FR3062396A1 - COMPOSITION OF METABOLITES FROM FERMENTATION OF BIO-SOURCE INGREDIENTS FROM MICROBIOTES OF CENTENARIANS HAVING ANTI-INFLAMMATORY PROPERTIES AND INCREASE IN MITOCHONDRIAL ACTIVITY - Google Patents

Abstract

A fermentation process for producing metabolites having anti-inflammatory and increasing mitochondrial activity characterized in that the bacteria used for culturing are extracted from microbiota of healthy centenarians. The process is carried out in several stages for a total duration of at least 120 hours comprising prior sterilization of the fermented ingredients, inoculation of the bacteria and culture of the mixture at a temperature of between 30 ° C and 45 ° C. The fermented ingredients are of bio-sourced origin only and are obtained without the use of pesticides.

Description

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NINAPHARM.

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(® Agent (s): NINAPHARM.

FR 3 062 396 - A1 (54) COMPOSITION OF METABOLITES ARISING FROM THE FERMENTATION OF BIO-SOURCE INGREDIENTS FROM CENTENARY MICROBIOTS HAVING ANTI-INFLAMMATORY AND MITOCHONDRIAL ACTIVITY PROPERTIES.

(57) Fermentation process intended to produce metabolites having anti-inflammatory properties and increasing mitochondrial activity, characterized in that the bacteria used for the culture are extracted from microbiota of hundred-year-old healthy individuals. The process takes place in several stages for a total duration of at least 120 hours including prior sterilization of the fermented ingredients, inoculation of bacteria and culture of the mixture at a temperature between 30 ° C to 45 ° C. The fermented ingredients are of bio-sourced origin only and are obtained without the use of pesticides.

The subject of the present invention is a fermentation process intended to produce metabolites having anti-inflammatory properties and of increasing mitochondrial activity, characterized in that the bacteria used for the culture are extracted from microbiota of hundred-year-old healthy individuals. The process takes place in several stages for a total duration of at least 120 hours including prior sterilization of the fermented ingredients, inoculation of bacteria and culture of the mixture at a temperature between 30 ° C to 45 ° C. The present invention also relates to a cosmetic or pharmaceutical composition containing the metabolites resulting from the fermentation process for their anti-inflammatory properties and for increasing the mitochondrial activity.

