ES2957341A1 - HERICIUM ERINACEUS STRAIN AND ITS USE IN NEURODEGENERATIVE DISEASES, CANCER AND DIGESTIVE DISEASES (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Hericium erinaceus strain and its use in neurodegenerative diseases, cancer and digestive diseases. The present invention relates to a strain of the Hericium erinaceus species, in particular to the DSM 34087 strain, to a mycelial extract of the H. erinaceus strain, to a method of producing a mycelial extract of the H. erinaceus strain , to compositions that comprise the strain or the extract, and to their nutritional and pharmaceutical applications. In particular, it refers to its activities (neuronal protective activity against oxidative stress, proapoptotic activity, anti-inflammatory activity and prebiotic activity) and/or its applications in neurodegenerative diseases, cancer and digestive diseases, among others; and/or its use in foods, as food supplements, its use as cosmetics, health products and/or pharmaceutical products. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Hericium erinaceus strain and its use in neurodegenerative diseases, cancer and digestive diseases

The present invention relates to a strain of the Hericium erinaceus species, in particular to the DSM 34087 strain, to a mycelial extract obtained from it, and to the uses of both the strain and the extract obtained from it in the treatment of diseases neurodegenerative, cancer and digestive diseases, among others; and/or its uses in food, cosmetics, health products and/or pharmaceutical products. Therefore, the present invention falls within the field of therapeutic applications of mycelial extracts obtained from fungi.

BACKGROUND OF THE INVENTION

Fungi are well established as a natural source of substances with medicinal applications. Several cellular components and secondary metabolites have been shown to have antitumor, antiproliferative, antimutagenic, antigenotoxic or immunomodulatory activity. Basidiomycetes (Basidiomycota) are a division of the kingdom Fungi of which some bioactive compounds are known, such as, for example, polysaccharides, triterpenes, ergosterol, ergothioneine, nucleic acids, proteins, polypeptides and sterols, some of them of high molecular weight.

Extracts from basidiomycete fungi have been used for a long time in the treatment of numerous diseases. Fungi are commonly used as biological response modifiers and immunostimulants. Basidiomycete fungi have the ability to generate a wide variety of active compounds that can subsequently be extracted to obtain products rich in certain molecules. For example, in organic extraction (also called lipophilic extraction) extracts rich in water-insoluble active compounds are obtained, such as secondary metabolites, which include triterpenes and isoflavones. The administration of organic extracts can be combined with aqueous extracts to enhance the effects of both extracts due to a synergistic action. Antitumor and anticancer activities of these extracts have been found when they were applied to tumor cell lines.

Basidiomycetes can be grown in submerged liquid culture medium. Patent document EP1451336B1 discloses a method of producing compounds with immunostimulating activity through mycelial culture in a liquid medium. The mycelial extracts obtained from these fermentation processes in liquid medium contain a large amount of secondary metabolites excreted by the fungi into the culture medium.

Methods for submerged fungal cultivation in bioreactors using a food-grade culture medium have been developed to improve the organoleptic and nutritional parameters of basidiomycetes. Patent documents WO2019226759A1 and US9427008B2 describe a method to obtain fermented vegetable substrates improving organoleptic and/or nutritional parameters.

Herícium erinaceuses is a basidiomycete of the Hericiaceae family and is considered a natural prebiotic that helps recover the microbiota.H. erinaceuses rich in some physiologically important components, especially p-glucan polysaccharides, which are responsible for the anticancer, immunomodulatory, antioxidant, neurostimulatory and neuroprotective activities of this species. It has also been described that H. erinaceus has antimicrobial, antihypertensive and antidiabetic properties, among other therapeutic potentials.

There are scientific studies on the action of H. erinaceusante digestive problems (inflammation, reflux, regularity, etc.), allergies and food intolerances. Its regenerative activity also supports its use in neurodegenerative diseases, memory loss and dementia.

There are several investigations related to the role of the polysaccharides of the mycelium of H. erinaceusen the intestinal microbiota. For example, the work of Shao S. et al., Int Immunopharmacol. 2019 Jun; 71: 411-422 describes a polysaccharide that improves acetic acid-induced ulcerative colitis in rats by modulating the composition of the intestinal microbiota, levels of short chain fatty acids and GPR41/43 receptors. Other polysaccharides described fromH. erinaceusattenuate colitis in C57BL/6 mice by regulating oxidative stress, inflammation-related signaling pathways, and modulating gut microbiota composition (Ren Y. et al. J Nutr Biochem. 2018 Jul;57:67 -76).

In other works an extract obtained fromH. erinaceusque may have potential as a neuroprotective and anti-inflammatory agent in the neuronal environment (Kushari N. et al., Antioxidants (Basel). 2019 Aug 1;8 (8):261). On the other hand, the work of Yang Y., et al., Molecules, 2018 Nov 30; 23(12): 3158 demonstrates the prebiotic activity of polysaccharides obtained fromH. erinaceus.

Patent document KR20190037037A refers to an extract of Hericium erinaceus and a culture medium of Schizophyllum commune as natural materials that can exhibit antioxidant activities, anti-inflammatory activities and skin aging inhibitory effects simultaneously.

There are a large number of documents that describe the neuroprotective, proapoptotic, anti-inflammatory and prebiotic activity of different strains of H. erinaceus. However, the current limitation of the state of the art is that no strain has been characterized that has all the activities (neuronal protective activity against oxidative stress, proapoptotic activity, anti-inflammatory activity and prebiotic activity).

Therefore, there is a need in the state of the art to provide new strains of H. erinaceusque have multiple activities that allow the development of a wide variety of therapeutic applications for different diseases.

DESCRIPTION OF THE INVENTION

The present invention relates to a strain of the genus Hericium, the species Hericium erinaceus and, in particular, the strain H. erinaceusDSM 34087, which can be used in a method for the production of mycelial extracts. In this way, the present invention also refers to a mycelial extract of H. erinaceus, particularly the strain H. erinaceusDSM 34087. Both the strain and the extract obtained from it may be part of a composition. Thus, the present invention also refers to compositions that comprise the strain or the extract derived from it.

One of the beneficial effects of the strain, the extract and/or the compositions object of the present invention is that they have the ability to improve neuronal viability in the face of oxidative stress, promote cell apoptosis and inhibit the secretion of inflammatory mediators. Furthermore, the strain, the extract and/or the compositions have prebiotic activity.

Thanks to this neuroprotective, proapoptotic, anti-inflammatory and prebiotic activity, the strain, the extract and/or the compositions object of the present invention are useful in the treatment of neurodegenerative diseases, cancer and digestive diseases, among others. These products could also be used in foods, food supplements (such as prebiotics or nutritional supplements), cosmetics, healthcare products and/or pharmaceutical products, both in humans and animals.

Therefore, one aspect of the present invention relates to the H. strain. erinaceusDSM 34087, hereinafter “strain of the invention” or “strain DSM 34087”.

In the present description, the strain of the invention is considered to refer to both the individual cell of H. erinaceusDSM 34087, as well as the hyphae or mycelium that comprise said cell. As understood by those skilled in the art, it is common for the strain of the invention to be preserved in its mycelial form and from said mycelium a mushroom or carpophore can be obtained.

