Probiotic composition for use as an antioxidant

Abstract

The present invention relates to a probiotic composition consisting of Lactobacillus rhamnosus, Lactobacillus casei and Bifidobacterium longum, and its use as an antioxidant, preferably the strains Lactobacillus rhamnosus CECT 8361, Lactobacillus casei CECT 9104 and Bifidobacterium longum CECT 7347. The composition is especially useful for the treatment and / or prevention of damage caused by oxidative stress at the molecular level, preferably during high intensity physical exercise.

Description

TECHNICAL FIELD

[0001] The present invention is in the field of medicine, preferably sports medicine and probiotic nutritional supplements with antioxidant effects. In particular, the present invention comprises in a probiotic pharmaceutical composition aimed at reducing oxidative stress, preferably induced by intense physical exercise. BACKGROUND

[0002] Reactive oxygen species (ROS), such as superoxide anion (O2"), hydrogen peroxide (H2O2) and hydroxyl radical (HO·), are free radical and non-radical oxygen species resulting from the partial reduction of oxygen. Intracellular ROS are endogenously produced in the process of mitochondrial oxidative phosphorylation or can come from interactions with exogenous agents, such as exogenous compounds. Oxidative stress occurs when ROS overwhelm the antioxidant cellular defense system, either by increasing their concentration or due to a decrease in cellular antioxidant capacity. This reaction damages nucleic acids, proteins and lipids. It is also involved in various pathological processes, such as carcinogenesis, neurodegeneration, atherosclerosis, diabetes and aging. In fact, there are many diseases associated with oxidative stress and the production of free radicals. Therefore, antioxidant therapies and antioxidant-rich foods help prevent or at least reduce the degradation of organic functions caused by excessive oxidative stress.

[0003] Fortunately, the human body has developed many enzymatic and non-enzymatic defense mechanisms to counteract the harmful effects of ROS. Thus, antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), play an important role in preventing the damage caused by free radicals to the organism.

[0004] Physical exercise, when performed regularly, has many health benefits, including reducing mortality and the risk of cardiovascular disease, cancer and diabetes. However, long and intense muscle-skeletal contractions generate free radicals and cause oxidative damage to cellular components. Thus, the induction of oxidative stress during physical exercise is considered a cause of damage to the myocardial cell membrane, leading to an exacerbated inflammatory response and, consequently, excessive post-exercise pain and muscle fatigue.

[0005] The World Health Organization defines probiotics as live microorganisms that, when administered in adequate amounts, have a beneficial effect on health. In recent years, the number of in vitro and in vivo studies related to the antioxidant properties of probiotics has significantly increased (Kleniewska, P., et al., 2016, Oxidative Medicine and Cellular Longevity, doi:10.1155 / 2016 / 1340903; Poljsak, B., 2011, Oxidative Medicine and Cellular Longevity, doi:10.1155 / 2011 / 194586; Hybertson, B. M., et al., 2011, Mol. Aspects Med., 32, 234-246; Banegas JR, et al., 2006, Rev Esp Cardiol., 6:3-12).

[0006] One example is the study described by Ali Akbar Mohammadi, et al. (Ali Akbar Mohammadi, et al., 2015 Int J Prev Med., 6:82) which analyzed and proved the antioxidant and anti-inflammatory effects of probiotic capsules, taken as a nutritional supplement, 1 capsule per day for 6 weeks, in workers of the petrochemical industry. Said capsules were composed of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Bifidobacterium breve, Bifidobacterium longum and Streptococcus thermophilus. The results of this study concluded that the probiotic capsules had a beneficial effect on oxidative stress biomarkers.

[0007] A specific strain of Lactobacillus casei described in CN101333505 is also proposed as an antioxidant product.

[0008] Moreover, the strain Bifidobacterium longum CECT7347 and Lactobacillus casei CECT9104 in combination with B. animalis subsp. Lactis (as part of a probiotic composition) have been described for the treatment and / or prevention of atopic dermatitis (EP3272396).

[0009] Another example of a probiotic composition comprising the strain B. animalis subsp. lactis together with the strains Bifidobacterium longum CECT7347 and Lactobacillus rhamnosus CECT8361 has been described in document EP3222282, wherein it is proposed for the treatment and / or prevention of psoriasis.

[0010] Finally, document EP3241893 describes a formulation comprising the strains Bifidobacterium longum CECT7347 and Lactobacillus rhamnosus CECT8361 for improving male fertility.

[0011] In short, it is desirable to have a probiotic ingredient that replaces the existing probiotic ingredients, with antioxidant properties, capable of reducing the harmful effects caused by oxidative stress at the molecular level, especially after intense exercise. SUMMARY

[0012] The present application relates to a probiotic composition consisting of Lactobacillus rhamnosus, Lactobacillus casei and Bifidobacterium longum, preferably the strains Lactobacillus rhamnosus CECT8361, Lactobacillus casei CECT9104 and Bifidobacterium longum CECT7347, together with a pharmaceutically acceptable carrier and / or excipient, for use as an antioxidant. The composition is particularly useful at the molecular level for the treatment and / or prevention of damage caused by oxidative stress during or after physical exercise.

[0013] The strains Lactobacillus rhamnosus CECT8361, Lactobacillus casei CECT9104 and Bifidobacterium longum CECT7347 were isolated from the faeces of a Spanish infant under 3 months of age, fed exclusively with breast milk. In all cases, the strains were isolated in selective culture media for lactic acid bacteria and bifidobacteria and were unequivocally identified by 16S rRNA gene sequencing.

