invention.

[0035] A "galenic form" or "pharmaceutical form" is a form in which the active ingredient and excipients are adapted to provide a pharmaceutical composition or 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 to be administered.

[0036] A "vehicle" or "carrier" is preferably an inert substance. The function of a carrier is to facilitate the incorporation of other ingredients or compounds, to allow better dosing and administration, and / or to give consistency and form to the pharmaceutical composition. Thus, a carrier is a substance used in a drug to dilute any of the ingredients or compounds of the pharmaceutical composition of the present invention to a given volume or weight; or, alternatively, a substance that allows better dosing and administration and / or can give consistency and form to the drug without diluting these ingredients or compounds. When the presentation is liquid, a pharmaceutically acceptable carrier is a diluent. The carrier may be natural or non-natural. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycol, gelatin, lactose, starch, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, flavor oils, mono- and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0037] Additionally, excipients and carriers must be pharmacologically acceptable, i.e., approved and evaluated as not damaging to the subject to which they are administered.

[0038] In each case, the presentation of the composition is adapted to the type of administration used.Therefore, the composition can be presented in a therapeutically effective amount in solution or any other clinically acceptable administration form.The composition of the present invention can be formulated into solid, semi-solid or liquid preparations such as tablets, capsules, powders, granules, solutions, suppositories, gels or microspheres.In certain embodiments, the composition of the present invention is formulated in liquid or solid form.

[0039] In another particular embodiment, the solid formulation is selected from the group consisting of tablets, lozenges, confectioneries, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles or coated tablets, tablets, pills, lozenges, gastroresistant tablets and capsules, and dispersible strips and films.

[0040] In another particular embodiment, the liquid formulation is selected from the group consisting of oral solutions, suspensions, drops, emulsions and syrups.

[0041] Similarly, various systems are known that can be used for sustained release administration of the composition of the present invention, including, for example, encapsulation in liposomes, microbubbles, microparticles or microcapsules.Suitable sustained release forms, as well as the materials and methods for their preparation, are well known in the art.Therefore, the orally administrable form of the composition of the present invention is a sustained release form that further comprises at least one coating or matrix.The sustained release coating or matrix includes, but is not limited to, natural, semi-synthetic or synthetic polymers, water-insoluble or modified waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic or synthetic plasticizers, or combinations of two or more thereof.Enteric coating can be applied using conventional processes known to those skilled in the art.

[0042] In addition to the above, the present invention also encompasses the possibility that the compositions of the present invention may be administered to a subject together with other components or compounds that are not part of the compositions of the present invention. Examples of such components or compounds are detailed in the preceding paragraphs.

[0043] In some embodiments of the present invention, the composition of the present invention is a nutritional composition. The nutritional composition of the present invention also comprises any suitable concentration of the non-viable bacteria B. longum CECT 7347 and L. rhamnosus CECT 8361 (and / or any strains and / or cellular components derived therefrom).

[0044] In some embodiments, compositions of the invention comprise any suitable concentration of L. rhamnosus CECT 8361 and B. longum CECT 7347 (and / or any strains and / or cellular components derived therefrom).

[0045] When the composition of the present invention is formulated as a nutritional composition, said nutritional composition may be a food product or may be incorporated into a food product or food product intended for human and / or animal consumption.

[0046] Thus, in some embodiments, the nutritional composition of the present invention is a food product or a nutritional supplement.

[0047] In some specific embodiments of the present invention, the nutritional composition of the present invention is a food for a specific nutritional and / or functional, biological and / or physiological purpose.In some specific embodiments of the present invention, the nutritional composition of the present invention is a medicinal food.

[0048] In some embodiments, the nutritional compositions of the present invention are nutritional or dietary supplements.

[0049] In the present invention, the term "nutritional composition" refers to any food product that beneficially affects one or more functions of the body, regardless of providing nutrients to the subject consuming it, thereby promoting and / or providing better health and wellness. In the present invention, said nutritional composition is intended to alleviate, reduce, treat and / or prevent anxiety disorders.

[0050] The term "supplement" is synonymous with any of the terms "dietary supplement", "nutritional supplement", "food supplement", or "nutritional supplement" or "nutritional ingredient" and refers to a product or preparation consisting of a concentrated source of nutrients or other substances having a nutritional or physiological effect, intended to supplement the normal diet of a subject. In the present invention, B. longum CECT 7347, L. rhamnosus CECT 8361, and / or any strains and / or cellular components derived therefrom are "substances" having a nutritional and / or physiological effect on a subject.

[0051] Dietary supplements may be in single or multiple forms and may be sold in dosage forms designed to be taken at a time, i.e., capsules, pills, tablets and other similar forms, sachets for powders, ampoules for liquids, dropper bottles and other similar forms of liquids and powders.

[0052] Nutrients and other elements present in nutritional ingredients are diverse, including vitamins, minerals, amino acids, essential fatty acids, fiber, enzymes, plants and plant extracts, among others.Since their role is to complement the supply of nutrients in the diet, they should not be used as a substitute for a balanced diet, and the intake should not exceed the daily intake explicitly recommended by a doctor or nutritionist.The composition of the present invention can also be part of so-called "foods for special groups", i.e. foods that meet specific nutritional needs.