The term "mycelium" refers to the set of hyphae that form the vegetative part of a fungus. As far as the present invention is concerned, the mycelium of Hericium erinaceuses not very dense, white and cottony.

The strain of the invention was isolated on November 5, 2015 from the carpophore of the species H. erinaceus collected in Portamuiños, (Bora), Pontevedra, Galicia (Spain) by Hifas da Terra S.L. The strain was deposited by the applicant (Hifas da Terra S.L.) under the Treaty of Budapest in the German Collection of Microorganisms and Cell Cultures GmbH (Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ); Inhoffenstra&e 7B, 38124 Braunschweig , GERMANY) on November 8, 2021, assigning deposit number DSM 34087.

The scientific classification of the strain of the invention is Kingdom Fungi; Basidiomycota Division; Class Agarimycetes; Russulales Order; Family Hericiaceae; GenusHeríciumand SpeciesHerícium erinaceus.

Within the present invention, those strains that derive from the strain of the invention and that maintain or improve their neuroprotective, proapoptotic, anti-inflammatory and prebiotic activity are also contemplated. Examples of strains derived from the strain of the invention may be mutants that present variations in their genome with respect to the genome of the strain of the invention but that do not affect the ability of the strain to act as a neuroprotective, proapoptotic, anti-inflammatory and prebiotic agent. . Thus, mutant strains derived from the strain of the invention that retain these activities are also contemplated within the present invention. Other examples of strains ofH. erinaceus derived from the strain of the invention may be hybrid strains obtained through hybrid breeding technology that maintain and/or improve the activities of the strain of the invention. Therefore, the present invention also relates to a strain derived from the strain of the invention with neuroprotective, proapoptotic, anti-inflammatory and prebiotic activity. An assay to determine whether a strain derived from the strain of the invention maintains or improves neuroprotective, proapoptotic, anti-inflammatory and prebiotic activity is, for example, a prebiotic activity assay by calculating the prebiotic index. According to the definition of Huebner et al. International Dairy Journal (2007 Jul 17:770-775), the prebiotic index reflects the ability of a given substrate to support the growth of an organism compared to that of other organisms, and growth on a non-prebiotic substrate, such as glucose or any other sugar used as a control. Therefore, the strains of H. erinaceus have a positive prebiotic activity score if: 1) they are metabolized and controlled by probiotic strains; and 2) they are selectively metabolized by probiotics, but not by other intestinal bacteria.

The strain derived from the strain of the invention can be produced naturally or intentionally by mutagenesis methods known in the state of the art, such as, for example, but not limited to, the growth of the original strain in the presence of agents. mutagenic or stress-causing, through hybridization methods or through genetic engineering aimed at modifying specific genes. Thus, within the present invention, genetically modified mutants derived from the strain of the invention that retain their neuroprotective, proapoptotic, anti-inflammatory and prebiotic activity are also contemplated.

As understood by those skilled in the art, the strain of the invention or the strain derived from it may be part of a composition. Thus, another aspect of the present invention refers to a composition, hereinafter "first composition of the invention", which comprises the strain of the invention and/or the strain derived from it.

The composition, generally defined, is a set of components that is formed by at least the strain of the invention in any concentration. The composition may comprise other components such as, for example, inert substances or bioactive components. Thus, in a particular embodiment, the composition of the invention comprises the strain of the invention and a vehicle or carrier (carrier).

The "vehicle" or "carrier" is preferably an inert substance. The functions of the carrier are to facilitate the incorporation of other components or compounds, allow better dosage and administration and/or give consistency and shape to the composition. When the presentation is liquid, the carrier is the diluent. The carrier can be natural or unnatural.

Optionally, in another particular embodiment, the first composition of the invention may also comprise at least one bioactive component (active substance, active ingredient or therapeutic agent), such as other food components, plant or fungal products and/or drugs.

The term "bioactive component" refers to a compound with biological activity within the scope of application of the patent that can improve or complement the activity of the strain of the invention, including ingredients or components of foods, probiotics, plants, fungi, extracts or components of plants, fungi and drugs.

The first composition of the invention can be formulated (i) for dietary administration, that is, on its own or as part of the foods consumed in the subject's diet, (ii) for administration as a cosmetic, that is, on its own. or forming part of cosmetic products or (iii) for pharmaceutical administration, that is, on its own or forming part of pharmaceutical products that are administered to the subject by any means of administration. Thus, in a particular embodiment, the first composition of the invention is a nutritional composition, a cosmetic composition or a pharmaceutical composition. Hereinafter, respectively, “first nutritional composition”, “first cosmetic composition” or “first pharmaceutical composition” of the invention.

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 (chickens, ostriches, chicks, geese, partridges, etc.), rabbits, hares, pets (dogs, cats, etc.), sheep , goats (goats, etc.), swine (boars, pigs, etc.), equine (horses, ponies, etc.), and cattle (bulls, cows, oxen, etc.); animals of hunting interest, such as deer, deer, reindeer, etc., animals with commercial interest in aquaculture such as fish (trout and salmon and any member of the salmonidae family, turbot, sole, sea bream and sea bass) and humans.

As mentioned, in some particular embodiments, the first composition of the invention is a nutritional composition.

The term "nutritional composition" of the present invention refers to that food that, regardless of providing nutrients to the subject who takes it, beneficially affects one or several functions of the organism, so as to provide a better state of health and well-being.

In the event that the first composition of the invention is formulated as a nutritional composition, said nutritional composition may be a food or incorporated into a food or food product intended for human and/or animal consumption.

Thus, in a particular embodiment, the first nutritional composition of the invention is a food or nutritional supplement.

In some particular embodiments, the first nutritional composition of the invention is a food for specific nutritional purposes. In some specific embodiments, the first nutritional composition of the invention is a food for special medical uses.

Foods for special medical purposes are those foods that have been specially prepared or formulated for the dietary management of patients under medical supervision. These foods are intended to fully or partially satisfy the nutritional needs of patients.

In some embodiments, the first nutritional composition of the invention is a nutritional or dietary supplement.

The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement", "food supplement", or "food supplement" or "food supplement", refers to products or preparations whose purpose is to complement the diet. normal of a subject and consisting of concentrated nutrient sources or other substances with a nutritional or physiological effect.

The nutritional supplement may be in single or combined form and marketed in dosage form, i.e. in capsules, pills, tablets and other similar forms, powder sachets, liquid ampoules and drop dispensing bottles and other similar forms of liquids and powders designed to be taken in a single amount. There is a wide range of nutrients and other elements that can be present in food supplements, including vitamins, minerals, amino acids, essential fatty acids, fiber, enzymes, plants, plant extracts, mushrooms and mushroom extracts. Since their function is to complement the nutrient intake of the diet, they should not be used as a substitute for a balanced diet and their intake should not exceed the daily dose expressly recommended by the doctor or nutritionist. The first composition of the invention can also be part of the so-called "foods for special medical groups", that is, foods that satisfy specific nutritional needs.

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

In some particular embodiments, the first composition of the invention is a pharmaceutical composition.

In the present invention, the term "pharmaceutical composition" refers to a set of components that is formed by at least the strain of the invention in any concentration, which has at least one application in improving the physical or physiological or psychological well-being of a subject and that implies an improvement in the general state of their health or reduction in the risk of disease.