[0014] The examples shown below demonstrate that the intake of the composition of the present application by humans, preferably daily and for six consecutive weeks, reduces the oxidative damage to lipids and DNA caused by high intensity and long duration physical exercise. Specifically, in the subjects who consumed the composition of the present application, the increase in the levels of serum malondialdehyde and oxidized LDL (indicative of oxidative lipid damage) and urinary 8-oxo-2-deoxyguanosine (indicative of oxidative DNA damage) was less. This indicates that the intake of the composition of the present application improves the antioxidant status of the subjects. Finally, these examples show that the administration of the composition of the present application is safe, given that no adverse effects related to the intake of the composition of the present application were observed during the clinical trial, nor were changes in the blood cell count or liver and kidney function of the study subjects observed.

[0015] Thus, one aspect of the present application relates to a composition, hereinafter referred to as "the composition of the present application", consisting of the bacteria Lactobacillus rhamnosus, Lactobacillus casei and Bifidobacterium longum, together with one or more carriers and / or additives and / or pharmaceutically acceptable carriers.

[0016] The composition of the present application is a probiotic composition. By "probiotic composition" it is meant a composition comprising at least one live microorganism or a fraction thereof which, when ingested, interacts with the metabolism of an individual and produces a beneficial effect on the individual.

[0017] In a preferred embodiment of the composition of the application, Lactobacillus rhamnosus is strain BPL0015 deposited in the Spanish Type Culture Collection with the accession number CECT 8361 ; Lactobacillus casei is strain BPL0004 deposited in the Spanish Type Culture Collection with the accession number CECT 9104; and Bifidobacterium longum is strain IATA-ES1 deposited in the Spanish Type Culture Collection with the accession number CECT 7347.

[0018] Lactobacillus rhamnosus is a bacterium that is mainly found in fermented products (dairy and plant-based) and in infant milk. The scientific classification of Lactobacillus rhamnosus is: Kingdom: Bacteria, Phylum: Firmicutes, Class: Bacilli, Order: Lactobacillales, Family: Lactobacillaceae, Genus: Lactobacillus, Species: Lactobacillus rhamnosus.

[0019] Strain Lactobacillus rhamnosus CECT 8361 was isolated from the feces of a healthy child under three months of age, exclusively breastfed. This strain was deposited in the Spanish Type Culture Collection (headquartered at Edificio 3CUE, Parc Científic Universitat de Valencia, C / Catedrático Agustín Escardino, 9, 46980 Paterna (Valencia), Spain) on May 27, 2013, as an international deposit authority under the Budapest Treaty. The assigned accession number is CECT 8361. In the present application, this strain will also be referred to as BPL0015.

[0020] Lactobacillus casei is a bacterium that is mainly found in fermented products (dairy and plant-based) and in infant milk. The scientific classification of Lactobacillus casei is: Kingdom: Bacteria, Phylum: Firmicutes, Class: Bacilli, Order: Lactobacillales, Family: Lactobacillaceae, Genus: Lactobacillus, Species: Lactobacillus casei.

[0021] Strain Lactobacillus casei CECT 9104 was isolated from the feces of a healthy child under three months of age, exclusively breastfed. This strain was deposited in the Spanish Type Culture Collection (headquartered at Edificio 3CUE, Parc Científic Universitat de Valencia, C / Catedrático Agustín Escardino, 9, 46980 Paterna (Valencia), Spain) on February 25, 2016, as an international deposit authority under the Budapest Treaty. The assigned accession number is CECT 9104. In the present application, this strain will also be referred to as BPL0004.

[0022] Bifidobacterium longum is a gram-positive, catalase-negative, double- shaped bacterium, commonly found in the gastrointestinal tract, where it mainly produces acetic and lactic acid. The scientific classification of Bifidobacterium longum is: Kingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Order: Bifidobacteriales, Family: Bifidobacteriaceae, Genus: Bifidobacterium, Species: Bifidobacterium longum.

[0023] Strain Bifidobacterium longum CECT 7347 was isolated from the feces of a healthy child under three months of age, exclusively breastfed, and deposited as an International Depository Authority under the Budapest Treaty on December 20, 2007, at the Spanish Type Culture Collection (headquartered at Edificio 3CUE, Parc Científic Universitat de València, C / Catedrático Agustín Escardino, 9, 46980 Paterna (Valencia), Spain). The assigned deposit number is CECT 7347. In the present invention, this strain will also be referred to as IATA-ES1 or ES1.

[0024] Another aspect relates to a composition comprising strain Lactobacillus rhamnosus BPL0015 deposited at the Spanish Type Culture Collection under accession number CECT 8361, strain Lactobacillus casei BPL0004 deposited at the Spanish Type Culture Collection under accession number CECT 9104 and Bifidobacterium longum IATA-ES1 deposited at the Spanish Type Culture Collection under accession number CECT 7347. In a preferred embodiment, the composition further comprises one or more pharmaceutically acceptable carriers and / or excipients. In a further preferred embodiment, the composition further comprises additional microorganisms, preferably additional bacteria. The additional bacteria can belong to the above-mentioned bacterial genera (Bifidobacterium and Lactobacillus), as well as other bacterial genera such as, but not limited to, Bacillus, Lactococcus, Pediococcus, Streptococcus or Veillonella, among others; and yeast species belonging to the genera Kluyveromyces, Pichia or Saccharomyces, among others. In a further preferred embodiment, the composition further comprises one or more active substances with antioxidant effect such as, but not limited to, fatty acids, glutathione, plant extracts (especially those rich in polyphenols such as anthocyanins, carotenoids, curcumin, lycopene, lutein, melatonin, resveratrol or zeaxanthin), peptides, selenium, vitamins (such as A, C or E), among others.