We know that the human being in his desire to stay healthy longer and extend his life tries to understand as best he can the aging mechanisms of the body. These mechanisms are complex and are not all fully understood at present. The aging of the organism is described by functional modifications of the organism resulting in a progressive decrease in its aptitudes and vital functions. This process is natural and is present in every human being. However, it can be accelerated or reinforced by various stresses experienced throughout life. The aging processes of the body are notably closely linked to an imbalance in the oxidative balance. An overproduction of reactive oxygen species (ROS) by the mitochondria and a decrease in antioxidant defenses causes this imbalance. This imbalance results in damage to the mitochondria and a decrease in their activity. The damaged mitochondria no longer fulfill their role, which has the effect of intensifying the process which gradually changes the organism with age. The consequences of the rise in oxidative stress with age are in particular cell aging, the decrease in the functions of the organism and the increase in the incidence of pathologies. Oxidative stress is also very linked to the phenomena of chronic inflammation. Inflammation occurs in the event of an infection or an injury to fight against pathogens, it is a vital mechanism. In the case of inflammation, pro-inflammatory chemical mediators are produced by the immune cells. During prolonged irritation, the inflammation becomes chronic, which damages the body's tissues in the long term. This irritation can also be caused by various external factors such as pollution, stress, tobacco or diet. The immune system, which becomes less efficient with age, loses its ability to manage immune responses. Chronic inflammation tends to increase at first without any apparent symptoms but plays a central role in the aging of the organism. Mitochondria and their activity are affected by inflammatory mechanisms. Mitochondria have a central role in the body by providing the energy necessary for it in the body. Their proper functioning protects the body against aging and obesity in particular. It is therefore essential to protect them. The structure of the mitochondria originally comes from a bacterium which would have integrated into an ancestral eukaryotic cell by endosymbiosis. Mitochondria and bacteria share a similar structure and mode of reproduction. The mitochondrion has a DNA genome on a circular chromosome, whose genes resemble those carried by bacteria. It should be noted that bacteria have a very important role in the body through microbiota. The microbiota is defined as the set of microorganisms, ie bacteria, yeasts, fungi or viruses living in a specific environment called the microbiome in a host. Numerous microbiota exist in humans and in particular the intestinal microbiota. The gut microbiota is better known as the gut flora. The intestinal microbiota is made up of all the microorganisms living in the intestine. Humans have around 100,000 billion. The microbiota brings together the genome of the different species that colonize it, the metagenome. The colonization of the organism by the microorganisms forming the microbiota comes essentially from the maternal vaginal microbiota which at birth will colonize the skin. The microbiota will be supplemented by that of breast milk during breastfeeding and by the environment of the individual throughout his life. The more diverse the microbiota is, the particular and pathogenic species is limited. This provides protection against inflammation and infection. The microbiota is a recent discovery, its understanding is subject to numerous studies. The microbiota functions as an ecosystem with a balance between beneficial bacteria and harmful bacteria. The microbiota makes it possible in particular to assimilate certain foods that only bacteria know how to degrade, to regulate the expression of some of our genes. An imbalance of the microbiota is called dysbiosis and can lead to many diseases such as obesity, diabetes, allergies, depression, cardiovascular disorders. Viral, bacterial or parasitic infections, a sudden change in the environment or diet, an immunodeficiency, certain drugs and in the first place antibiotics can lead to dysbiosis. Studies have recently shown that the microbiota communicates with mitochondria through metabolites and is able to modulate the gene expression of proteins necessary for the functioning of mitochondria and also to directly modulate their activity. In this way, the microbiota is also involved in the aging process (Caroline Heintz and William Mair, You Are What You Host: Microbiome Modulation of the Aging Process, Cell 156, January 30, 2014 Elsevier Inc.). Indeed, the elderly and more particularly centenarian individuals have demonstrated having a different microbiota containing species of bacteria not present in the microbiota of conventional individuals. These microbiota from centenarians produce metabolites with antioxidant and anti-inflammatory properties that contribute to the maintenance of good health and longevity (Elena Biagil, Lotta Nylund, Marco Candela, Rita Ostan, Laura Bucci, Elisa Pini, Janne Nikkla, Daniela Monti, Reetta Satokari, Claudio Franceschi, Patrizia Brigidi, Willem De Vos, Through Aging, and Beyond: Gut Microbiota and Inflammatory Status in Seniors and Centenarians, PLoS ONE, May 2010, Volume 5, Issue 5, el0667). One solution to benefit from the benefits of the centenary microbiota and prevent the effects of aging would be to transplant a microbiota from a centenary individual to another individual. Microbiota transplantation consists of the introduction of a suspension of faeces from a healthy individual, that is to say having a varied microbiota in the intestine of the target individual. Transplantation of the Fecal Microbiota (or TMF) is often used for curative purposes in patients suffering from dysbiosis. The fecal microbiota is used because it is directly linked to the intestinal microbiota which is not available directly from the sample. The donor fecal sample collected on the day of the transplant is diluted in a solution such as water or milk. In order to avoid oral administration, the solution obtained is transplanted using different types of gastric tubes. However, this technique arouses strong disgust both in the person treated and in the doctor. TMF is applied from a single donor on all of its fecal microbiota. Its effects are therefore very dependent on the state of the donor's microbiota and the species that make it up. Indeed, the donor can be healthy while possessing harmful species for the recipient. Once in the intestine of the target individual, the micro-organisms resulting from the TMF complete the recipient's microbiota and possibly bridge an imbalance or provide it with beneficial missing species. These microorganisms are exposed to the whole food bowl of the recipient, that is to say to everything that the individual consumes. An imbalance of the intestinal microbiota can be the result of a dietary imbalance: a diet too rich in meats, sugars, fat or starches for example. In this case, the efficiency of the transplant may be limited or only effective for a short time. In addition, TMF poses the problem that this technique is expensive, that it is complicated to perform and that it requires medical intervention. This explains why its use is not widespread.

It is by studying the properties of these species of bacteria peculiar to centenarian individuals that the Applicant has discovered quite unexpectedly that the problems associated with TMF can be improved as well as the problems of inflammation and loss of blood. mitochondrial activity easily through the use of metabolites from fermentation by bacteria

extracted of microbiota individuals centenarians in healthy. For is Do the plaintiff at developed a protocol of collection of bacteria microbiota individuals centenarians. The collection of bacteria take place at go from fecal microbiota

individuals between the ages of 90 and 110. The donors follow a Mediterranean type diet and have never had a digestive pathology during their life. Donors must not have received antibiotics for at least three months before collection. In order to avoid the reduction in the number of bacteria due to the drop in pH after collection, the samples must be treated and cultured at most one hour after collection. The samples are collected and then mixed at 20% with a phosphate-buffered saline buffer at pH 7 and at a concentration of 1 mol / L. After centrifugation, the supernatant is collected. The supernatant is cultured in the presence of an agent stimulating bacterial growth. Culture bacteria are used in a fermentation process. The object of the present invention is defined as a fermentation process intended to produce metabolites having anti-inflammatory properties and increasing mitochondrial activity, characterized in that the bacteria used for the culture are extracted from microbiota of healthy centenarians.