The term "subject" has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

The pharmaceutical composition may comprise one or more components or compounds that have some biological, pharmacological and/or veterinary activity useful in the treatment of neurodegenerative diseases, cancer and/or digestive diseases in a subject. As understood by those skilled in the art, the additional components or compounds must be compatible with the strain of the composition of the invention. The term "pharmaceutical composition" also encompasses veterinary compositions.

The term “pharmaceutical composition” also encompasses medical devices, provided that the term “medical product” refers to a product intended for use in humans, separately or in combination, for any of the following specific medical purposes: prevention, treatment or alleviation of a disease or treatment, alleviation or compensation of an injury or disability and its main mechanism of action is not pharmacological, metabolic or immunological. The first pharmaceutical composition of the invention may include compounds or components useful in the treatment of neurodegenerative diseases, cancer and/or digestive diseases including, but not limited to, Alzheimer's disease, Parkinson's, breast cancer, colon cancer, bowel syndrome irritable or histamine intolerance.

In some embodiments, the first pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier and/or excipient.

The term "excipient" refers to a substance that helps the absorption of any of the components or compounds of the first composition of the invention, specifically, of the strain of the invention, or stabilizes the components or compounds and/or helps the preparation of the pharmaceutical composition in the sense of giving it consistency or flavors to make it more pleasant. Thus, excipients may have the function, by way of example, but not limited to, of binding the components (for example, starches, sugars or cellulose), sweetening, coloring, protecting the active ingredient (for example, to isolate it from the air and/or humidity), fill a pill, capsule or any other presentation or a disintegrating function to facilitate the dissolution of the components, without excluding other excipients not listed in this paragraph. Therefore, the term "excipient" is defined as that material that, included in the pharmaceutical forms, is added to the active ingredients or their associations to allow their preparation and stability, modify their organoleptic properties or determine the physical and chemical properties of the pharmaceutical composition and its bioavailability.

The “pharmaceutically acceptable” excipient must allow the activity of the components or compounds of the pharmaceutical composition, that is, be compatible with the strain of the invention.

The “galenic form” or “pharmaceutical form” is the configuration to which the active ingredients and excipients are adapted to provide a pharmaceutical composition or a medicine. It is defined by the combination of the way the pharmaceutical composition is presented by the manufacturer and the way it is administered.

The term “vehicle” or “carrier” has been defined or explained in previous paragraphs, and said definition is applicable to the present particular embodiment of the first pharmaceutical composition of the invention. In the particular case of the first pharmaceutical composition of the invention, the carrier is a substance used in the drug to dilute any of the components or compounds of the first composition to a certain volume or weight; or that even without diluting these components or compounds, it is capable of allowing better dosage and administration and/or giving consistency and shape to the drug. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, starch, amylose, magnesium stearate, talc, surfactants, salicylic acid, viscous paraffin, perfume oil , monoglycerides and diglycerides of fatty acids, esters of petroethral fatty acids, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Additionally, the excipient and carrier must be pharmacologically acceptable, that is, the excipient and carrier are permitted and evaluated not to cause harm to the subject to whom it is administered.

As indicated above, in some particular embodiments, the first composition of the invention is a cosmetic composition.

In the present invention, the term "cosmetic composition" refers to any substance or mixture intended to be brought into contact with the superficial parts of the human body (epidermis, hair and capillary system, nails, lips and external genital organs) or with the teeth and oral mucous membranes, with the exclusive or main purpose of cleaning them, perfuming them, modifying their appearance, protecting them, keeping them in good condition or correcting body odors. The first cosmetic composition of the invention may comprise one or more components or compounds.

The first composition of the invention, which can be a nutritional, pharmaceutical and/or cosmetic composition, can be administered by different routes of administration. Thus, in some particular embodiments, the first composition of the invention can be administered orally, topically, parenterally, rectally, vaginally, ophthalmicly, otically and/or nasally, among others.

The presentation of the first composition of the invention will be adapted to the type of administration used. The composition can be formulated in solid, semi-solid or liquid preparations, such as tablets, capsules, powders, granules, solutions, suppositories, gels, microspheres and/or any other form known in the art of food, health products, cosmetics and/or or pharmacy. In a particular embodiment, the first composition of the invention is formulated for administration in solid or liquid form.

In another particular embodiment, the solid formulation is selected from the group consisting of tablets, lozenges, candies, chewable tablets, chewing gum, capsules, sachets, powders, granules, coated particles or coated tablets, tablets, pills, gastro-resistant tablets and capsules and strips. and dispersible films.

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

Likewise, various systems are known that can be used for sustained release administration of the first composition of the invention, including, but not limited to, encapsulation in liposomes, microbubbles, microparticles or microcapsules and the like. Suitable forms of sustained release, as well as the materials and methods for their preparation, are well known in the state of the art. Thus, the orally administrable form of the first composition of the invention is in a sustained release form that further comprises at least one coating or matrix. The sustained release coating or matrix includes, without limitation, natural semi-synthetic or synthetic, water-insoluble or modified polymers, waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic or synthetic plasticizers or a combination of two or more thereof . Enteric coatings can be applied by conventional processes known to those skilled in the art.

In addition to what is described above, the present invention also encompasses the possibility that the first composition of the invention can be administered to a subject together with other components or compounds, although these are not part of the first composition of the invention.

In another aspect, the present invention relates to the use of the strain of the invention or the first composition of the invention for the production of cosmetic products.

Another aspect of the invention refers to the use of the strain of the invention or the first composition of the invention for the production of human and/or animal food products. Examples of said human and/or animal food products are detailed in the previous paragraphs.

Another aspect of the invention relates to the strain of the invention or the first composition of the invention for use as a medicament.

The term "drug", as used herein, has a more limited meaning than the meaning of "pharmaceutical composition", since the drug necessarily implies a preventive or therapeutic effect. The term "medicine" and the term "drug" are considered equivalent in the present invention and may be used interchangeably. The medicine to which the present invention refers can be for human or veterinary use. "Medicament for human use" is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in human beings or that can be used in human beings or administered to human beings for the purpose of restoring, correcting, or modify physiological functions by exercising a pharmacological, immunological or metabolic action, or to establish a medical diagnosis. The "medicine for veterinary use" is any substance or combination of substances that is presented as having curative or preventive properties with respect to diseases. animals or that can be administered to the animal in order to restore, correct or modify its physiological functions by exercising a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis. "Premixes for medicated feed" prepared to be incorporated into feed will also be considered "veterinary medicines".

The pharmaceutical composition or medicament may be presented under any clinically permitted form of administration and in a therapeutically effective amount.

The strain of the invention must be present in the first composition of the invention in a therapeutically effective amount to exert its neuroprotective, proapoptotic, anti-inflammatory and prebiotic effect.

In the present invention, the term "therapeutically effective amount" is any amount of the component or compound of the composition that, when administered to a subject, is sufficient to produce the desired effect. Said component or compound of the composition refers to the strain of the invention. The therapeutically effective amount may vary depending on, for example, the age, body weight, general health, sex and diet of the subject, as well as the mode and timing of administration, excretion rate or any possible co-treatment with other medications.

The term "neuroprotective" refers to any agent that possesses pharmacological activity that promotes the survival and function of neurons. Medications that offer neuroprotection can be used to slow or prevent the loss of brain function in a neurodegenerative disease.