[0025] In the present application, it is also contemplated the bacteria from Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus casei (or their corresponding strains Lactobacillus rhamnosus BPL0015 CECT8361, Lactobacillus casei BPL0004 CECT9104 and Bifidobacterium longum IATA-ES1 CECT7347) and these bacteria can constitute part of the probiotic composition of the application as an alternative to the specific indicated strains, provided that they retain the ability to prevent, alleviate and / or improve the damage caused by oxidative stress in an organism. Examples of strains derived from the strains specifically mentioned in the present application can be mutants and transgenic organisms, which have variations in their genome compared to the genome of the strains described in the present application, but these variations do not affect the ability of the strain to prevent, alleviate and / or improve the damage caused by oxidative stress in an organism. The strains from Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus casei (or their corresponding strains Lactobacillus rhamnosus BPL0015 CECT8361, Lactobacillus casei BPL0004 CECT9104 and Bifidobacterium longum IATA-ES1 CECT7347) can occur naturally or intentionally by mutagenesis methods known in the state of the art, such as, but not limited to, growth of the original strain in the presence of mutagens or stress agents, or by genetic engineering aimed at obtaining the desired mutation. Transgenic organisms from Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus casei (or their corresponding strains Lactobacillus rhamnosus BPL0015 CECT8361, Lactobacillus casei BPL0004 CECT9104 and Bifidobacterium longum IATA-ES1 CECT7347) are also contemplated, which maintain the ability to prevent, alleviate and / or improve the damage caused by oxidative stress in the body and therefore can be used to treat and / or prevent oxidative stress. A method for detecting whether a microorganism has the ability to prevent, alleviate and / or improve the damage caused by oxidative stress in the body is described in the example attached to this description.

[0026] In addition, the present application also encompasses cellular components, metabolites and / or molecules secreted by Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus casei (or their corresponding strains Lactobacillus rhamnosus BPL0015 CECT8361, Lactobacillus casei BPL0004 CECT9104 and Bifidobacterium longum IATA-ES1 CECT7347), as well as compositions comprising said cellular components, metabolites and / or secreted molecules, and their use for the treatment and / or prevention of oxidative stress. "Cellular components" can include cell wall components (e.g. peptidoglycans), nucleic acids, membrane components or other components (e.g. proteins, lipids and carbohydrates) and combinations thereof (e.g. lipoproteins, glycolipids or glycoproteins). "Metabolites" include any molecule produced or modified by the bacteria as a result of its metabolic activity during growth, for use in a process or product (composition of the application) 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 and nucleic acids. "Secreted molecules" include any molecule secreted or released by the bacteria during growth, for use in a process (e.g. pharmaceutical processing) or product storage (composition of the application). 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, minerals, salts and nucleic acids.

[0027] The term "excipient" refers to a substance that aids the absorption of any of the ingredients contained in the composition of the application (i.e. the strains of the application), or that aids in the stabilization of said ingredients and / or that aids in the preparation of the composition, as it provides consistency or flavour, making it more palatable. Thus, excipients can act to bind ingredients (e.g. starches, sugars or cellulose), sweeten, colour, protect active ingredients (e.g. isolate them from air and / or moisture), form the content of a pill, capsule or any other dosage form or break it down to facilitate the dissolution of the ingredients, without excluding other types of excipients not mentioned in this paragraph. Thus, the term "excipient" is defined as a material added to the active ingredients to facilitate their manufacture and stability, to modify their organoleptic properties and / or to determine the physicochemical properties of the composition and its bioavailability. A "pharmaceutically acceptable" excipient must not hinder the activity of the active ingredients of the composition, i.e. it must be compatible with the activity and functionality of the strains of the application.

[0028] A "vehicle" or "carrier" is "preferably an inert substance. The function of the carrier is to facilitate incorporation of other ingredients or compounds, facilitate dosing and / or administration and / or to give consistency and shape to the composition. Thus, a carrier is a substance used to dilute to a certain volume or weight any of the ingredients contained in the composition of the application; or, even if it does not dilute these ingredients, it is able to facilitate dosing and / or administration and / or to give consistency and shape to the composition. When the dosage form is liquid, the carrier is a diluent. Examples of pharmacologically acceptable carriers include, but are not limited to, water, saline solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, starch, amylose, magnesium stearate, talc, surfactants, silicic acid, mucilage, stearic acid, essential oils, fatty acid mono- and di-glycerides, petroleum tetra-fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and similar substances.

[0029] In addition, the excipients and carriers must be pharmacologically acceptable, i.e. they must be evaluated and approved so that they do not harm the subject who ingests the composition of the application. In addition, the excipients and / or carriers can be natural, i.e. they exist in nature, or non-natural, if they are not found in nature in combination with the bacteria of the application.

[0030] The bacteria Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus casei, preferably the strains Lactobacillus rhamnosus BPL0015 CECT 8361, Lactobacillus casei BPL0004 CECT 9104 and Bifidobacterium longum IATA-ES1 CECT 7347, must be present in the composition of the application in a therapeutically effective amount, so that it exerts its effect of preventing, alleviating and / or improving the damage caused by oxidative stress in the organism.

[0031] In the present application, "therapeutically effective amount" means the amount sufficient to produce the desired effect when administered to a subject. As known by the person skilled in the art, the therapeutically effective amount can vary depending on factors such as the age, weight, general health condition, diet and sex of the subject, as well as the mode and time of administration or excretion rate. Thus, in a more preferred embodiment of the composition of the application, the concentration of Lactobacillus rhamnosus is 45%, the concentration of Lactobacillus casei is 45% and the concentration of Bifidobacterium longum is 10% compared to the total concentration of bacteria contained in said composition.