The Applicant has carried out its studies from 15 healthy centenarian subjects. Species common to all subjects' microbiota are assigned to be the species responsible for patient health and longevity. These species have the most effective anti-inflammatory and mitochondrial activity enhancing properties. The species were identified by PCR then sequencing of the 16s rRNA. PCR or polymerase chain reaction is a technique for amplifying DNA fragments in vitro. Amplification of DNA allows easier sequencing and better identification of species. The 16s rRNA is a fragment of RNA that all species of bacteria possess, with sequence variations characteristic of each species. The sequences obtained for the patient's microbiota are compared with a ribosomal sequence database. The samples used therefore contain at least Lactobacillus fermentum, Lactobacillus casei, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescentis,

Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus Jensenii, Lactobacillus paracasei subsp. Paracasei, Lactobacillus gasseri, Lactobacilllus reuteri, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus helbeticus, Lactococcus lactis, Lactobacillus casei subsp. Casei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. Delbrueckii, Enterococcus faecium and Steptcoccus thermophiles. This selection makes it possible to control the species used for fermentation and therefore to control their numbers, their concentrations and their properties. It is also possible to select bacteria with interesting properties from several different individuals. The object of the present invention is also defined as a fermentation process characterized in that the microorganisms used for the culture include the bacteria common to the microbiota of the centenary individuals studied and are selected by PCR then sequencing of the 16s rRNA. The Applicant describes below a slow fermentation process in several stages. The fermented ingredients used are first soaked and then sterilized at high temperature. This operation makes it possible to eliminate the germs and bacteria potentially present on their surface in order to ensure better control of the fermentation. The fermented ingredients are cultured with the bacteria derived from microbiota of centenary individuals collected as described above at a temperature between 30 ° C to 45 ° C for at least 3 days. The result thus obtained is once again sterilized at high temperature in order to remove the bacteria from the culture. The final product of fermentation is obtained after filtration and drying at 40 ° C for 24 hours. The total duration of the process is at least 120 hours. The result is a mixture of at least 200 different types of metabolites. They are either created by slow fermentation or their concentration has been amplified during this process. Prior fermentation outside helps avoid direct contact between the fecal sample and the individual. This process makes it possible to choose both the species of bacteria used for fermentation and the fermentation substrate. An end product which is practical for consumption is obtained while retaining the properties of the metabolites derived from centenary microbiota bacteria with controlled conditions. The object of the present invention is therefore further characterized in that the fermentation process takes place in several stages. for a total duration of at least 120 hours including prior sterilization of the fermented ingredients, inoculation of bacteria and culture of the mixture at a temperature between 30 ° C to 45 ° C.

The fermented ingredients used during the process are all of bio-sourced and edible origin, that is to say all plants such as fruits, vegetables and algae and also certain compounds of animal origin such as milk. The fermented ingredients can be, for example, without limitation, the fruits, leaves or flowers of Figs, Olive, Rosemary, Thyme, Beans, Sage, Melon, Pomegranate, Soybeans, Watermelon, Cabbage, Green or black tea. The materials are selected to be grown without the use of pesticides. A mixture of several materials without limit of number or only one can be used for fermentation. This freedom of choice of fermentation substrate makes it possible to select only ingredients favorable to the diversity of the microbiota. The variation of the fermented ingredients will have a consequence on the final composition of metabolites and a modification of their concentration, of their bioavailability. It should be noted that all the compositions fermented by said process have in common at least 200 different types of metabolites with variable concentrations. Depending on the desired use, the ingredients will be chosen for their ability to produce the appropriate metabolites by fermentation. The advantage of selecting one or more ingredients is to avoid the in-vivo fermentation in the stomach of the entire food bolus and the decrease in the production of functional metabolites due to the presence of foods unfavorable to fermentation such as fatty foods. The fermentation process is therefore finally characterized in that the fermented ingredients are of bio-sourced origin only and obtained without the use of pesticides.