The term “proapoptotic” refers to any substance or mechanism that promotes apoptosis and hinders the appearance and progression of cancer.

The term “anti-inflammatory” refers to any medication or medical procedure used to prevent or decrease tissue inflammation.

The term “prebiotic” refers to compounds, usually present in foods, that induce the growth or activity of beneficial microorganisms such as bacteria and fungi. Prebiotics are typically compounds that pass undigested through the upper gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the large intestine by acting as a substrate for them.

Another aspect of the invention refers to the strain of the invention or the first composition of the invention for use in the treatment of neurodegenerative diseases, cancer or digestive diseases, among others.

The term "treatment", as used in the present invention, refers to the treatment of a disease or medical condition in a subject, preferably human, including:

(a) prevent the onset of the disease or medical condition, that is, the prophylactic treatment of a subject;

(b) improve the disease or medical condition, that is, cause regression of the disease or medical condition in a subject;

(c) suppress the disease or medical condition, that is, slow the development of the disease or medical condition in a subject; either

(d) alleviating the symptoms of the disease or medical condition in a subject.

The term "prevention" refers to preventing the occurrence of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particular, when said subject has a predisposition due to the pathological condition.

The term "subject" has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

The term "neurodegenerative diseases" refers to a type of disease that groups together a genus of cognitive disorders, such as Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease and multiple sclerosis. These cognitive disorders are due to an increase in of cell death, reducing the number of neurons and generating changes in behavior.

The term "cancer" refers to a group of related diseases in which some of the body's cells begin to divide without stopping and spread to surrounding tissues, such as breast cancer, colorectal cancer, and lung cancer. Division may begin locally and spread to other surrounding tissues.

The term “digestive diseases” refers to different disorders of the digestive system. These conditions can range from mild to severe. Some common problems include heartburn, cancer, irritable bowel syndrome, histamine intolerance, and lactose intolerance. Others Digestive diseases include: gallstones, cholecystitis and cholangitis, rectal problems (anal fissure, hemorrhoids, proctitis and rectal prolapse), esophageal problems (stenosis, achalasia and esophagitis), stomach problems (gastritis, gastric ulcers usually caused by Helycobacter Pylori infection and cancer ), liver problems (hepatitis B, hepatitis C, cirrhosis, liver failure and alcoholic and autoimmune hepatitis), pancreatitis and pancreatic pseudocyst, intestinal problems such as polyps and cancer, infections, celiac disease, Crohn's disease, ulcerative colitis, diverticulosis, malabsorption, syndrome of the short intestine and intestinal ischemia, gastroesophageal reflux disease (GERD), peptic ulcer disease and hiatal hernia, among others.

As mentioned at the beginning of the description, the strain of the invention can be used for the production of mycelial extracts by means of a production method for these extracts. Thus, another aspect of the invention refers to the use of the strain of the invention for the production of mycelial extracts.

In another aspect, the present invention relates to a method for the production of a mycelial extract, hereinafter the "method of the invention", which comprises the following steps:

(a) cultivating under aerobic conditions the strain of the invention, or the first composition of the invention, in a liquid culture medium comprising brown sugar, potato flour, carrot flour, rice protein, oat flour and oil of virgin olive, to obtain a fermented product, and (b) extract, filter and/or concentrate the fermented product obtained in step (a) to obtain a mycelial extract.

As indicated at the beginning of this description, the strain of the invention is considered to refer to both the individual cell of H. erinaceusDSM 34087, as well as the hyphae or mycelium that comprises said cell.

In the present invention the term "fermented product" refers to a composition obtained from the aerobic fermentation of the culture medium by the strain of the invention or the composition of the invention, and comprising active compounds as a result of the enzymatic activity of the strain on the culture medium.

The term "mycelial extract" refers to the extract obtained after a process of extraction, filtration and/or concentration of the fermented product, and which includes active compounds, exopolysaccharides and endopolysaccharides of the strain.

In a first stage, the method of the invention comprises cultivating the strain of the invention, or the composition containing it, under aerobic conditions, in a liquid culture medium comprising brown sugar, potato flour, carrot flour, rice protein , oat flour and virgin olive oil.

However, in a particular embodiment, prior to step (a), the method of the invention may comprise the cultivation of the strain or the composition of the invention in a nutrient medium, preferably liquid, in order to generate an initial amount of mycelium in liquid medium that will subsequently be used to inoculate the liquid culture medium of step (a) of the method of the invention. In a particular embodiment, the initial inoculum ratio versus the final volume to be added to the fermenter is 1:50. Preferably the same liquid culture medium as that used in step (a) is used.

Step (a) of the method of the invention is carried out under aerobic or aerobic conditions, which involves the provision of oxygen to the liquid culture medium. Under these conditions, the strain of the invention (or the composition that comprises it) will carry out the industrial fermentation of the compounds present in the liquid culture medium.

The term “industrial fermentation” refers to a process by which a culture is carried out in a fermenter or bioreactor, with various purposes such as, for example, increasing cellular biomass, producing enzymes or producing metabolites. Industrial fermentations usually occur in the presence of oxygen to obtain the maximum cellular metabolic performance of the biological system used.

The term “liquid culture medium” means a liquid formulation that contains nutrients and other conditions necessary for the growth of the strain and/or the mycelium obtained from it. In the present invention, the liquid culture medium comprises brown sugar, potato flour, carrot flour, rice protein, oat flour and virgin olive oil.

In a particular embodiment, the liquid culture medium of step a) of the method of the invention comprises the following concentrations: from 1 to 20 g/L of brown sugar; from 0.5 to 6 g/L of potato flour; from 0.5 to 6 g/L of carrot flour; 0.5 to 6 g/L rice protein; from 0.5 to 6 g/L oat flour; and from 1 to 6 g/L virgin olive oil.

In a more particular embodiment, the liquid culture medium of step a) of the method of the invention comprises the following concentrations: from 1 to 6 g/L of brown sugar; from 1.5 to 3 g/L of potato flour; from 0.5 to 2 g/L of carrot flour; from 0.5 to 2 g/L of rice flour; from 0.5 to 2 g/L of oat flour; and from 1.5 to 3.5 g/L of virgin olive oil.

In another particular embodiment, the cultivation of step a) of the method of the invention is done for a time of between 3 and 7 days (both ends included), at a temperature of between 20°C and 30°C (both ends included). ) with constant stirring, a source of oxygen, and a pH between 5.0-8.0 (both ends included). In a more particular embodiment, the cultivation of stage a) is carried out between 4 and 6 days (both ends included), at a temperature of between 25° and 28°C (both ends included), and at a pH of 6.0.

Once the cultivation in aerobic conditions of the liquid culture medium is completed, a fermented product is obtained that comprises exopolysaccharides, endopolysaccharides and other active compounds with therapeutic properties. In the present invention, the term "fermented product" refers to the biological material generated from the consumption of the liquid culture medium by the strain of the invention thanks to the enzymatic action of the strain, and the consequent growth of the mycelium of the strain. The fermented product thus obtained will give rise to a specific and unique composition different from the initial one that will comprise not only active compounds but also the mycelium itself.

Once stage (a) of the method of the invention is completed and the fermented product is obtained, we proceed to stage (b) which comprises the extraction, filtration and/or concentration of said fermented product to obtain a mycelial extract.