[0053] Examples of food products that can be included in the composition of the present invention include, but are not limited to, feed, dairy products, vegetable products, meat products, snacks, chocolates, beverages, baby foods, cereals, fried foods, industrial bakery products and biscuits.Examples of dairy products include, but are not limited to, fermented milk products (such as, but are not limited to, yogurt or cheese) or non-fermented milk products (such as, but are not limited to, ice cream, butter, margarine or whey).Examples of vegetable products include, but are not limited to, cereals in any presentation form, fermented (such as, for example, soy yogurt, oat yogurt, etc.) or non-fermented, and snacks.Beverages include, but are not limited to, non-fermented milk.

[0054] In some embodiments, the food product or food product is selected from the group consisting of fruit or vegetable juice, ice cream, infant formula, milk, yogurt, cheese, cultured milk, milk powder, cereals, baked goods, dairy-based products (such as milkshakes or smoothies), meat products, and beverages.

[0055] In some embodiments, compositions of the invention may include other microorganisms in addition to B. longum CECT 7347 and L. rhamnosus CECT 8361.

[0056] As will be appreciated by those skilled in the art, the B. longum and L. rhamnosus strains must be present in the compositions of the present invention, either together or separately, in therapeutically effective amounts in order to exert their effects, such as the effects of alleviating, mitigating, treating and / or preventing anxiety disorders.

[0057] In the context of the present invention, a "therapeutically effective amount" is any amount of a component or compound of a nutritional or pharmaceutical composition that is sufficient to produce a desired effect when administered to a subject. Said component or compound of a nutritional or pharmaceutical composition refers to the bacterial strains B. longum CECT 7347 and L. rhamnosus CECT 8361. A therapeutically effective amount may vary depending, for example, on the subject's age, weight, general health, sex and diet, as well as the mode and time of administration, excretion rate or the possibility of co-treatment with other drugs. For the avoidance of doubt, said drugs may be prescribed for the prevention and treatment of anxiety disorders or for other conditions.

[0058] In some embodiments of the composition of the present invention, the total concentration of B. longum CECT 7347 and L. rhamnosus CECT 8361 microorganisms is 10 3 From 10 12 cfu or cells, preferably between 10 9 cfu or cells.

[0059] In some specific embodiments of the compositions of the present invention, the amount of B. longum CECT 7347 and L. rhamnosus CECT 8361 microorganisms is 10 3 From 10 12 cfu or cells, preferably between 10 9 cfu or cells. In some specific embodiments, the nutritional composition of the present invention is a food or nutritional supplement for humans.

[0060] In some specific embodiments, the nutritional compositions of the present invention are foods or nutritional supplements for livestock and / or pets.

[0061] In some specific embodiments, the nutritional composition of the present invention is a food or nutritional supplement for the prevention of anxiety disorders in healthy humans.

[0062] In some specific embodiments, the nutritional compositions of the present invention are foods or nutritional supplements for the prevention of anxiety disorders in healthy livestock and / or pets.

[0063] In another preferred embodiment, the composition of the present invention further comprises a microorganism or component thereof selected from the group consisting of Bacillus spp., Lactobacillus spp., Streptococcus spp., Bifidobacterium spp., Saccharomyces spp., Kluyveromyces spp., and combinations thereof.

[0064] As mentioned above, the use of probiotics for various symptoms or to improve general health is well established, but the same cannot be said for the use of postbiotics.In particular, while many probiotic strains and mixtures have been reported to have effects on anxiety and other behavioral indicators, there is less evidence of positive results on anxiety associated with the use of postbiotics.This application provides data for the first time that demonstrate the effectiveness of postbiotic compositions containing non-viable L. rhamnosus and B. longum strains in treating anxiety disorders.

[0065] Therefore, in another aspect, the present invention relates to a composition of the invention for use as a medicament.

[0066] The term "pharmaceutical product" as used herein refers to any composition / substance used for the prevention, diagnosis, mitigation, treatment or cure of disease in a subject, or any composition / substance that can be administered to a subject for the purpose of restoring, correcting or altering physiological function by exerting a pharmacological, immunological or metabolic effect.

[0067] In another embodiment, the present invention relates to a composition of the present invention for use in the treatment and / or prevention of an anxiety disorder in a subject.

[0068] As used herein, the term "treat" or "treatment" includes inhibiting a disease or pathological condition, i.e., arresting its development; alleviating a disease or pathological condition, i.e., causing regression of a disease or pathological condition; and / or stabilizing a disease or pathological condition in a subject. In the present invention, the disease or pathological condition is at least one anxiety disorder.

[0069] As used herein, the term "prevention" refers to avoiding the occurrence of a disease or pathological condition in a subject, particularly when the subject has a predisposition to the pathological condition but has not yet been diagnosed. In the present invention, the disease or pathological condition is at least one anxiety disorder.