The fermentation products obtained are used in cosmetic or pharmaceutical compositions.

For the sake of clarity of the explanations which follow, we decide to call Centibiota a cosmetic or pharmaceutical composition containing metabolites produced by the process described here resulting from the fermentation of one or more ingredients bio-sourced by bacteria extracted from microbiota of centenarian individuals in good health for at least 120 hours.

Centibiota compositions, thanks to their properties, have several cosmetic and pharmaceutical uses. Centibiota metabolites have an average size of between 100

Da and .200 Da which allows them to be easily assimilated by the body without being damaged by digestion. They will therefore interact with the organism, the mitochondria and the microbiota of the individual. The mitochondrion is a central organelle of the eukaryotic cell. It is the only part of animal cells to have its own DNA, in addition to the nucleus. The mitochondrion is the place of cellular respiration. Thanks to the ATP synthases enzymes, it converts the organic molecules resulting from digestion into energy directly usable by the cell, the ATP energy molecule. ATP is the cell's energy reserve. The process of cellular respiration in the mitochondria is called the Krebs cycle. ATP production decreases in parallel with the increase in free radicals and the oxidation of mitochondria.

The object of the present invention therefore relates in particular to cosmetic or pharmaceutical compositions containing Centibiota used for increasing mitochondrial activity in that it modulates the respiratory chain of the mitochondria and increases the energy produced.

There are many pro-inflammatory markers in the body that are secreted in response to certain immune reactions. Among them, we can cite the super family of interleukins. In particular, interleukin IL-18 is a pro-inflammatory cytokine produced by macrophages in response to a microbial infection and in particular in response to stimuli from Lipopolysaccharide. Lipopolysaccharide or LPS is one of the major components of the outer surface of the membrane of pathogenic bacteria. LPS induces strong inflammation. Hydrogen sulfide, H2S, is a toxic compound that causes irritation and inflammation. It is excreted for example by the decomposition of organic matter by bacteria.

Remember that oxidative stress is caused by a species called reactive oxygen derivative (ROS). ROS when present in excess in the body will oxidize key molecules and proteins in tissues. For example, low density lipoproteins (LDL) transport cholesterol in the blood are prime targets for oxidation. A high level of oxidized LDL is one of the important markers of oxidative stress. Another important marker of oxidative stress is lipid peroxidation, i.e. the oxidation of unsaturated lipids by radical oxygen species. The body has a defense mechanism against oxidative stress. In particular, superoxide dismutases (SOD) is a protein with enzymatic activity. It catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. Likewise glutathione peroxidase (GPx) ensures the transformation of organic hydroperoxides. The metabolites of Centibiota have the ability to regulate the production of free radicals at the source, which helps prevent oxidative stress. The defense mechanism against oxidative stress is therefore less stressed, the production of antioxidant proteins decreases. The mitochondria have more energy available and the body is relieved and can function normally and defend itself against other threats. Centibiota helps decrease inflammation and oxidation factors and supports the body's defense system against oxidative stress. In particular, the subject of the present invention relates to cosmetic or pharmaceutical compositions containing Centibiota used to maintain a normal level of inflammatory markers and oxidative stress, maintain the good health of the individual, reduce the risk of inflammation and stress. oxidative in that it decreases tissue inflammation and oxidative stress and reduces the plasma concentration of Lipopolysaccharide, Interleukin IL-18 and Hydrogen Sulfide.

Centibiota users have reported a decrease in the frequency of infection as well as a decrease in symptoms of dysbiosis such as stomach upset, digestive problems, cramps, diarrhea and inflammation. Centibiota acts as a substitute for the normal production of metabolites of the intestinal microbiota allowing it to recover. Thanks to the good diversity of the microbiota, the development of pathogenic microorganisms is slowed down. Centibiota is used to help maintain the balance of the microbiota in that it limits the risk of colonization of tissues, in particular the intestinal tract by pathogens, and promotes the development of good bacteria.

When eating, the body stores the energy it does not need, including lipids. Burning lipids provides energy in the form of ATP. The fat burning mechanism is dependent on mitochondrial activity. As a result, Centibiota users have felt a loss of fat and reported feeling in better shape. The object of the present invention is defined as being able to be used for the contribution to the overall health of the individual in that it promotes the burning of fats and reduces the storage of lipids.