[0032] In a further preferred embodiment, the total amount of bacteria in the composition of the application is 10 9 CFU.

[0033] The composition of the application can be formulated for pharmaceutical administration, i.e. to form part of a medicinal product to be administered to a subject (e.g. orally, topically, etc.). Thus, in a further preferred embodiment, the composition of the application is a medicament.

[0034] A "pharmaceutical composition" or "drug" is a set of active ingredients or compounds, consisting at least of the microorganisms Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus casei, preferably Lactobacillus rhamnosus BPL0015 CECT 8361, Lactobacillus casei BPL0004 CECT 9104 and Bifidobacterium longum IATA-ES1 CECT 7347, in any concentration, preferably the above-mentioned concentrations, and, in addition, can comprise one or more ingredients or compounds with certain biological and / or pharmacological activities that can increase, enhance and / or potentiate the activity of the strains contained in the composition of the present application upon administration to a subject. As understood by the person skilled in the art, the other components or compounds must be compatible with the bacteria in the composition of the present application. In the context of the present application, veterinary compositions are also included within the term "pharmaceutical composition".

[0035] The composition of the present application can also be part of a so-called "specific group nutritional supplement", i.e. a supplement that meets specific nutritional needs. In particular, the composition of the present application is preferably for use by individuals who perform intense physical exercise, more preferably on a regular basis.

[0036] In a further preferred embodiment, the composition of the present application is formulated for oral administration.

[0037] The dosage form of the composition of the present application should be suitable for the administration route used. Thus, the composition can be formulated as a solution, suspension, emulsion, syrup or any other clinically permissible dosage form. Given that the preferred administration route is oral, the composition of the present application is preferably presented in solid, semi-solid or liquid form, more preferably solid, for oral administration. Examples of solid formulations include tablets, capsules, powders, granules or granulated products, granules or coated tablets, suppositories, tablets, pills, gels, dispersible films or microspheres. More preferably, the composition of the present application is presented in the form of a capsule.

[0038] Alternatively, a sustained release form can be used to deliver the composition of the present application, including, for example, its encapsulation in liposomes, microvesicles, microparticles or microcapsules, and the like. Suitable sustained release forms, as well as materials and methods for their preparation, are widely known in the state of the art. Thus, the oral form of the composition of the present application can be a sustained release form additionally comprising a coating or matrix. The sustained release coating or matrix includes, but is not limited to, water-insoluble or modified, natural, semi-synthetic or synthetic polymers, proteins, waxes, fats, fatty alcohols, fatty acids, semi-synthetic or synthetic natural plasticizers, or a combination of two or more of the above. The enteric coating can be applied using conventional processes known to experts in the field.

[0039] Another aspect of the present application relates to the use of the composition of the present application as a medicinal product

[0040] The term "drug" as used in the present specification means any substance used to prevent, alleviate, treat, reduce or cure a disease or clinical condition in an animal, preferably a human. In the context of the present invention, the disease or clinical condition is oxidative stress or molecular damage to the body, preferably to lipids and DNA, caused by oxidative stress.

[0041] Another aspect of the present invention relates to the composition of the present invention for use in the treatment and / or prevention of oxidative stress in an individual, or for use in the treatment and / or prevention of molecular damage, preferably lipid and DNA damage, caused by oxidative stress in an individual. Preferably, the oxidative stress is caused by physical activity or physical exercise.

[0042] "Oxidative stress" is a condition caused by an imbalance between the production of reactive oxygen species and the body's ability to repair the damage caused. This imbalance in the normal redox state of the cell produces peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA, resulting in toxic effects. In humans, oxidative stress, as well as so-called reactive oxygen species (ROS), are involved in the main pathogenic mechanisms or consequences of more than a hundred diseases of important clinical and social significance, such as atherosclerosis, Parkinson's disease, myalgic encephalopathy, multiple chemical sensitivity, periodontitis, varicocele, and Alzheimer's disease can also play an important role in aging.

[0043] Oxidative stress is caused by an imbalance between the production of reactive oxygen species and the ability of biological systems to recover intermediates and / or repair damage caused. The impact of oxidative stress depends on the degree of change that occurs, and whether the cell is able to overcome small disturbances and return to its original state. Moderate oxidation can trigger apoptosis, while severe oxidative stress can lead to cell necrosis and even death. One particularly destructive aspect of oxidative stress is the production of reactive oxygen species, which include free radicals and peroxides. Most reactive oxygen species are produced at low levels under normal aerobic metabolic conditions, and the cell damage they cause is constantly repaired. However, at levels of severe oxidative stress caused by necrosis, damage leads to ATP depletion, prevents cell death by controlling apoptosis, and causes cell death by releasing large amounts of cytotoxic compounds into the culture medium.

[0044] As used in the present invention, the term "physical exercise" or "physical activity" refers to any physical movement by skeletal muscles that requires energy expenditure, also including planned, structured, repetitive bodily exertion with the aim of improving or maintaining one or more components of physical fitness, such as sports. This term includes professional as well as recreational or leisure physical exercise. Furthermore, the energy expenditure of daily tasks (work, household care and maintenance, household care, etc.) is often comparable to the energy expenditure after targeted physical exercise. Such duties are therefore also included in the term "physical activity" as used in the present invention. The term includes the strenuous physical wear and tear caused by high intensity daily household chores (high intensity physical labor, household care, etc.). The physical exercise or physical activity referred to in the present invention can be aerobic or anaerobic, preferably aerobic.