[0070] In the context of the present invention, the term "subject" refers to any animal of 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, caprine livestock (goats, etc.), swine (wild boars, pigs, etc.), equine livestock (horses, ponies, etc.), cattle (bulls, cows, oxen, etc.); animals of hunting interest, such as stags, deer, reindeer; and humans.

[0071] In some embodiments, the subject is a mammal, and in some preferred embodiments, the mammal is a human of any race, sex, or age.

[0072] In some other preferred embodiments, the mammal is a farm animal, including but not limited to a cow, horse, goat, llama, sheep, pig, chicken, or any animal kept or bred for commercial purposes. In other preferred embodiments, the mammal is a pet, including but not limited to a dog, cat, rabbit, etc.

[0073] In some embodiments, the compositions of the invention are administered to a subject through the diet.

[0074] In some embodiments of the invention, the administration regimen of the compositions of the invention is at least once daily; twice daily; or three times daily, once with each major food intake (breakfast and / or lunch and / or dinner).

[0075] The use of postbiotics is highly relevant for their industrial applicability, especially in their production, which allows the use of products in harsh environments, such as exposure to heat or moisture, that compromise the stability of conventional probiotics, but also the use of harsh matrices or processes (e.g. extrusion of feed) that compromise the viability of the probiotic cells.

[0076] Additionally, the use of both postbiotics and probiotics offers numerous advantages over traditional anxiety disorder treatments, avoiding the typical side effects of standard drugs such as fluoxetine, including but not limited to insomnia, muscle tension, nausea, and sexual dysfunction.

[0077] As explained above, one aspect of the present invention relates to a postbiotic composition comprising the non-viable B. longum CECT 7347 and L. rhamnosus CECT 8361 strains for use in the treatment and / or prevention of anxiety disorders.

[0078] Anxiety is an emotion characterized by feelings of tension, anxious thoughts, and physical changes such as elevated blood pressure. Although anxiety is a normal response to stress and may be beneficial in some circumstances, anxiety disorders involve excessive fear or apprehension, as opposed to normal feelings of tension or anxiety. Anxiety can be experienced not only by humans, but also by other animals, particularly other non-human mammals.

[0079] Anxiety disorders are the most common of human mental disorders, affecting nearly 30% of adults at some point in their lives. Anxiety disorders include, but are not limited to, generalized anxiety disorder, panic disorder, specific phobias, agoraphobia, social anxiety disorder (formerly known as social phobia), noise aversion, and separation anxiety disorder.

[0080] Generalized anxiety disorder involves persistent and excessive worry that interferes with daily life. It may be accompanied by physical symptoms such as restlessness, irritability, or fatigue, difficulty concentrating, muscle tension, or problems sleeping.

[0081] Panic disorder is usually characterised by recurrent panic attacks, a severe combination of physical and psychological distress accompanied by quite severe symptoms. The attacks may be expected, such as a response to a feared object, or unexpected, occurring for no apparent reason. It may also occur together with other mental illnesses, such as depression or post-traumatic stress disorder (PTSD).

[0082] A specific phobia is an excessive and persistent fear of a particular, usually harmless, object, situation, or activity.

[0083] Agoraphobia is the fear of being in a difficult or embarrassing situation from which it is difficult to escape, or from which help may not be available if panic attacks occur. The fear is out of proportion to the situation, typically lasts for more than six months, and causes impairment.

[0084] Social anxiety disorder is characterized by significant anxiety and discomfort about being embarrassed, humiliated, rejected, or looked down upon in social interactions. The fear or anxiety interferes with daily life and lasts for at least six months.

[0085] The causes of anxiety disorders are currently unknown, but they probably involve a combination of genetic, environmental, psychological, and developmental factors.

[0086] Although each anxiety disorder has its own unique characteristics, most respond well to psychotherapy and drug therapy. Drug therapy does not cure anxiety disorders, but it does significantly reduce symptoms. The most commonly used drugs are antianxiety medications (which are generally prescribed only for short periods of time) and antidepressants. Beta-blockers, used for heart conditions, may also be used to control the physical symptoms of anxiety.

[0087] In the present invention, the term "anxiety disorder" includes not only the anxiety disorders described above, but also related disorders, including, but not limited to, post-traumatic stress disorder (PTSD), acute stress disorder, obsessive-compulsive disorder, and adjustment disorder.

[0088] PTSD is a mental disorder that can occur in people who have experienced or witnessed a traumatic event, such as a natural disaster, a serious accident, an act of terrorism, or who have been threatened with death, sexual violence, or serious injury. PTSD can affect people of any ethnicity, nationality, or culture, and at any age. People with PTSD have intense and disturbing thoughts and feelings related to the experience that last long after the traumatic event has ended. People with PTSD may also experience sadness, fear, or anger, and feel detached or alienated from others.

[0089] Acute stress disorder, like PTSD, develops in response to a traumatic event and has similar symptoms. However, symptoms occur 3 days to 1 month after the event and cause significant distress and problems in daily life. About half of people with acute stress disorder progress to PTSD. Both psychotherapy and medication can help control symptoms and prevent acute stress disorder from progressing to PTSD.