The final product obtained after filtration is in the form of a liquid. It can either be dried or used as is. It is to be used directly alone for high concentration applications or in mixture with other liquids or additives in single-phase or multi-phase products with in the presence of surfactants. The creation of a mixture containing various types of emulsion according to the steric and electromagnetic properties of the surfactant (s) in solution. In the case where the final product is dried, a powder is obtained. This powder is used either as is or as a mixture in dry formulations. These properties allow compositions of Centibiota types a wide variety of formatting accessible according to the desired use. The cosmetic or pharmaceutical composition containing Centibiota is in the form of an oil-in-water or water-in-oil emulsion, multiple emulsion, microemulsion, nano-emulsion, twin phase emulsion, PIT emulsion, stable dispersion of two immiscible phases by means of gelling agent, stable dispersion of two immiscible phases by means of one or more surfactants, a liquid, aqueous gel, fatty gel, hydroalcoholic gel, fatty phase, suspension, foaming solution or not, gel, lyophilized emulsion or powder.

Depending on the form chosen and the properties of the final formulation, Centibiota can be included in various cosmetic and pharmaceutical compositions for the uses mentioned above. Centibiota comes in the form of liquids, foams, pastes, ready-to-use drink, lotions, emulsions, oils, gels, syrups, solids, powders, masks, stick, tablets, capsules, sprays, aerosol, capsules, jellies, syrups, creams, patches, shower gels, shampoos.

These compositions are either consumed orally or applied topically. In addition, the oral route and the topical route can be used jointly, successively or separately according to the desired uses.

The Applicant will now give, by way of example, studies on compositions according to the subject of the invention as well as examples of formulations without these examples being limiting. Recall that for clarity of the explanations which follow we decide to call Centibiota a cosmetic or pharmaceutical composition containing metabolites produced by the process described here resulting from the fermentation of one or more ingredients bio-sourced by bacteria extracted from microbiota of individuals centenarians on good for at least 120 hours.

Example 1: Lists of metabolites

The different types of Centibiota compositions as mentioned in the present invention were analyzed by HPLC chromatography. Analysis of the results has demonstrated that the Centibiota compositions formulated with different fermented ingredients all contain at least the following metabolites: glycine, acetoacetamide (3-oxobutanamide), l-amino-2-propanol, galactosamine (chondrosamine), alanine, palmitoylethanolamide, L-noradrenaline glucosamine, serine stearoylethanolamide, cyclohexylamine mannosamine, pyroglutamic acid proline, acetylcholine, acetylglucosamine, Baline, 5aminolevulinique acid, phytosphingosine, ethyl glucuronide, threonine, Nacetyl-B-aranine, oleoylethanolamine, D-glucuronic acid, Lcystéine, N- acetyl-L-aspartic, cadaverine, sucrosephosphate, isoleucine acid N-carbamoyl-L-aspartic, Nacetylputrescine, eleutheroside E, leucine, N- acetylglutamic acid, N8-acetylspermidine, myoinositol diophosphate di-phosphate, 2methylshydroxy -hydroxy-acetophenone, phytic acid, lysine, N-methylproline, sphingenine (sphingosine), methylthioadenosine, a L-glutamic acid, thioproline, cholic acid (coralyne), uridine diphosphate glucose, L-methionine, 0acetyl - L- serine, glycodeoxycholic acid, xanthine, histidine, N6methyllysine, androstane-3,17-diol, 7-methylguanine, phenylalanine, L-theanine, campesterol thiamine, arginine, D-glucosamic acid, sitosterol thiamine, monophosphate, tyrosine, carboxymethyl-lysine, cycloartenol, riboflavin, tryptophan, 5-hydroxy-L-tryptophan, sphingomyelin, nicotinamide (niacinamin), glutathione 2-azetidinecarboxylic, hexanoic acid, nicotinic acid (niacin), glutathione (GHS), glycochenodeoxycholic acid, octanoic acid, pantothenic acid, 4-hydroxy-L-proline, O-succinyl-Lhomoserine, trilaurine, pyridoxine, L-asparagine -alanyl-D-alanine, citraconic acid, pyridoxamine, L-glutamine, cystathionine, glutaconic acid, glutaric acid, L-citrulline, ophthalmic acid, dihydrotachysterol, pelargonic acid, β-alanine, glutamylglycine, α-tocopherol, acid α-lipoic, Y-aminobutyric acid, S-Dlactoylglutathione, tocopherol acetate, malronic acid, betaine, N-acetyl-L-leucine biotin, trigonelline, L-homoserine, 2aminobutanoic acid, palmitoleic acid, lobelin, ornithine, acid