[0045] The physical exercise or physical activity referred to in the present invention is strenuous physical exercise, i.e. high intensity and long duration (preferably at least 30 minutes, more preferably at least 60 minutes; or > 6 METs). Intensity reflects the speed at which the activity is performed, or the degree of effort required to perform such exercise or activity. The intensity of different forms of physical activity varies from person to person. The intensity of physical activity depends on the amount of exercise and physical fitness of each individual. METs are used to express physical activity. METs is the ratio between a person's working metabolic rate and resting metabolic rate (1 MET = energy cost of sitting quietly, equivalent to consuming 1 kcal / kg / hour). In comparison to this, the calorie consumption is estimated to be about 3-6 times higher (3-6 METs) when performing a moderate intensity activity, and more than 6 times higher (> 6 METs) when performing a strenuous activity.

[0046] The composition of the present invention can be administered before, during or after physical exercise, preferably before or during, more preferably before.

[0047] Most preferably, the composition of the present invention is administered once a day, preferably at breakfast, even more preferably for 6 weeks.

[0048] In a further preferred embodiment, the composition of the present invention is used for the treatment and / or prevention of a disease or clinical condition associated with or related to oxidative stress, such as, but not limited to, cancer, neurodegenerative diseases, atherosclerosis and diabetes.

[0049] "Prevention" means preventing molecular damage associated with oxidative stress in an individual, preferably lipid and DNA damage, especially when the individual is susceptible to oxidative stress, for example, because he or she regularly performs strenuous physical exercise.

[0050] The term "treat" or "treatment" includes inhibiting or alleviating molecular damage associated with oxidative stress, preferably to lipid and DNA.

[0051] Another aspect of the application relates to the use of the composition of the application as an antioxidant. This use preferably refers to a non-therapeutic use, i.e. a cosmetic use, more preferably for treating and / or preventing aging of a subject.

[0052] The term "subject", "individual" or "organism" used in the present application refers to any animal, preferably a healthy animal, preferably a mammal, more preferably a human, belonging to any species. Examples of subjects include, but are not limited to, animals of commercial interest such as poultry (hens, ostriches, chickens, geese, partridges, etc.), rabbits, hares, domestic animals (dogs, cats, etc.), sheep and goat domestic animals (sheep, goats, etc.), pig domestic animals (wild boars, pigs, etc.), horse domestic animals (horses, ponies, etc.), cattle or bovine domestic animals (oxen, cows, castrated oxen, etc.), game or quarry animals (such as deer, reindeer, etc.), and humans. In particular embodiments, the subject is a mammal, preferably a human of any race, gender or age.

[0053] Throughout the description and claims, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other technical features, additives, components, or steps. The following examples and drawings are provided by way of example and are not intended to limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 Serum malondialdehyde of each group (placebo and probiotic) for each trial and for the initial and final phases of each trial.*p < 0.05 comparison between initial and final phases of each trial.

[0055] Figure 2 Increase in serum malondialdehyde during the physical exercise trial for each group (placebo and probiotic).*p < 0.05 trial comparison.

[0056] Figure 3 Serum oxidized LDL of each group (placebo and probiotic) for each trial and for the initial and final phases of each trial.*p < 0.05 comparison between initial and final phases of each trial.

[0057] Figure 4 Increase in serum oxidized LDL during the physical exercise trial for each group (placebo and probiotic).*p < 0.05 trial comparison.

[0058] Figure 5 8-oxo 2'-deoxyguanosine (pg / ml) in 24-hour urine of each group (placebo and probiotic) for each trial and for the initial and final phases of each trial.*p < 0.05 comparison between initial and final phases of each trial.

[0059] Figure 6: Increase in 8-oxo-2'-deoxyguanosine (pg / ml) in 24h urine during the physical exercise trial in each group (placebo and probiotic). *p<0.05 trial comparison. DETAILED DESCRIPTION

[0060] Below, we will show the present application using a nutritional clinical trial carried out by the inventors, which highlights the effectiveness of the composition of the present application in reducing the molecular damage caused by oxidative stress during high intensity long duration physical exercise.

[0061] Example 1 Clinical trial to determine the effectiveness of the composition of the present application in reducing oxidative stress during high intensity long duration physical exercise compared to placebo, as well as to determine the tolerability and safety of the composition.

[0062] 1.1 Study Design

[0063] A double-blind and single-center, randomized, placebo-controlled clinical trial based on the product consumed (experimental or placebo) was carried out in two parallel study groups, designed to assess the effect of the product on reducing oxidative stress generated by high intensity long duration physical exercise.

[0064] The selected subjects were healthy Caucasian male subjects between 18 and 45 years of age, selected from the general population, who performed between 2 and 4 times per week of aerobic physical exercise. Excluded from the study were subjects with any history of chronic illness, especially digestive, who had undergone abdominal surgery in the three months prior to the study, with a history of bronchial asthma or chronic obstructive pulmonary disease, reactive airway disease, such as bronchial asthma, sinus bradycardia, second or third degree atrioventricular block, significant heart failure or cardiogenic shock, hypersensitivity or poor tolerance to any of the ingredients of the study product, participation in another clinical trial in the three months prior to the study, subjects diagnosed and / or being treated for hypertension, smokers (> 10 cigarettes per day), subjects with a body mass index greater than 35 kg / m 2 (BMI > 30), subjects with a history of drug or alcohol abuse or other substances or other factors that limit their ability to cooperate during the study.