[0090] Obsessive-compulsive disorder (OCD) is a disorder characterized by recurrent, unwanted thoughts, ideas, or sensations (obsessions) that cause recurrent urges to do things (compulsions). These compulsions may significantly interfere with a person's daily life and social interactions, cause significant distress, and impair work or social functioning.

[0091] Adjustment disorders also develop in response to a stressful life event (or events). The emotional or behavioral symptoms a person experiences in response to a stressor are more severe or intense than would be reasonably expected for the type of event that occurred. Symptoms may include sadness, hopelessness, and physical symptoms such as tremors, palpitations, and headaches. These symptoms cause significant distress or impairment in important areas of the subject's life and usually begin within three months of the stressful event. These symptoms subside within six months after the stressor or its consequences have ended. The disorder is usually treated with psychotherapy.

[0092] As mentioned above, animals, especially mammals, can also develop anxiety. For example, separation anxiety is common in pets. Separation anxiety is triggered when a dog or cat is separated from its guardian. Escape attempts by animals are often extreme and may even lead to self-harm. There is evidence that social stress also plays an important role in the welfare of pets. Furthermore, there is evidence that social stress, transportation, and high density on farms may also play an important role in disease prevention and farm welfare in livestock, and that common management practices may be mediators that increase disease risk. Social factors such as deprivation of social contact ("social isolation"), reduced space ("crowding"), and disruption of social order ("social instability") induce physiological and behavioral indicators of stress in livestock.

[0093] In another embodiment, the present invention encompasses a method for treating or preventing an anxiety disorder in a subject ("a therapeutic or prophylactic method of the present invention") comprising the step of administering to the subject a composition of the present invention.

[0094] Another aspect of the present invention relates to the use of a composition of the present invention in the manufacture of a medicament for the treatment or prevention of an anxiety disorder in a subject ("Use of the Invention").

[0095] All preferred embodiments and terms used in the treatment or prevention method and use of the present invention are as explained above. All specific terms, definitions and embodiments of the above aspects of the present invention are applicable to the treatment method and use of the present invention.

[0096] In addition to the relevant use in the treatment and / or prevention of anxiety disorder, the composition of the present invention can also be used to improve non-pathological anxiety.As mentioned hereinabove, anxiety is a normal response to stress and can be beneficial in some circumstances.Therefore, normal feelings of tension or anxiety can be differentiated from anxiety disorder.

[0097] Thus, another aspect of the invention relates to the non-therapeutic use of the compositions of the invention in ameliorating anxiety.

[0098] Anxiety is a typical response of an individual to stress and new situations and encompasses, but is not limited to, feelings of worry, restlessness, or tension in a subject.

[0099] Another aspect of the invention relates to the non-therapeutic use of a composition of the invention in ameliorating tension and / or anxiety in a subject.

[0100] Furthermore, the composition of the present invention can be used as a food supplement. Accordingly, another aspect of the present invention relates to the use of the composition of the present invention as a food supplement.

[0101] The term "dietary supplement" is defined above and the specific embodiments associated with that definition apply here as well.

[0102] Throughout the specification and claims, the word "comprises" and variations thereof are not intended to exclude other technical features, additional objects, components or steps.

[0103] Description of the drawings Figure 1. Body weights (mean ± SEM) of animals in each experimental group at the start of the experiment (t = 0).

[0104] Figure 2. Image of the template generated by Noldus Ethovision® used to analyze specimen swimming, with two arenas, top and bottom.

[0105] Figure 3. Animal length and weight (mean ± SEM) at t = 2mpi and t = 4mpi in each experimental group. Asterisks indicate statistically significant differences. * (p>0.050).

[0106] Figure 4. Total swimming distance and fish speed (mean ± SEM) for each experimental group at t = 2 mpi and t = 4 mpi.

[0107] Figure 5. Zone preference expressed as seconds spent in the upper zone of the aquarium (mean ± SEM) and normalized percentage during NTT recordings (mean ± SEM) for each experimental group at t = 2 mpi and t = 4 mpi.

[0108] Figure 6. Percentage of fish (dark dots) that spent less than 30 s in the upper zone during the NTT (A) 2 months (2 mpi) and (B) 4 months (4 mpi) after the start of the experiment.

[0109] Figure 7. Latency (seconds) to first entry into the upper zone. Asterisks indicate fish that spent all of their NTT in the upper area of ​​the tank. Hashes indicate fish that spent all of their NTT in the lower area of ​​the tank.

[0110] Figure 8. Merged heatmap of all trajectories for each experimental group at 2 mpi sampling.

[0111] Figure 9. Merged heatmaps of all trajectories for each experimental group at 4 mpi sampling.

[0112] Figure 10. Reaction times of C. elegans to octanol shown in control condition (OP50 added), control anxiety condition (food deprivation), and anxiety nematodes treated with a mixture of heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1).