5-aminovaleric, linolenic acid, papaverine, choline, L2,4-diaminobutyric acid, linoleic acid, imidazoleacetic acid, putrescine, L-pipecolic acid, oleic acid, imidazolelactic acid, spermidine, 6-aminohexanoic acid, ricinoleic acid, imazaquine , stachydrine (proline betaine), Y-homolinolenic acid, indole-3-carboxaldehyde, tyramine, guanidobutyric acid (Y-guanidobutyric acid), icosadienoic acid, lumichrome, D- glucose-l-phosphate L-carnitine, 11-oleicosanoic acid, 5-Oxo2-tetrahydrofuran acid, glucose-6-phosphate, 5hydroxylysine, erucic acid, chelidonic acid, fructose-6phosphate, methionine sulfoxide, linolelaidic acid, methyl kojic acid, cytosine 3-methylhistidine, lauric acid, pyridine, cytidine, N-acetylornith , myristic acid, 4-pyridoxic acid, uridine, homoarginine, pentadecanoic acid, ectony, adenosine, homocitrulline, palmitic acid, l-methyl-2-pyrrolidone, guanosine, 2-6-diami acid nopimelic, heptadecanoic acid, syringaldehyde, cytidine 5'-phosphate, saccharopine, stearic acid, rhaponticin (rhapontine), uridine phosphate, argininosuccinic acid, eicosanoic acid, syringin (eleutheroside B), adenylic cyclic acid, N-acetyl-tyr heneicosanoic acid, syringic acid, 3'5-cyclic guanyl diphosphate, thyroxine, trico acid, 1phenylethylamine, adenosine 5'-phosphate, trimethylamine, 24-carbon chain fatty acid, 2-phenylethylamine, guanosine pentaphosphate, phosphocholine (choline phosphate ), 25 carbon hydrocarbon chain fatty acid, gemfibrozile, uridine diphosphate, O-acetylcarnitine, 26 carbon hydrocarbon chain fatty acid, diethyltoluamide, adenosine diphosphate, ethanolamine, sphinganine, benzoic acid, guanosine diphosphate, glycerol, amonoamine N -acylsphingosine, ceramide, 3hydroxybenzoic acid, uracil, urea, palmityl carnitine, p-coumaric acid, adenine, isobutylamine (paramine), glycerophosphocholine, terephthalic acid, hypoxanthine, triethanolamine, lecithin, phloretic acid, guanine, trimethylamine-N-oxide, linoleylethanolamide, vanillic acid, 3-methyl-2-oxobutanoic acid, neoeriocitrine, D-galiduronic acid, rutinoside, fumaric acid, vitexin (apigenin-8-glucoside), 3-hydroxymyristic acid, peonidin3-glucoside, 2-oxoglutaric acid, chrysoerol, prostaglandin B2, biochanin A, cis - aconitic acid, Datiscentine, 2-butenoic acid, cupressuflavone glycolic, 5methoxyindoleacetic acid, 2-hydroxyglutaric acid, formononetin, pyruvic acid, dimethoxyflavone, puerarin, mevalonic acid, 5,7tartaric acid, acidic

3-hydroxy-3apigenin-7 butyric acid, L-lactic acid, saponarin, 3hydroxybutyric, luteorin, ononin, 2-hydroxybutyric acid, shikimic, isoorientine (homoorientine), homovanillic acid, dalbergine, myricitrine, quinic acid, threonic acid, citric, liquiritigenin, D-galaturonic acid, citramalic acid, isocitric acid, acacetin, galactaric acid (mucic acid), methylglutaric, glucoside, succinic acid, glucoluteoline, L-malic, myricitrine, acid acid glucaric acid acid, D-gluconic acid, prunine (naringenine7-0-BD-

glucoside), D-glyceric acid, abietic acid, 3-phospho-D- acid glyceric, eriodictyol, 2-hydroxyvaleric acid, trans- aconitic, glycerophosphoric acid, rutin, baicalin, acid

isovaleric (methylbutyric acid), saikosaponin A, resveratrol, kaempferol, naringenin.

Example 2: Mitochondrial activity

A study has been carried out to show that the supplementation of Centibiota described in the present invention increases mitochondrial activity. This study was carried out using Centibiota obtained from Olive fruits, Rosemary and Beans. This study was carried out with 30 healthy volunteers without distinction of sex between 30 and 60 years of age, without any known disease.