[0065] As a control, a placebo was selected with the same organoleptic characteristics and the same visual appearance as the trial product.

[0066] The characteristics of the study product were as follows:

[0067] - Pharmaceutical form: capsules, trial product and placebo.

[0068] - Content: excipients that in no case modify the pharmacokinetics or pharmacodynamics of the active substance, their addition being only for technical reasons.

[0069] - Route of administration: oral.

[0070] - Dose: 1 capsule / day.

[0071] - Dose regimen: 6 weeks.

[0072] The composition of the application consists of the following mixture:

[0073] - Lactobacillus rhamnosus BPL0015 (CECT 8361) (45%)

[0074] - Lactobacillus casei BPL0004 (CECT 9104) (45%), and

[0075] - Bifidobacterium longum IATA-ES1 (CECT 7347) (10%)

[0076] The final product contains 10 9 CFU / capsule.

[0077] 1.2. Study Protocol

[0078] To demonstrate the proposed objective, the study subjects undergo an oxidative stress model, including a performance of high intensity long duration physical activity (90 minutes). This increases the oxidative stress of the study subjects and shows the efficacy of the product (composition of the application) in improving the oxidative state of the subjects compared to placebo. The proposed oxidative model consists of a preliminary test and two non-maximal stress tests of high and constant intensity, hereinafter referred to as Test 1 and Test 2. Each one is described below.

[0079] - Preliminary test: the aim of this test is to be able to calculate, individually, the intensity at which the subjects (cyclists) should perform the subsequent physical activity, i.e. tests 1 and 2. During the test implementation, the subjects did not ingest any of the products used in the study (probiotics or placebo). This test was performed on a bicycle roller (Technogym Spin Trainer) with electromagnetic resistance, the bicycle of which has a starting load simulating a speed of 12 km / h, with an increase of 2 km / h per minute, maintaining a constant slope of 2%. The cyclist adopts a free way. In order to calculate the intensity of the subsequent physical activity, the subjects underwent ergo- spirometry and electrocardiographic monitoring. Therefore, prior to this, the subjects were prepared to perform a respiratory gas analysis (open circuit, Jaeger Oxicom Pro brand gas analyzer) while performing the test. The main variable evaluated during this test is the maximum / peak consumption of absolute and relative oxygen (VO2 max), i.e. the maximum volume of oxygen measured in the test, in milliliters / minute or milliliters / kilogram x minute, or the maximum value of this variable, after which, even with an increase in the intensity of the effort, it does not increase. Then, after a 7-day washout period from the oxidative stress generated in the preliminary test, the following tests were performed.

[0080] - First stress test (test 1): one week after the first test, the study subjects performed the following high-intensity physical activity for 90 minutes. The study subjects performed a constant intensity stress test on a bicycle roller with electromagnetic resistance where the bicycle was placed. The maximum load maintained corresponded to a heart rate corresponding to 75% of the maximum oxygen consumption of the subjects calculated in the preliminary test, maintaining a constant slope of 2%. The aim of this test was to generate a high oxidative stress in the subjects in order to evaluate the antioxidant action of the test products and the placebo. During the test, the study subjects did not consume any products, only drinking water ad libitum. After the completion of test 1 by the subjects, a 6-week period of product consumption (probiotics or placebo) began.

[0081] - Second stress test (test 2): after the 6-week period of product consumption, the study subjects performed test 2, which included the same high-intensity physical activity performed in test 1.

[0082] Before and after tests 1 and 2, the study subjects performed blood draws and 24-hour urine collections. Blood samples were collected half an hour before the subjects performed tests 1 and 2 and half an hour after each test. Likewise, 24-hour urine collections were performed the day before and the day after each test. After measuring the total amount of urine excreted in 24 hours, a 9 ml sample was extracted and frozen at -80°C in three different cryovials until further analysis.

[0083] 1.3. Analysis of Study Variables

[0084] All variables were analyzed at baseline and after 6 weeks of continuous use of the product.

[0085] 1.3.1. Oxidative damage variables caused by high-intensity, long-duration physical exercise.

[0086] Both aerobic and anaerobic physical exercise increase the production of free radicals. Certain levels of these oxidative compounds have positive effects on the body's immune function, tissue replacement and cellular resistance, and even on muscle contraction and adaptation to systemic movement. However, physical exercise can trigger an imbalance between free radical generation and antioxidant defense mechanisms in the body, leading to different types of molecular damage, which can be demonstrated by different biomarkers of damage to lipids, proteins, and DNA molecules. In this study, subjects were subjected to oxidative stressors (Trial 1 and Trial 2) to assess the antioxidant effects of probiotics versus placebo—specifically, their ability to mitigate oxidative damage induced by high-intensity, prolonged physical exercise, which may outweigh antioxidant defense mechanisms.

[0087] Lipid oxidative damage

[0088] - Serum malondialdehyde (MDA) analysis. Serum MDA was analyzed using the MDAoxLDL ELISA (MDA(Malondialdehyde)ELISAKITELABSCIENCE Houston, Texas (USA)). This analysis was performed on serum obtained from blood extracts taken half an hour before and after each stress test.

[0089] – Oxidized LDL Analysis. Oxidized serum LDL was quantified using the MDAoxLDL ELISA (Human OxLDL (Oxidized Low Density Lipoprotein) ELISA KIT ELABSCIENCE Houston, Texas (USA)). This analysis was performed on serum obtained from blood extracts taken half an hour before and after each stress test.