[0113] Figure 11. Analysis of dispersal by quantifying the distribution of nematodes on an agar plate. Nematodes were stressed (cholinergic stimulants) and treated with a mixture of heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1). The different grey scales indicate the difference in nematode position (cm) from the center of the plate.

[0114] Figure 12. Effects of blend HT-ES1+HT-BPL15 (10 8 Cells) and other blends formulated with alternative strains (BPL33, BPLA3, BPL31, BPL8). One-way ANOVA was applied. Data are the mean of two independent experiments. ** p<0.01.

[0115] [Example] Example 1. Effects of oral administration of six different probiotic / postbiotic supplements on adult zebrafish behavior material and method Six different probiotic supplements in a novel tank test (NTT) * To evaluate the effect of oral administration of on adult Danio rerio (zebrafish). The swimming patterns of the fish will be analyzed at the individual level, and the results and conclusions will be compiled in a report.

[0116] * Blended preparation 004009 HT (heat treated) blend formulation 004009 Bifidobacterium longum ES1(CECT 7347) HT Bifidobacterium longum ES1 Lactobacillus rhamnosus BPL15(CECT 8361) HT Lactobacillus rhamnosus BPL15 All procedures described in this report were approved by the Institutional Animal Care and Use Committee of the University of Leon (Spain). All animals were handled standardly in accordance with the Spanish regulations for the use of laboratory animals (RD / 2013) and in accordance with the European Union Council Guidelines (2010 / 63 / EU).

[0117] A batch of 70 4-month post-fertilization (mpf) zebrafish (AB strain) sibling fish was homogenously divided into seven groups (n = 10) taking into account the fish body weight (Figure 1, Table 1).

[0118] [Table 2]

[0119] Each fish was anesthetized with 110 mg / L buffered tricaine methanesulfonate (MS222) and individually tagged with a visible implant elastomer (Northwest Marine Technology, WA, USA) for individual identification and animal tracking. Zebrafish were housed in 3 L aquaria with constant water exchange from a recirculating system equipped with mechanical, chemical, and biological filters. Water was kept at an average temperature of 26°C, and the room photoperiod was a 14 / 10 light / dark cycle.

[0120] Probiotic and / or postbiotic supplements and placebo were compared for 10 9The animals were provided in capsules containing cfu or cells. The capsule contents were divided into two portions and, to ensure intake, each dose (half of the contents) was given to the experimental males in their water 30 min before each scheduled feeding (twice daily). This feeding condition was maintained for the duration of all experiments (4 months). Technical staff in charge of maintenance, feeding and animal welfare were blinded to the capsule contents. The experimental groups were blinded and numbered from 1 to 7.

[0121] To study the exploratory behavior of animals, we used the novel tank test (NTT), which opposes "safe" diving behavior with "exploratory" swimming behavior and therefore allows to investigate the strength of the gravitactic escape diving instinct of fish under specific experimental conditions. Under normal controlled laboratory conditions, fish generally tend to spend more time at the bottom of the tank during the first few minutes of monitoring, exhibiting a higher rate of erratic movements and immobility phenomena. After an acclimatization period, fish gradually begin to explore the upper part of the water column.

[0122] The protocol was performed twice, 2 and 4 months post-initiation (mpi), as follows: each animal was individually placed in the bottom of a rectangular glass tank (20 × 8 × 18 cm, length × width × depth, swimming volume: 3.5 L). Fish were filmed for 6 min (1920 × 1080 px). The first minute was for habituation to the new environment, and the remaining 5 min were for behavioral analysis. Individual swimming behavior was analyzed using Noldus Ethovision® tracking software, which generated a virtual grid that divided the tank into two arenas (upper and lower; Figure 2).

[0123] For each animal, the following estimates of exploratory behavior were quantified: Distance traveled. · Speed. Zone preferences. · Waiting time before first entry into the upper zone.

[0124] Results are expressed as mean ± SEM. Percentage data were normalized using an arcsine square root transformation. Statistical differences between the mean values ​​of each variable at 2 and 4 mpi were determined using a one-tailed unpaired t-Student test (normally distributed variables) or a one-tailed Mann-Whitney test (non-parametric variables). All statistical analyses were performed using Prism 9 (GraphPad Software, San Diego, CA, USA). A P value < 0.0500 was considered statistically significant. Postbiotic experiments were performed using the Danio rerio model of anxiety. This model is based on the preference of zebrafish to swim close to the bottom when first placed in a novel aquarium. Differences in the time it takes for fish to leave the bottom of the aquarium and start exploring elsewhere may reflect the anxiety experienced by the fish. Parameters related to swimming pattern, mean swimming speed, and distribution within the aquarium were analyzed.

[0125] biometrics The effect of probiotic and postbiotic intake on biometric parameters is shown in Figure 3. Fish body length was uniform at 2 mpi. At 4 mpi, mean values ​​were higher in all postbiotic-fed animals except for HT L. rhamnosus BPL15, which was similar to placebo, but no statistically significant differences were reported (Table 2).