Techniques and conditions for extracting, filtering and/or concentrating a composition or a fermented product are widely known in the state of the art, and any of them can be used in the context of the present invention.

In the method of the invention, "extract" or "extraction" is understood as that process by which the active compounds retained inside the cell, in particular from the strain of the invention, are released into the medium. This extraction can be carried out by mechanical procedures, such as pressure or ultrasound.

In another particular embodiment of the method of the invention, the extraction in step b) is carried out by applying pressure and temperature, at 0.5-1.5 bars and at 115 125 ° C, for 8-12 minutes (including the extremes of all ranks). Preferably, step b) is carried out at 1 bar and 121°C, for 9 - 11 minutes (including both ends).

In another particular embodiment of the method of the invention, the extraction in step b) is carried out by ultrasound-assisted extraction at a frequency of 20-30kHz, amplitude of 18-22%, a flow rate of 88-95 mL. /minute and at a temperature of 10-15°C (including the extremes of all ranges). Preferably, in step b) ultrasound is used at a frequency of 20kHz, an amplitude of 20%, a flow rate of 92 mL/minute and a temperature of 12°C.

The term “ultrasound” refers to mechanical waves, generally longitudinal, whose frequency is above the hearing capacity of the human ear. Ultrasound equipment operates at frequencies higher than 20 kHz. Ultrasound has a large number of applications in the medical, therapeutic, food and technological fields. In the present invention, ultrasound is applied in an ultrasound-assisted extraction that allows the release of the bioactive compounds present in the fermented product. Ultrasonic-assisted extraction is achieved when high-power, low-frequency ultrasound waves are coupled into a mixture of material and solvent. In the present invention, the material would be the fermented product and the solvent would be water and/or ethanol.

After extraction, filtration of the resulting product is carried out. Thus, in another particular embodiment of the method of the invention, filtration is carried out through a 190 μm filter and subsequently through a 22 μm filter to eliminate residual biomass. Filtration makes it possible to eliminate most of the mycelium that is insoluble in the liquid medium.

Finally, the product obtained after filtration is concentrated. Techniques for concentrating a composition are widely known to those skilled in the art and any of them can be used in the method of the invention. However, in another particular embodiment of the method of the invention, the concentration is carried out by the rotary evaporator between 5 and 20 times and at a temperature between 30 and 50°C (including the extremes of all ranges). Preferably 20 times and at 45°C.

After putting the method of the invention into practice, a mycelial extract is obtained. Thus, in another aspect, the invention relates to a mycelial extract, hereinafter "extract of the invention", obtained by the method of the invention.

As understood by those skilled in the art, the extract of the invention may be part of a composition. Thus, another aspect of the present invention refers to a composition, hereinafter "second composition of the invention" that comprises the extract of the invention.

The terms "composition" and "mycelial extract" have been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

The composition, generally defined, is a set of components that is made up of at least the extract of the invention in any concentration. The composition may comprise other components such as, for example, inert substances and/or bioactive components. Thus, in a particular embodiment, the second composition of the invention comprises the extract of the invention and a vehicle or carrier (carrier). The terms vehicle and carrier have been previously defined for the first composition of the invention, and their definition is applicable to the second composition of the invention.

Optionally, in another particular embodiment, the second composition of the invention may also comprise at least one bioactive component (active substance, active ingredient or therapeutic agent), such as other food components, plant or fungal products and/or useful drugs. in the treatment of different diseases.

As was the case with the first composition of the invention, the second composition of the invention can be formulated for dietary administration, that is, on its own or as part of the foods consumed in the subject's diet, for administration as a cosmetic. that is, on its own or as part of cosmetic products or for pharmaceutical administration, that is, on its own or as part of pharmaceutical products that are administered to the subject by any means of administration. Thus, in some embodiments, the second composition of the invention is a nutritional composition, a cosmetic composition, or a pharmaceutical composition. Hereinafter, second nutritional, cosmetic or pharmaceutical composition of the invention.

The term "subject" has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

As discussed, in some embodiments of the present invention, the second composition of the invention is a nutritional composition. The term "nutritional composition" has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

In the event that the second composition of the invention is formulated as a nutritional composition, said second nutritional composition of the invention can be a food or incorporated into a food or food product intended for human and/or animal consumption.

Thus, in some particular embodiments, the second nutritional composition of the invention is a food or nutritional supplement.

In some particular embodiments of the invention, the second nutritional composition is a food for specific nutritional purposes. In some particular embodiments of the invention, the second nutritional composition of the invention is a food for specific medical uses.

In some particular embodiments, the second nutritional composition of the invention is a nutritional or dietary supplement.

The terms “supplement” and “food supplement” have already been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

In some embodiments of the present invention, the second composition of the invention is a pharmaceutical composition.

The term "second pharmaceutical composition" refers to a set of components that is formed by at least the extract of the invention in any concentration, that has at least one application in improving the physical or physiological or psychological well-being of a subject and that It implies an improvement in the general state of your health or a reduction in the risk of disease. The term "pharmaceutical composition" also encompasses veterinary compositions. The term “pharmaceutical composition” also encompasses medical devices, provided that the term “medical product” refers to a product intended to be used on people separately or in combination, for any of the following specific medical purposes: prevention, treatment or relief of a disease or treatment, relief or compensation of an injury or disability and its main mechanism of action is not pharmacological, metabolic or immunological.

The second pharmaceutical composition of the invention may comprise one or more components or compounds that have some biological, pharmacological and/or veterinary activity useful in the treatment of neurodegenerative diseases, cancer and/or digestive diseases in a subject. As understood by those skilled in the art, the additional components or compounds must be compatible with the extract of the second composition of the invention.

In some embodiments, the second pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier and/or excipient.

The terms “excipient”, “pharmaceutically acceptable”, “vehicle” or “carrier” have been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect. Examples of such excipients, vehicles and carriers are detailed in the preceding paragraphs.

In some particular embodiments, the second composition of the invention is a cosmetic composition.

The term “cosmetic composition” has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect. The second cosmetic composition of the invention may comprise one or more components or compounds.

As occurred in the first composition of the invention, the presentation of the second composition of the invention will be adapted to the type of administration used. The second composition of the invention can be administered orally, topically, parenterally, rectally, vaginally, ophthalmically, otically and/or nasally, among others.

The composition may be formulated in solid, semi-solid or liquid preparations, such as tablets, capsules, powders, granules, solutions, suppositories, gels or microspheres. Thus, in a particular embodiment, the second composition of the invention is formulated for administration in solid or liquid form.

In another particular embodiment of the second composition of the invention, the solid formulation is selected from the group consisting of tablets, lozenges, candies, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles or coated tablets, tablets. , pills, troches, gastro-resistant tablets and capsules and dispersible strips and films.

In another particular embodiment of the second composition of the invention, the liquid formulation is selected from the group consisting of oral solutions, suspensions, emulsions and syrups.

Various systems are known that can be used for sustained release administration of the second composition of the invention, including, for example, encapsulation in liposomes, microbubbles, microparticles or microcapsules and the like. Suitable forms of sustained release, as well as the materials and methods for their preparation, are well known in the state of the art. Thus, the orally administerable form of the second composition of the invention is in a sustained release form that further comprises at least one coating or matrix. The sustained release coating or matrix includes, without limitation, natural semi-synthetic or synthetic, water-insoluble or modified polymers, waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic or synthetic plasticizers or a combination of two or more thereof . Enteric coatings can be applied by conventional processes known to those skilled in the art.