The 30 patients were randomized into 2 groups of 15 people. The first group ingested a 500 mg dose of Centibiota described by the present invention and the second group a placebo every day for 60 days. Participants underwent adipocyte sampling at the start and end of the study. The samples taken were observed under an optical microscope in order to visualize the size of the adipocytes. It was observed that in the case of the placebo group the size of the adipocytes remains of the same order of magnitude between the start and the end of the study. In the case of the Centibiota group, there is a decrease in the size of the adipocytes at the end of the study compared to the size observed at the start of the study. The samples were then marked with 2 different markers and observed by fluorescence microscope. The first marker is the Green MitoTracker which selectively marks the mitochondria. The second marker is the red MitoTracker which colors the viable mitochondria in activity. The observation resulting from the green MitoTracker shows a substantially similar volume of mitochondria at the start and at the end of the study and between the two groups. However, the observation resulting from the red MitoTracker shows a quantity of viable mitochondria in activity significantly higher at the end of the study in the Centibiota group compared to the placebo group when they were similar at the start of the study. It is concluded from these observations that Centibiota targets the mitochondria and effectively increases their activity which results in an increase in energy production and an increase in fat burning.

Example 3: Decrease in inflammation

A study was carried out to assess the effect of ingestion of a food supplement containing Centibiota on the markers of inflammation TNF-α, H ₂ S and IL-18. This study was carried out from Centibiota obtained from Pomegranate fruits with 40 volunteers in good health without distinction of sex of age between 40 years and 60 years, without any known disease. The 40 patients were randomized into 2 groups of 20 people. The first group ingested a single 600 mg dose of Centibiota described by the present invention and the second group a placebo each day for 60 days. The participants underwent at the start and at the end of the study a blood sample and an analysis of the markers of inflammation mentioned above. It should be noted that no significant variation was noted in the placebo group. Only the average results for the Centibiota group are therefore presented in the following table:

Day 0 Day 60 Change (%) H2S (μΜ) 31.9 ± 9.4 18.4 + 5.7 -42.3 IL-18 (pg / ml) 5.21 ± 0.23 2.55 ± 0.14 -51.1 TNF-ot (pg / ml) 2.12 ± 0.10 0.84 ± 0.09 -60.3

It is observed that the blood levels of TNF-α, H2S and IL-18 decrease significantly at the end of the study. These results show that Centibiota effectively helps reduce markers of inflammation and at the same time reduces the harmful effects of LPS production by pathogens.

EXAMPLE 4 Antioxidant Activity

A study was carried out to assess the effect of ingestion of a food supplement containing Centibiota on oxidation markers (lipid peroxidation and LDL oxidation) and on the enzymes responsible for antioxidant activity (SOD and GPx). This study was carried out from Centibiota obtained from Pomegranate fruits with 40 healthy volunteers without distinction of sex of age between 40 years and 60 years, without any known disease. The 40 patients were randomized into 2 groups of 20 people. The first group ingested a single 600 mg dose of Centibiota described by the present invention and the second group a placebo each day for 60 days. The participants underwent at the beginning and at the end of the study a blood sample and an analysis of the oxidation markers and on the enzymes responsible for the antioxidant activity. It should be noted that no significant variation was observed in the placebo group. Only the average results for the Centibiota group are therefore presented in the following table:

UI / gHb Day 0 Day 60 Change (%) SOD 1502.9 ± 3.3 925.7 ± 3.1 -38.4 GPx 46.2 ± 0.9 29.6 ± 1.0 -35.9 Oxidized LDL 305.2 ± l, 3 234.5 + 1.5 -23.2 Peroxidation lipids 75, l ± 0.8 58.5 + 0.9 -22.1

We know that the reference value of SOD in a patient not suffering from oxidative stress is between 758 and 1570 IU / gHb and that of GPx between 20 and 58 IU / gHb. It is observed that the patients at the start of the study are within the upper limit of these reference values, that is to say that their antioxidant defense system is active and that they suffer from a slight oxidative stress which shows that the defense system is less active. At the end of the study, we notice that the blood levels of SOD and GPx decrease significantly towards a low level of the reference values. It is also observed at the end of the study that the levels of oxidized LDL and lipid peroxidation decrease significantly. The simultaneous decrease in antioxidant enzymes and markers of oxidative stress shows that Centibiota targets the production of free radicals at the source before the need for reaction of the antioxidant defense system. Centibiota effectively helps to balance the oxidative balance.