[0126] [Table 3]

[0127] Statistical analysis showed that HT B. longum ES1 group enhanced body weight gain at 2 mpi (p = 0.0402) and 4 mpi (p = 0.0140) compared to the placebo group. Similar to body length, the remaining experimental groups also showed non-significantly higher mean values ​​compared to the control group at 4 mpi, except for HT L. rhamnosus BPL15 (Table 3).

[0128] [Table 4]

[0129] dynamics The dynamics of the studied fish showed similar results between the groups (Figure 4). Interestingly, the total distance swam by the fish was greater in the 2-month sampling compared to the 4-month sampling, ranging from approximately 1800 to 2100 cm (Table 4). The greatest distances were observed in the groups supplemented with Blend 004009 and HT Blend 004009 in both samplings.

[0130] [Table 5]

[0131] Similar to the distance data, analysis of fish speed revealed no statistically significant differences between groups. Correlatively, the highest values ​​of this parameter were in the group of fish supplemented with the blend (Table 5).

[0132] [Table 6]

[0133] Zone Preference Given the previous results about the similarities between groups in speed and distance traveled, the following data are even more interesting.

[0134] Zonal preferences of fish showed statistically significant preferences in several experimental groups examined (Figs. 5, 6, 7).

[0135] As can be seen in the graphs (both in terms of total seconds spent in the upper zone and normalized percentage), the data from the two-month sampling is indeed promising. Four of the groups fed probiotics or postbiotics showed significant differences compared to the control group: Blend formulation 004009|p=0.0112 HT blend formulation 004009|p=0.0024 B. longum ES1|p=0.0165 · L. rhamnosus BPL15|p=0.0112 At 2 mpi, the HT blend formulation 004009 group showed the best results with a mean upper zone value of 106.2 ± 14.98 seconds compared to 34.64 ± 12.48 seconds reported in the placebo group (Figure 8, Table 6).

[0136] [Table 7]

[0137] At 4 months, statistically significant differences were only observed for the HT blend 004009 group (p=0.0423) and the B. longum ES1 group (p=0.0429). These two experimental groups contained the highest percentage of fish in the upper zone of the tank at sampling (Figure 8, Table 7).

[0138] [Table 8]

[0139] [Table 9]

[0140] Zone preference data were analyzed by focusing on the number of animals spending little or no time in the upper part of the tank, with a threshold of 30 s (10%) of the total NTT (Table 8).

[0141] [Table 10]

[0142] After two months of probiotic or postbiotic intake, the lowest percentage of animals exhibiting this type of behavior was in the Blend 004009 and HT Blend 004009 groups, whereas the highest percentage of these individuals was in the placebo group.

[0143] Four months after the start of the experiment, these two groups maintained this trend, although the proportions of B. longum ES1 and L. rhamnosus BPL15 had decreased compared to the previous sampling (Fig. 9).

[0144] Waiting time before first entry into the upper zone Typically, fish introduced into a new tank will initially prefer to live on the bottom of the tank while they get used to their new environment.

[0145] After 1 min of acclimation, the latency of animals in each experimental group was quantified. As can be seen in Figure 9, in the 2-month sampling, 4 fish in the placebo group did not enter the upper zone, and 2 of them had a latency delay of more than 2 min to rise. In the other groups, especially Blend 004009, HT Blend 004009 and B. longum ES1, the latency of fish to first enter was generally lower.

[0146] After 4 months of probiotic or postbiotic supplementation, the trend was abating in the placebo group, but the aforementioned groups continued to show low values, especially in the HT blend formulation 004009, where up to four fish had already entered the upper zone at the start of recording after 1 min.

[0147] Example 2. C. elegans experiments C. elegans has become a suitable model for studying longevity, obesity, and metabolic syndrome. In addition, it has been used as a simple model to analyze many behaviors that reveal cognitive dysfunction (learning and memory, AD, PD) and to model aspects of various psychiatric disorders (Dwyer DS. Crossing the Worm-Brain Barrier by Using Caenorhabditis elegans to Explore Fundamentals of Human Psychiatric Illness. Mol Neuropsychiatry. 2018, 3:170-179). In C. elegans, neurotransmitters and neuropeptides are similar to those in the mammalian nervous system (e.g., dopamine, GABA, acetylcholine, serotonin), and worms exhibit remarkable behavioral plasticity similar to mammalian learning and memory.

[0148] Basic functions such as growth, feeding, and movement are controlled by the nervous system.

[0149] Here, a heat-treated postbiotic mixture composed of B. longum CECT7347 (ES1) and L. rhamnosus CECT8361 (BPL15) was evaluated in the C. elegans anxiety and stress model. The mixture was administered at a final concentration of 1 × 10 8 Cells / plate were assessed (0.5 × 10 8 Cells / plate HT-BPL15+0.5×10 8 cells / plate HT-ES1).

[0150] Anxiety model The perception of stress and aversive stimuli is the initial component of anxiety behavior in humans, which in turn is controlled by the amygdala, the HPA (hypothalamic-pituitary-adrenal) axis, and neuromodulators (eg, serotonin).