In addition to what is described above, the present invention also encompasses the possibility that the second composition of the invention can be administered to a subject together with other components or compounds, although these are not part of the second composition of the invention.

According to the understanding of the person skilled in the art, the extract of the invention must be present in the second composition of the invention in a therapeutically effective amount to exert its neuroprotective, proapoptotic, anti-inflammatory and prebiotic effect.

The terms "neuroprotective", "proapoptotic", "anti-inflammatory" and "prebiotic" have been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

The expression “therapeutically effective amount” has also been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect. In the present inventive aspect, the component or compound of the composition that, when administered to a subject, is sufficient to produce the desired effect, refers to the extract of the invention and not to the strain of the invention, as occurred in the first composition of the invention.

In some particular embodiments, the total concentration of the extract of the invention in the second composition of the invention varies between 100 μg / mL and 100 mg / mL, both ends included.

Another aspect of the present invention refers to the use of the extract of the invention or the second composition of the invention for the production of cosmetic products.

Another aspect of the invention refers to the use of the extract of the invention or the second composition of the invention for the production of human and/or animal food products. Examples of said human and/or animal food products are detailed in the previous paragraphs.

Another aspect of the invention relates to the extract of the invention or the second composition of the invention for use as a medicine.

The terms “medicine”, “medicine for human use” and “medicine for veterinary use” have been described or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

The pharmaceutical composition or medicament may be presented under any clinically permitted form of administration and in a therapeutically effective amount. The term “therapeutically effective” has already been defined in previous paragraphs, and said definition is applicable to the present inventive aspect.

Another aspect of the invention refers to the extract of the invention or the second composition of the invention for use in the treatment of neurodegenerative diseases, cancer or digestive diseases, among others.

The terms “treatment”, “prevention”, “neurodegenerative diseases”, “cancer” and “digestive diseases” have already been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

As shown in the examples, the extract of the invention is capable of improving neuronal viability in the face of oxidative stress, of activating the caspase pathway, thus promoting cell apoptosis, and of inhibiting the secretion of inflammatory mediators. Furthermore, the extract of the invention has prebiotic activity in the presence of different strains of probiotic bacteria.

Thus, another aspect of the invention relates to the extract of the invention or the second composition of the invention for use as a neuroprotective medication.

Another aspect of the present invention relates to the extract of the invention or the second composition of the invention for use as a proapoptotic medication.

Another aspect of the invention relates to the extract of the invention or the second composition of the invention for use as an anti-inflammatory medication.

Another aspect of the invention relates to the use of the extract of the invention or the second composition of the invention as a prebiotic.

Finally, another aspect of the invention refers to the extract of the invention or the second composition of the invention for simultaneous use as a neuroprotective, proapoptotic, anti-inflammatory and prebiotic medication.

The terms "neuroprotective", "proapoptotic", "anti-inflammatory" and "prebiotic" have been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.

DESCRIPTION OF THE FIGURES

Fig. 1. MTT assay of neuronal viability in the presence of hydrogen peroxide with a control or in combination with the mycelial extract obtained using the Hericium erinaceus DSM 34087 strain.

Fig. 2. Caspase 3 activity assay in the presence and absence of the mycelial extract obtained using the Hericium erinaceus DSM 34087 strain.

Fig. 3. Anti-inflammatory activity test based on IL-6 in the presence of the mycelial extract obtained using the Hericium erinaceus DSM 34087 strain.

EXAMPLES

Next, the invention will be illustrated through tests carried out by the people responsible for the invention, which demonstrate the effectiveness of the product of the invention.

Results of characterization of protective activity of neuronal viability against oxidative stress

The assay is based on the metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) to formazan, which is a blue compound, quantifiable spectrophometrically at 570 nm. wavelength. The metabolic activity of intracellular NAD(P)H-oxide-reductases converts MTT into formazan, allowing the resulting functionality of the treated cells to be determined. The amount of live cells is proportional to the amount of formazan produced and absorbance measurements determine the percentage of viable cells after treatment.

Hydrogen peroxide (H2O2) is used as a stress factor to induce oxidative stress in experimental models and to stimulate apoptotic and necrotic pathways, evaluating the protective effect of certain compounds against this damage.

For the growth of the BE neuroblastoma cell line (ATCC CRL-2267™), a culture medium resulting from a 1:1 mixture of EMEM media (Eagle's Minimum Essential Medium, ATCC-302003) and F 12 medium ( Gibco Ham's F-12). The mixture was enriched with 10% FBS (fetal bovine serum) and 1% antibiotic (solution of 10,000 units of penicillin and 10 mg streptomycin/mL). The culture was carried out at 37 °C and 5% CO2 and the cells were kept growing in monolayer in 25 cm2 flasks.

To perform the assay, the cells were seeded in 96-well plates at a concentration of 2x10<5>cells/ml, incubated for 24 hours and subjected to the treatment required for the assay. Once it was verified by microscope observation that adequate confluence was reached, they were treated with 2 concentrations (25 and 50 mg/mL) of the mycelial extract (ME) and incubated for 1 hour at 37 °C. After this time they were exposed to a concentration of H2O2 of 150 μM and a control was maintained without hydrogen peroxide. The cell plates were incubated again at 37 °C for 24 hours and after this time cell viability was analyzed using the commercial MTT Proliferation Assay kit (CaymanChemical, MI, USA).

The treatment was carried out with the mycelial extract with the objective of evaluating its protective capacity against damage induced by hydrogen peroxide. The results shown in Figure 1 show that concentrations of 25 and 50 mg/mL of the mycelial extract (ME) improve the viability (higher MTT value) of the BE neuronal cells treated with H2O2150 μM compared to the control without extract.

Apoptosis results in neuroblastoma cell lines by determination of caspase 3 activity

The assay is based on determining the activity of caspase 3. The enzyme caspase-3 is a member of the family of endoproteases that regulate inflammation and apoptosis signaling networks. Caspase-3 is known as the executioner caspase in apoptosis due to its role in coordinating the destruction of cellular structures, such as DNA fragmentation or the degradation of cytoskeletal proteins. Its activation involves cell apoptosis.

The caspase detection protocol was carried out using the Caspase 3 Assay Kit (CASP-3-C, Sigma-Aldrich, MO USA). This colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), by the enzyme caspase 3, resulting in the release of p-nitroaniline (pNA). The concentration of pNA released from the substrate is calculated from the absorbance values determined at 405 nm, and a higher absorbance value is indicative of a greater activity of the caspase 3 enzyme. The assay was carried out on neuroblastoma cells BE (ATCC CRL-2267™) that were subjected to treatment with the mycelial extract for 24 hours of exposure. The BE neuroblastoma cell line was cultured under the same conditions as in the previous example. After treatment, cell lysates were obtained by applying the lysis buffer provided by the kit. Next, the caspase 3 enzyme activity of the samples was determined in comparison with a control using a specific substrate for this enzyme. A single experiment was carried out (in triplicate), expressing the results as an average of the absorbance values in percentage in relation to the value obtained in untreated cells (control).