[0090] DNA oxidative damage

[0091] Analysis of 8-oxo-2-deoxyguanosine in 24-hour urine. The 24-hour urine 8-oxo-2-deoxyguanosine was analyzed using the DNA / RNA Oxidative Damage EIA Kit (80HdG(8-Hydroxideoxyguanosine) ELISA KIT ELABSCIENCE Houston, Texas (USA)). This analysis was performed on 24-hour urine samples collected before and after each stress test.

[0092] 1.3.2. Safety Variables

[0093] Blood biochemistry profile was analyzed to determine GOT, GPT, GGT, LDH enzyme values, bilirubin to assess liver function, urea and creatinine among other biomolecules to assess kidney function. Blood count was also performed to assess red blood cells, white blood cells and platelets. Two blood samples were taken at baseline and at the end of the study, respectively.

[0094] Adverse events were also recorded and evaluated.

[0095] 1.4. Statistical Analysis

[0096] Descriptive analysis (mean and standard deviation) was performed on all variables in the study, including baseline for each variable and its evolution. This analysis was performed on the total group of subjects participating in the study.

[0097] Homogeneity in the population at baseline was also analyzed in terms of demographic variables, medical history and other clinical parameters. For quantitative variables, a t-Student comparison was performed between the two study groups. For qualitative variables, a homogeneity test based on the chi-square distribution was performed when the expected value was possible, otherwise an exact Fisher test was performed.

[0098] To analyze the differences between groups (experimental and control) in the trend of the different variables, an analysis of variance was performed on the repeated measurements of the two factors of interest within (trial: before consumption and after 8 weeks of consumption, time: before and after each trial) and between (product: experimental product and placebo product) the groups. In this way, the differences in each of the variables analyzed were determined taking into account these factors. Tukey or Bonferroni tests were used for post hoc analysis. The comparison of these significant effects was performed assuming or not assuming equal variances.

[0099] In a set of statistical tests, a significance level of 0.05 was used. Statistical analysis was performed with SPSS 21.0 software.

[0100] 1.5. Results

[0101] This study started with 45 subjects, one of whom was excluded before the first trial. The remaining 44 subjects were randomly divided into two study groups. During the study, one subject from the placebo group dropped out due to non-attendance at follow-up. Therefore, 43 subjects were analyzed: 22 subjects who took the probiotic product and 21 subjects who took the placebo product.

[0102] In the group that consumed the probiotic product, the average age was 25.3 ± 7.2 years, while in the placebo group, the average age was 27.1 ± 8.4 years.

[0103] 1.5.1. Serum malondialdehyde analysis

[0104] The descriptive statistics are shown in the following table:

[0105] Table 1 : Statistical levels of serum malondialdehyde (ng / ml) (mean, standard error, mean difference, P1 statistical significance level of the difference between the levels before and after each test and P2 significance level of the difference between the placebo and probiotic level increase).

[0106]

[0107] The following conclusions were reached in the comparative analysis:

[0108] - Comparison of the variable values at the initial state. When comparing the values of this variable at the initial stage, no significant differences were found, so it can be said that the groups were homogeneous in relation to this variable at the initial stage of each test.

[0109] - Placebo group. During the first test, the results showed no significant increase in serum malondialdehyde levels (P < 0.094), secondary to the damage caused by high intensity and long duration physical exercise. When performing the second test, the increase in the levels of this parameter produced by physical exercise after ingesting the placebo product was the same as in test 1 (p < 0.149). Therefore, we cannot be sure that the consumption of placebo changed the trend of this variable during the stress test. Figure 1 ).

[0110] - Experimental group. During the first test, serum malondialdehyde levels increased significantly (P < 0.001). When performing the second test, the increase in this parameter caused by physical exercise after ingesting the probiotic product was much lower than in test 1 (p < 0.623). When comparing the change in this parameter in test 1 with the change obtained in test 2, a significant difference was observed (p < 0.005), i.e. the increase in malondialdehyde in the subjects who consumed the probiotic product in the second test was lower than in the subjects in the first test Figure 1 ). Therefore, it can be said that the consumption of the probiotic product changed the trend of this variable during the stress test.

[0111] When comparing the trends between the two groups Figure 2 ), a significant difference was observed (p = 0.047), i.e. it can be stated that the trends of the two products were different, so it can be concluded that the intake of the probiotic product for 6 weeks produced a significant improvement in this variable compared to the placebo.

[0112] 1.5.2. - Oxidized LDL analysis

[0113] The descriptive statistics are shown in the following table:

[0114] Table 2: Statistical levels of oxidized LDL in serum (ng / ml) (average, standard error, average difference, P1 statistical significance level of the difference between the levels before and after each test and P2 significance level of the difference between the placebo and probiotic level increase).

[0115]

[0116] The following conclusions were reached in the comparative analysis:

[0117] - Comparison of the variable values at the initial state. When comparing the values of this variable at the initial stage, no significant differences were observed, so it can be said that, at the initial stage of each test, the groups were homogeneous with respect to this variable.

[0118] - Placebo group. During the first test, a significant increase in serum oxidized LDL levels was observed (P < 0.001), secondary to the damage caused by high intensity and long duration physical exercise. When performing the second test, the increase in the parameter level produced by physical exercise after ingesting the placebo product was the same as in test 1 (p < 0.001) Figure 3 ). Therefore, it cannot be said that the ingestion of the placebo changed the trend of this variable in the stress test.