[0151] The serotonin pathway underlies aversive behavior in C. elegans and mediates avoidance of the repellent odorant octanol, and we therefore used avoidance behavior as an anxiety-related behavior in C. elegans.

[0152] Anxiety assay was performed by avoidance behavior assay. Experiments were performed using the "smell-on-a-stick" assay as previously described (Chao, MY, et al. 2004, Proc Natl Acad Sci US A. 26;101(43):15512-7. doi: 10.1073 / pnas.0403369101. Epub 2004 Oct 18. PMID: 15492222; PMCID: PMC524441). "Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit." Proceedings of the National Academy of Sciences of the United States of America 101: 15512-15517). For the octanol avoidance assay, the blunt end of a Loew-Cornell (Teaneck, NJ) 9000 Kolinsky 7 paintbrush was taped to a Pasteur pipette, dipped in freshly prepared 30% octanol, and placed in front of a moving nematode. The latency for the nematode to move backwards (octanol avoidance) was measured using a laboratory timer.

[0153] In the anxiety condition, worms were cultured on NGM plates without OP50. This food deprivation caused increased anxiety due to a decrease in serotonin levels (Chao, MY, et al. 2004, Proc Natl Acad Sci US A. 26;101(43):15512-7. doi: 10.1073 / pnas.0403369101. Epub 2004 Oct 18. PMID: 15492222; PMCID: PMC524441). This anxiety condition induced an increase in the time required for worms to move backwards in response to an octanol stimulus.

[0154] Figure 10 shows the time it took for nematodes to start moving backwards when they smelled octanol. As mentioned earlier, nematodes in the food-deprived anxious control took longer to avoid octanol than nematodes in the control condition with added OP50. Nematodes cultured with a mixture of heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1) significantly reduced the avoidance time under food deprivation (p-value < 0.05). Thus, the mixture of heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1) suppressed anxiety-related behaviors due to food deprivation.

[0155] Stress Model C. elegans locomotion is characterized as a sinusoidal wave of muscle contractions and is mediated by acetylcholine, an excitatory neurotransmitter.

[0156] In this experiment, to analyze the exploratory behavior of nematodes under stress conditions, we provided cholinergic stimulation using the drug Denubil and assessed how the heat-treatment mixtures heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1) affect this behavioral pattern.

[0157] The plate was divided into three concentric regions: <0.9 cm, 0.9–1.8 cm, and 1.8–2.8 cm. Worms were placed in the center of the plate and their position was scored after 2 min at each location.

[0158] Figure 11 shows the distribution of nematodes in the feeding conditions, including both control (unstressed) and stressed populations. Under control conditions, nematodes tend to congregate in the center of the plate, whereas when stressed with psychostimulant drugs, nematodes tend to disperse to the edges of the plate. Furthermore, nematodes fed mixed heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1) recovered the behavioral pattern observed in control-unstressed (p-value < 0.001) and again congregated in the center of the plate (did not disperse).

[0159] Example 3. Comparative assay between different blends of heat-treated L. rhamnosus and B. longum strains Lactobacillus rhamnosus and Bifidobacterium longum strains Two strains belonging to the species Bifidobacterium longum (BPL33 and BPL31) and two belonging to the species Lactobacillus rhamnosus (BPLA3 and BPL8) were obtained from the applicant's Biopolis, SL collection and were evaluated in the anxiety C. elegans model. Both strains were evaluated in heat-treated form.

[0160] Four different combinations were tested: · B. longum HT-BPL33 + L. rhamnosus HT-BPLA3 · B. longum HT-BPL33+ L. rhamnosus HT-BPL8 · B. longum HT-BPL31 + L. rhamnosus HT-BPLA3 · B. longum HT-BPL31+ L. rhamnosus HT-BPL8 For comparative analysis, an additional condition was added in which the anxiety blend of the present invention, B. longum HT-ES1 + L. rhamnosus HT-BPL15, was added.

[0161] Anxiety model in C. elegans Octanol avoidance was used as an anxiety-related behavioral assay in C. elegans. Experiments were performed using the "smell-on-a-stick" assay. In the octanol avoidance assay, the blunt end of a hair (Loew-Cornell (Teaneck, NJ) 9000 Kolinsky 7 paintbrush) taped to a Pasteur pipette was dipped into freshly prepared 30% octanol in EtOH (vol / vol) and placed in front of a moving nematode. The time it took for the nematode to move backwards was measured. In the control condition, nematodes were reared in NGM supplemented with E. coli OP50. The nematodes were transferred to an NGM plate seeded with 100 μL of fresh overnight E. coli OP50. The nematodes were incubated on this plate for 20 min before testing. In the anxiety condition, the nematodes were incubated on an NGM plate without OP50 for 20 min. This food deprivation induced anxiety-related behavior by decreasing serotonin levels. To evaluate the treatments, wild-type worms were cultured with a blend of the corresponding two heat-treated strains for 10 min. 8 Age-synchronized nematodes were placed on NGM plates containing cells (final dose). At the young adult stage, nematodes were transferred to NGM plates containing each treatment but were not allowed to feed for 30 min. Then, nematodes were transferred to NGM plates containing no OP50 or each treatment and tested after 20 min. The percentage of time to react to octanol compared to the control is shown.