The results shown in Figure 2 show that the mycelial extract (Extract) at a concentration of 100 mg/ml has greater caspase 3 activity than the controls. The observed results suggest that the mycelial extract activates the caspase pathway, which plays a fundamental role in promoting cell apoptosis.

Results of anti-inflammatory activity by determining the effects on the production of proinflammatory cytokines

The assay is based on determining proinflammatory cytokines. The anti-inflammatory capacity was tested using a murine macrophage model cell line (Raw 264.7, ATCC® TIB-71™). The Raw 264.7 cell line was cultured in DMEM medium (ATCC-30-2002) enriched with 10% FBS (fetal bovine serum) and 1% antibiotic (10,000 U/mL of penicillin and 10 mg/mL of streptomycin), at 37 °C and in the presence of 5% CO2, in 6-well microplates. At the initial time, an inflammation process was induced in the cell line by administering a bacterial lipopolysaccharide (LPS) at a dose of 100 ng/mL per well, and 30 minutes later the treatment was carried out with the different concentrations of the mycelial extracts. and a positive control was also incorporated. Concentrations of 1 mg/mL and 100 μg/mL of mycelial extract were used and were filtered through 0.22 μm in order to avoid possible contamination in the cellular assay. Hydrocortisone (Hdc) at 100 mM was used as a positive control to be able to make a comparison and evaluate its anti-inflammatory capacity. This compound is the main steroid hormone secreted by the adrenal cortex, it is administered as a drug for its anti-inflammatory or immunosuppressive action.

The study conditions were the following:

> Treatment with extract (EM) (1 mg/mL).

> Treatment with extract (EM) (100 μg/mL).

> Treatment with LPS (100 ng/ml) extract (1 mg/mL).

> Treatment with LPS (100 ng/ml) extract (100 μg/mL).

> Treatment with LPS (100 ng/mL): positive inflammation control.

> Treatment with LPS (100 ng/mL) and Hydrocortisone (100 μM): This treatment is the positive control for anti-inflammatory activity.

All treatments have been tested in triplicate. After 24 hours, the cell supernatant was collected and the cytokines released by the cells in the different treatments were quantified.

The determination of cytokine levels (Interleukin 6 or IL-6) was carried out using commercial ELISA kits (IL-6 Mouse ELISA -KMC0061) following the instructions established by the manufacturer. The ELISA (Enzyme-Linked-Immunosorbent Assay) is a highly sensitive solid-phase immunoenzymatic assay. Briefly, it consists of a 96-well analysis plate whose surface has been previously coated with an IL-6 antibody (anti-IL-6) with known concentrations that the kit itself provides or the cell supernatant samples. The IL-6 present in the cell supernatant samples or in the standards binds to the antibodies attached to the surface of the plate. Next, a second antibody (anti-mouse IL-6) conjugated to biotin is added, leaving the target cytokine immobilized between the two antibodies. After an incubation period, the biotinylated antibody that has not managed to bind to its target is removed from the plate through a washing buffer. Finally, a substrate containing streptavidin is added, which reacts with biotin, giving rise to a colorimetric reaction that can be quantified by spectrometry by measuring the absorbance at 450 nm. The intensity of the color generated by this reaction is directly proportional to the amount of IL-6 present in the serum sample.

The results shown in Figure 3 show that the mycelial extract (ME) inhibits the secretion of IL-6 at doses of 100 μg/ml and 1 mg/ml in the conditions where inflammation is induced in the presence of LPS. Furthermore, this inhibition (anti-inflammatory activity) is higher than the positive control of anti-inflammatory activity with Hydrocortisone.

Prebiotic activity results

The probiotic bacteria tested were the species Lactobacillus plantarum CECT 9567 and Lactobacillus rhamnosus ATCC53103. They were grown in MRS medium (Peptone 10.0 g/L, Beef Extract 8.0 g/L, Yeast Extract 4.0 g/L Ammonium Citrate 2.0 g/L Sodium Acetate 3.0 g/L, Magnesium Sulphate 0.1 g/L, Manganese Sulphate 0.05 g/L, Dipotassum Phosphate 2.0 g/L; Final pH 6.2 ± 0.2 at 25°C) supplemented with the mycelial extracts at 37°C for 24-48 hours.

The conditions tested were:

MRS Control Medium without glucose (VWR Life Science, Liofilchem, 610144) MRS Control Medium supplemented with glucose (Sigma G7021) (1mg/mL);

MRS control medium supplemented with 95% chicory inulin (CarboSynth, 3D-YI01274-500G) (1mg/mL);

MRS control medium supplemented with mycelial extract (1mg/mL).

The different supplements: Glucose, inulin and mycelial extracts were diluted and sterilized by filtration with a 0.22 μm STERIFLIP® filter. Sterile distilled water is added to the MRS control medium to maintain the same proportion of nutrients in all treatments.

The enteric bacteria tested were Escherichia coli strains 343 and 516. They were grown in M9 medium (dibasic sodium phosphate 6 g/L, monobasic potassium phosphate 3 g/L, sodium chloride 0.5 g/L, ammonium chloride 1 g/L). L) at 37°C for 24-48 hours and the following conditions were tested:

M9 control medium (VWR LifeScience) supplemented with 0.1mM Ca2Cl and MgSO<4>2mM without glucose;

M9 control medium supplemented with glucose (Sigma G 7021) (1 mg/mL);

M9 control medium supplemented with 95% chicory inulin (CarboSynth, 3D-YI01274-500G) (1mg/mL) (M9-INU);

M9 control medium supplemented with mycelial extract (1 mg/mL).

The different treatments were inoculated at 1% (100 μL of microorganism preculture in 9.9 mL of liquid medium) with the bacterial cultures previously adjusted to a turbidity of 0.5 Mac Farland. Each treatment was tested in triplicate. Two tubes of each treatment were incubated without inoculation to control possible contamination of the culture media and/or supplements. At the different sampling times (0, 24 and 48 hours), an aliquot of 250 μL was removed from each tube and the absorbance was measured at 600 nm in a 96-well plate. The prebiotic activity index is calculated according to the formula: A-B (Huebner et al., International Dairy Journal.

2007 Jul 17:770-775). Being: A= (Probiotic Abs in sample 24 hours - Probiotic Abs in sample 0 hours) / (Probiotic Abs in glucose 24 hours - Probiotic Abs in glucose 0 hours): and B= (Enteric Abs in sample 24 hours -Enteric Abs in sample 0 hours) / (Enteric Abs in glucose 24 hours -Enteric Abs in glucose 0 hours).

The prebiotic index reflects the ability of a given substrate to support the growth of an organism compared to that of other organisms, and growth on a non-prebiotic substrate, such as glucose or any other sugar used as a control. Therefore, samples have a positive activity score if: 1) they are metabolized as well as the control by the probiotic strains; and 2) they are selectively metabolized by probiotics, but not by other intestinal bacteria (such as enteric bacteria).

The results show that the mycelial extract (EM1 and EM2) presents a higher prebiotic activity index than Inulin in 8 different scenarios, the result of the combination of 2 different strains of probiotic bacteria, L. plantarumyL. rhamnosus, and 2 different strains of E. coli(Table 1).

Table 1. Prebiotic activity index results of the mycelial extract obtained from the Hericium erinaceus DSM 34087 strain and using inulin as a control.