Example 5: O / W gel emulsion for the anti-inflammation body

- Carbopol 981 ............................................... .0.6g

- Ethyl alcohol ............................................. 15g

- Volatile silicone oil ................................... 3g

- Purcellin oil ............................................ 3g

- Centibiota Olive .............................................. 1g

- Perfume ................................................ ...... 0.4g

- Triethanolamine ............................................. 0.2g

- Preservatives .............................................. 0, 3g

- Demineralized water qs ....................................... 100g

This gel emulsion, applied once a day helps relieve skin inflammation.

Example 6: Capsules for the form

- Centibiota grapes .......................................... 0.6g

- Proto-milk .............................................. .... 2.0g

- Concentrated grape juice ..................................... 0.7g

- Skim milk powder ..................................... 0.05g

- Glucose oligosaccharides ................................ 0.5g

- Dietary fiber 0.1g

This food supplement comes in the form of capsules to be used 2 times a day. Users have noticed a loss of fat after a month of use and report feeling more energetic.

Example 7: Anti-infective drink

- Centibiota Fig ......................................... 0.2-0, 8g

- Proto-milk .............................................. .0.2-6.0g

- Concentrated orange juice ..................................... 0.1-1g

- Citric acid ............................................ 0.5 -2g

- Dietary fiber ..................................... 0.01-0.1g

- Glucose oligosaccharides ............................ 0.05-0.5g

- Aroma ................................................ 0.30-0.40g

- Water ................................................ ....... 20-30g

This formula is. present in the form of a drink to be consumed once a day. The users noted after 1 month of use a decrease in infections and better transit.

Claims (10)

1.) Fermentation process intended to produce metabolites having anti-inflammatory properties and increasing mitochondrial activity, characterized in that it is carried out in several stages comprising: i. a step of extracting bacteria from the faecal microbiota of healthy centenarian individuals between 90 and 110 years of age, who have never had digestive illnesses and who have not consumed antibiotics for a period of at least less than three months, ii. a step of fermentation of one or more ingredients at a temperature between 30 ° C and 45 ° C for a total duration of at least 120 hours by the bacteria obtained in step i. 2.) A fermentation process according to claim 1 characterized in that the bacteria used for the culture include those common to microbiota of centenarian individuals selected by PCR then sequencing of the 16s rRNA in particular comprising at least Lactobacillus fermentum, Lactobacillus casei. Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium adolescent girls, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus Jensenii, Lactobacillus paracasei subsp. Paracasei, Lactobacillus gasseri. Lactobacilllus reuteri, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus helbeticus, Lactococcus lactis, Lactobacillus casei subsp. Casei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. Delbrueckii, Enterococcus faecium and Steptcoccus thermophiles. 3.) Fermentation method according to one of claims 1 and 2 characterized in that the ingredient or ingredients fermented in step (ii) are of bio-sourced origin only, are obtained without the use of pesticides and are previously sterilized . 4.) Pharmaceutical composition comprising the metabolites resulting from the fermentation process according to one of claims 1 to 3. 5.) Pharmaceutical composition according to claim 4 for its use for increasing the mitochondrial activity in that it modulates the respiratory chain of the mitochondria and increases the energy produced. 6.) Pharmaceutical composition according to claim 4 for its use for maintaining a normal level of inflammatory markers and oxidative stress, maintaining good health for the individual and reducing the risk of inflammation and oxidative stress. in that it decreases tissue inflammation and oxidative stress and reduces the plasma concentration of Lipopolysaccharide, Interleukin IL-18 and Hydrogen Sulfide. 7.) Pharmaceutical composition according to claim 4 for its use for the contribution of the balance of the microbiota in that it modifies the quality, the diversity and the quantity of the microbiota. 8.) Cosmetic composition comprising the metabolites resulting from the fermentation process according to one of claims 1 to 3. 9.) Composition according to one of claims 4 and 8 characterized in that the composition is in the form of an oil-in-water or water-in-oil emulsion, multiple emulsion, microemulsion, nano-emulsion, twin phase emulsion, PIT emulsion, stable dispersion of two immiscible phases by means of gelling agent, stable dispersion of two immiscible phases by means of one or more surfactants, a liquid, aqueous gel, fatty gel, hydro-alcoholic gel, fatty phase, suspension, foaming solution or not, gel, lyophilized emulsion or powder. 10.) Composition according to one of claims 4 and 8 characterized in that the composition is in the form of liquids, J foams, pasta, ready-to-use drink, lotions, emulsions, oils, gels, syrups, solids, powders, masks, stick, tablets, capsules, sprays, aerosol, capsules, jellies, syrups, creams, patches, shower gels , shampoos.