[0162] result Different blends of alternative B. longum and L. rhamnosus strains were evaluated using the anxiety-related behavior, octanol avoidance, in C. elegans.

[0163] In this assay, anxious control nematodes take longer to avoid octanol than control condition nematodes (NGM, non-anxious control). Results represent the relative percentage of each treatment to the anxious control population (Figure 12).

[0164] As a result, the blend of heat-treated strains using ES1 and BPL15 (the composition of the present invention) showed a high effect of reducing anxiety behavior, shortening the reaction time by 34% when octanol was presented to the nematodes, while the other four blends only reduced the reaction time by 8 to 14% (Figure 12 and Table 9).

[0165] [Table 11]

[0166] Thus, these results demonstrate that the blend of strains of the present invention has surprisingly superior efficacy to other blends of strains of these species. [Brief description of the drawings]

[0167] [Figure 1] Body weights (mean ± SEM) of animals in each experimental group at the start of the experiment (t = 0). [Diagram 2] An image of the template generated by Noldus Ethovision® used to analyze the specimen's swimming, with two arenas, top and bottom. [Diagram 3] Animal length and weight (mean ± SEM) at t = 2mpi and t = 4mpi in each experimental group. Asterisks indicate statistically significant differences * (p > 0.050). [Figure 4] Total swimming distance and fish speed (mean ± SEM) for each experimental group at t = 2 mpi and t = 4 mpi. [Diagram 5] Zone preference expressed as seconds spent in the upper zone of the aquarium (mean ± SEM) and normalized percentage during NTT recordings (mean ± SEM) for each experimental group at t = 2 mpi and t = 4 mpi. [Figure 6]Percentage of fish (dark dots) that spent less than 30 s in the upper zone during the NTT (A) 2 months (2 mpi) and (B) 4 months (4 mpi) after the start of the experiment. [Figure 7] Latency (seconds) before first entry into the upper zone. An asterisk indicates that the fish spent all of its NTT in the upper area of ​​the tank. A hash indicates that the fish spent all of its NTT in the lower area of ​​the tank. [Figure 8] Merged heatmap of all trajectories for each experimental group at 2 mpi sampling. [Figure 9] Merged heatmap of all trajectories for each experimental group at 4 mpi sampling. [Figure 10] Reaction times of C. elegans to octanol are shown for control conditions (OP50 added), control anxiety conditions (food deprivation), and anxiety nematodes treated with a mixture of heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1). [Figure 11] Analysis of dispersal by quantifying the distribution of nematodes on an agar plate. Nematodes were stressed (cholinergic stimulant) and treated with a mixture of heat-treated L. rhamnosus CECT8361 and heat-treated B. longum CECT7347 (HT-BPL15+HT-ES1). The different grey scales indicate the difference in nematode position (cm) from the center of the plate. [Figure 12] Effect of the blend HT-ES1+HT-BPL15 (108 cells) and other blends formulated with alternative strains (BPL33, BPLA3, BPL31, BPL8) on avoidance behavior (anxiety) in C. elegans. One-way ANOVA was applied. Data are the mean of two independent experiments. **p<0.01.

Claims

1. A postbiotic composition comprising non-viable Bifidobacterium longum strain CECT 7347 and non-viable Lactobacillus rhamnosus strain CECT 8361.

2. 10. The postbiotic composition of claim 1, wherein the non-viable Bifidobacterium longum and Lactobacillus rhamnosus strains are heat-treated.

3. The postbiotic composition of claim 1 or 2, wherein the postbiotic composition is a pharmaceutical composition or a nutritional composition.

4. The postbiotic composition of claim 3 , wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

5. The postbiotic composition of claim 3 , wherein the composition is formulated in a liquid or solid form.

6. 6. The postbiotic composition of claim 5, wherein the solid formulation is selected from the group consisting of tablets, lozenges, confectioneries, chewable tablets, chewing gum, capsules, sachets, powders, gels, granules, coated particles or coated tablets, tablets and gastro-resistant tablets and capsules, and dispersible strips and films.

7. 6. The postbiotic composition of claim 5, wherein the liquid formulation is selected from the group consisting of oral solutions, suspensions, drops, emulsions and syrups.

8. 4. The postbiotic composition of claim 3, wherein the nutritional composition is a food product or a nutritional supplement.

9. 9. The postbiotic composition of claim 8, wherein the food product 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, dairy-based products, meat products and beverages.

10. The total concentration of B. longum and L. rhamnosus in the composition is 10 ３ From 10 １２ The postbiotic composition of claim 1 or 2, which is intercellular.

11. 3. A postbiotic composition according to claim 1 or 2 for use as a pharmaceutical.

12. 3. A postbiotic composition according to claim 1 or 2 for use in the treatment and / or prevention of anxiety disorders.

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