Conditions of the method to obtain the mycelial extract

The method includes the cultivation of fungal mycelium in a liquid culture medium containing plant substrates to obtain a fermented product. Subsequently, the fermented product is subjected to an extraction and concentration process by which the mycelial extract is obtained.

In a first step, the mycelium of the DSM 34087 strain fungus is transferred to plates with a solid culture medium containing potato, dextrose and agar (PDA) that favors its growth. The incubation time varies between 14 and 24 days and the temperature varies between 20 and 30 °C.

The plates with PDA medium on which the mycelium has grown are used to inoculate the liquid culture medium. 1000 mL of liquid culture medium is added to a shaking flask. The liquid culture medium contains nutritional compounds that promote the growth of the fungus. The liquid culture medium comprises 1 to 6 g/L of brown sugar; from 1.5 to 3 g/L of potato flour; from 0.5 to 2 g/L of carrot flour; from 0.5 to 2 g/L of rice flour; from 0.5 to 2 g/L of oat flour; and from 1.5 to 3.5 g/L of virgin olive oil.

The mycelium is transferred from the PDA plate to the liquid culture medium in a flask under aseptic conditions. The shaken flasks are inoculated with the fungus and incubated while shaking. The incubation time is 15 days, in the temperature range of 25 to 28°C. The flask is kept under constant agitation, preferably at 120 to 150 rpm.

The contents of the bottle can be used to inoculate 50 liters of the liquid bioreactor medium (inoculum/final volume ratio of 1:50). The composition of the preferred liquid medium comprises 1 to 6 g/L of brown sugar; from 1.5 to 3 g/L of potato flour; from 0.5 to 2 g/L of carrot flour; from 0.5 to 2 g/L of rice flour; from 0.5 to 2 g/L of oat flour; and from 1.5 to 3.5 g/L of virgin olive oil. The incubation time is 5 days, at a temperature range of 25 to 28°C. The bioreactor is kept under constant agitation, preferably in the range of 120-150 rpm and with an air flow of 0.5 VVM. The pH of the medium is adjusted to pH 6. The pH is adjusted before inoculation. The liquid medium is sterilized before inoculation so that the fermentation process is sterile.

The fermented product resulting from submerged culture in the bioreactor includes the culture medium, mycelium, bioactive exopolysaccharides, intracellular polysaccharides and other active compounds resulting from the fermentation process. The extraction is preferably carried out at 1 bar at 121 °C for 10 minutes allowing the release of the polysaccharides from the intracellular mycelium, followed by sequential filtration through 190 μm and 22 μm to eliminate residual biomass.

Extraction can also be carried out by ultrasound at a frequency of 20-30 kHz, amplitude of 18-22%, flow rate of 88-95 mL/minute and a temperature of 10-15°C.

After filtration, the liquid extract is concentrated 20 times in a rotary evaporator at a temperature of 45 °C.

Claims (34)

1. Strain isolated from the species Hericium erinaceus with deposit number DSM 34087. 2. Composition comprising the strain according to claim 1. 3. Composition according to claim 2, where the composition is nutritional, cosmetic or pharmaceutical. 4. Composition according to any of claims 2 or 3, wherein the composition is administered orally, topically, parenterally, rectally, vaginally, ophthalmicly, otically and/or nasally. 5. Composition according to any one of claims 2 to 4, wherein the composition is formulated for administration in liquid or solid form. 6. Composition according to claim 3, wherein the nutritional composition is a food or a nutritional supplement. 7. Composition according to claim 3, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or excipient. 8. Use of the strain according to claim 1 or the composition according to claim 2, for the production of cosmetic products. 9. Use of the strain according to claim 1 or the composition according to any one of claims 2 to 5, for the production of human and/or animal food products. 10. Strain according to claim 1 or composition according to any one of claims 2 to 5, for use as a medicine. 11. Strain according to claim 1 or composition according to any one of claims 2 to 5, for use in the treatment of neurodegenerative diseases, cancer or digestive diseases. 12. Use of the strain according to claim 1 or the composition according to claim 2, for the production of a mycelial extract. 13. Method for the production of a mycelial extract that includes the following steps: (a) cultivating under aerobic conditions, a strain according to claim 1, or a composition according to claim 2, in a liquid medium comprising jaggery, potato flour, carrot flour, rice protein, oat flour and oil of virgin olive, to obtain a fermented product, and (b) Extract, filter and concentrate the fermented product obtained in step (a) to obtain a mycelial extract. 14. Method according to claim 13, wherein the liquid medium of step (a) comprises the following concentrations: 1 to 20 g/L of brown sugar; from 0.5 to 6 g/L of potato flour; from 0.5 to 6 g/L of carrot flour; 0.5 to 6 g/L rice protein; from 0.5 to 6 g/L oat flour; and from 1 to 6 g/L virgin olive oil. 15. Method according to any of claims 13 or 14, wherein the cultivation of step (a) is done for a time of between 3 and 7 days, at a temperature of between 20° and 30°C with constant stirring, a source of oxygen, and a pH of between 5.0-8.0. 16. Method according to any one of claims 13 to 15, wherein the extraction of step (b) is carried out at 0.5-1.5 bars and at 115-125 °C, for 8-12 minutes. 17. Method according to any one of claims 13 to 15, wherein the extraction of step (b) is done by ultrasound at a frequency of 20-30 kHz, amplitude of 18-22%, a flow rate of 88-95 mL/minute and at a temperature of 10-15°C. 18. Mycelial extract obtained by the method according to any of claims 13 to 17. 19. Composition comprising the extract of claim 18. 20. Composition according to claim 19, wherein the composition is nutritional, cosmetic or pharmaceutical. 21. Composition according to any of claims 19 or 20, wherein the composition is administered orally, topically, parenterally, rectally, vaginally, ophthalmicly, otically and/or nasally. 22. Composition according to any one of claims 19 to 21, wherein the composition is formulated for administration in liquid or solid form. 23. Composition according to any one of claims 19 to 22, comprising an extract concentration of 100 μg / mL to 100 mg / mL. 24. Composition according to claim 20, wherein the nutritional composition is a food or a nutritional supplement. 25. Composition according to claim 20, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or excipient. 26. Use of the extract according to claim 18 or the composition according to claim 19, for the production of cosmetic products. 27. Use of the extract according to claim 18 or the composition according to any one of claims 19 to 23 for the production of human and/or animal food products. 28. Extract according to claim 18 or composition according to any one of claims 19 to 23 for use as a medicine. 29. Extract according to claim 18 or composition according to any one of claims 19 to 23 for use in the treatment of neurodegenerative diseases, cancer or digestive diseases. 30. Extract according to claim 18 or composition according to any one of claims 19 to 23 for use as a neuroprotective medication. 31. Extract according to claim 18 or composition according to any one of claims 19 to 23 for use as a proapoptotic medication. 32. Extract according to claim 18 or composition according to any one of claims 19 to 23 for use as an anti-inflammatory medication. 33. Use of the extract according to claim 18 or composition according to any one of claims 19 to 23 as a prebiotic. 34. Extract according to claim 18 or composition according to any one of claims 19 to 23 for simultaneous use as a neuroprotective, proapoptotic, anti-inflammatory and prebiotic medication.