[0119] - Experimental group. During the first test, a significant increase in serum oxidized LDL levels was observed (P < 0.001). When performing the second test, the increase in the parameter produced by physical exercise after ingesting the probiotic product was much lower than in test 1 (p < 0.467) Figure 3 ). Comparing the change in this parameter in test 1 with the change in the parameter obtained in test 2, a significant difference was found (p < 0.042), i.e. in the second test, the subjects who consumed the probiotic product had a lower increase in oxidized LDL than in the first test. Therefore, it can be said that the consumption of the probiotic product changed the trend of this variable during the physical test.

[0120] When comparing the trends between the two groups Figure 4 ), a significant difference was observed (p = 0.05), i.e. the trends of the two products can be said to be different, so it can be concluded that the intake of the probiotic product for 6 weeks produced a significant improvement in this variable compared to the placebo.

[0121] 1.5.3. Analysis of 8-oxo-2-deoxyguanosine in the urine after 4 hours

[0122] The descriptive statistics are shown in the following table:

[0123] Table 3: Statistical levels of 8-oxo-2'-deoxyguanosine in 24-hour urine (pg / ml) (mean, standard error, mean difference, P1 significance level of the difference in levels before and after each test, and P2 significance level of the difference in the increase in levels of placebo and probiotics).

[0124]

[0125]

[0126] The comparative analysis yielded the following conclusions:

[0127] - Comparison of variable values ​​in the initial state. No significant differences were observed when comparing the values ​​of the variable in the initial stage, so it can be said that the groups are homogeneous for the variable in the initial stage of each test.

[0128] – Placebo group. During the first trial, a significant increase in 24-hour urinary 8-oxo-2'-deoxyguanosine levels was observed (p<0.001), attributed to damage from prolonged high-intensity physical exercise. In the second trial, the increase in this parameter following physical exercise after placebo ingestion was the same as in trial 1 (p<0.001). Figure 5 Therefore, it cannot be said that placebo intake altered the trend of this variable in stress tests.

[0129] – Experimental group. During the first trial, a significant increase in 24-hour urinary 8-oxo-2'-deoxyguanosine levels was observed (15.7 pg / ml; p < 0.001). In the second trial, after probiotic product intake, physical exercise led to a much smaller increase in this parameter than in trial 1, but it was still significant (4.8 pg / ml; p < 0.007). Figure 5 Comparing the changes in this parameter in Experiment 1 with those in Experiment 2 revealed a significant difference (p<0.001), meaning that in the second experiment, the increase in 24-hour urinary 8-oxo-2'-deoxyguanosine levels in subjects consuming probiotic products was lower than that in the first experiment. Figure 5 Therefore, we can say that consumption of probiotic products altered the trend of this variable during the stress test.

[0130] When comparing trends between two groups ( Figure 6 A significant difference was observed (p = 0.001), indicating that the trends of the two products were arguably different. Therefore, it can be concluded that 6 weeks of probiotic product intake significantly improved this variable compared to placebo.

[0131] In summary, consuming the probiotic products of this invention for 6 weeks can reduce oxidative damage to lipids and DNA caused by high-intensity, long-duration physical exercise.

[0132] High intensity long duration physical exercise induces oxidative damage to lipids, proteins and DNA. This can be evidenced by the analysis of different metabolites: oxidative damage to lipids results in an increase in serum malondialdehyde and serum oxidized low-density lipoprotein cholesterol, while oxidative damage to DNA results in an increase in the levels of 8-oxo-2'- deoxyguanosine in urine. The intake of probiotics reduces the increase in serum malondialdehyde, serum oxidized cholesterol LDL and 8-oxo-2'-deoxyguanosine in 24-hour urine. That is, the results show that the intake of probiotics reduces the oxidative damage to lipids and DNA induced by high intensity long duration physical exercise; it has been previously demonstrated that this exercise induces oxidative damage to lipids and DNA.

[0133] 1.5.4. Safety variables

[0134] No adverse events related to the intake of the probiotics of the application were observed in any of the study subjects. No changes were found in the blood count, liver or kidney function of the subjects. Therefore, the intake of the composition of the application is safe.

[0135] In summary, the daily consumption of the probiotics of the application for 6 weeks:

[0136] - reduces the oxidative damage to lipids and DNA induced by high intensity long duration physical exercise.

[0137] - improves the antioxidant status of the subjects.

[0138] - no adverse events related to its intake were observed in any of the study subjects, no changes in liver or kidney function were observed in the subjects, therefore it can be concluded that it is safe.

Claims

1. A composition comprising the bacteria Lactobacillus rhamnosus, Lactobacillus casei, and Bifidobacterium longum together with one or more pharmaceutically acceptable carriers and / or excipients; wherein Lactobacillus rhamnosus is strain BPL0015, which is deposited at the Spanish Center for Type Culture Collection, with accession number CECT8361; Lactobacillus casei is strain BPL0004, deposited at the Spanish Center for Type Culture Collection, accession number CECT9104; and Bifidobacterium longum is strain IATA-ES1, deposited at the Spanish Center for Type Culture Collection, with accession number CECT7347.

2. The composition according to claim 1, wherein the total bacterial count in the composition is 10. 9 CFU.

3. The composition according to claim 1, wherein the composition is a pharmaceutical composition.

4. The composition according to claim 1, formulated for oral administration.

5. The composition according to claim 1, wherein the composition is in solid form.

6. The composition according to claim 5, wherein the composition is presented in capsule form.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a medicament for treating and / or preventing lipid and DNA damage caused by oxidative stress in an individual, oxidative stress caused by physical exercise.

8. The use according to claim 7, wherein the composition is applied once daily.

9. The use according to claim 7, wherein the composition is applied for 6 weeks.

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