FR2873038A1 - COMPOSITION COMPRISING A MARINE MICROORGANISM CULTURE MEDIUM AND / OR PHOTOSYNTHETIC FRESHWATER - Google Patents

Abstract

The present invention relates to a cosmetic, dermatological, pharmaceutical or food composition, characterized in that it comprises a culture medium of at least one marine microorganism and / or fresh photosynthetic water, said medium being clarified.

Description

COMPOSITION COMPRISING A CULTURE MEDIUM

  MARINE MICROORGANISM AND / OR FRESHLY PHOTOSYNTHETIC FRESH WATER.

  The present invention relates to a composition comprising, as active ingredient, a clarified culture medium of marine microorganism and / or fresh photosynthetic water.

  The use of microorganisms in the field of cosmetics or pharmacy is known from the prior art. The document EP 0 876 813 describes a cosmetic or pharmaceutical composition comprising, as active principle, an effective amount of culture medium of at least one non-photosynthetic filamentous bacterium, said medium being clarified and stabilized. In particular, it has been found in document EP 0 876 813 that the clarified and stabilized culture medium has properties similar to those of biomass.

  The search for biologically active ingredients is a field in full development because it is a way to increase the performance of products having to present a biological activity which is necessary for the development of cosmetic, dermatological, pharmaceutical and food products. The biological activity is measured through biological models that will allow to compare several ingredients in order to retain only one or the best on the model considered.

  For example, biologists have developed many models that can select active extracts and molecules on important biological mechanisms to regulate cutaneous metabolism. The active ingredients, selected by these models, can thus be incorporated into cosmetic and / or dermatological products that will be beneficial for cutaneous parameters that have been previously targeted. The same approach is valid for the development of pharmaceutical, dietary and food products that also rely on biological activity demonstrated on models.

  The search for biologically active ingredients relies on screening of many ingredients on biological models judiciously chosen by those skilled in the art. Generally, the ingredients come from plants that have been previously selected by various methods. Ethnobotany is the most used approach. It consists in studying preferentially plants whose use is already existing in traditional uses or by ancient medicines. The selected plant is tested on appropriate biological models. For each plant many tests are to be performed on different organs of the plant and different modes of extraction. In the case of plants selected by the ethnobotanical approach, traditional use directs the researcher towards the biological model (s) to be used. The ethnobotanical approach is very effective for the selection of terrestrial plants, which is however not the case for marine plants that are little or not at all known for traditional uses.

  The present invention relates to the discovery, development and use of a new family of assets particularly interesting. This new family comes from the marine world and / or fresh water and more specifically marine and / or freshwater containing microorganisms. These microorganisms belong to the different families of microalgae and marine bacteria (= cyanobacteria), widely distributed in the seas, oceans, lakes, rivers and ponds, commonly called phytoplankton.

  The present invention more particularly relates to a cosmetic, dermatological, pharmaceutical or food composition, characterized in that it comprises a culture medium of at least one marine microorganism and / or fresh photosynthetic water, said medium being clarified.

  Generally, microorganisms are grown under standardized conditions to provide a biomass that is usually obtained by centrifugation. This biomass can then undergo the same extraction techniques as those used for terrestrial plants, and can therefore result in molecules and biologically active extracts.

  However, in the case of the present invention, it is not the biomass that is used as a source of new active substances but it is the medium freed of biomass. This culture medium is called clarified because it represents the result of a clarified culture by removing the biomass, that is to say by eliminating all the microorganisms and cell debris present in the culture medium. Surprisingly, the Applicant has discovered that the properties of the clarified culture medium are different from those of the biomass. Thus, when the clarified culture medium of the present invention has particularly advantageous cosmetic, dermatological, pharmaceutical and food properties, this is not necessarily the case with biomass.

  The clarified culture medium used in the composition of the invention refers to the culture medium obtained after amplification of the microorganism (s) cultured, and after separation of all the microorganisms and cell debris from the culture medium. The clarified culture medium then contains only the substances resulting from the culture of the microorganism (s), also called active substances.

  The active substances denote in particular the primary and secondary metabolites synthesized and released by the cells of said marine microorganism (s) and / or fresh water, said metabolites being able to be excreted during cell activity or else released after lysis. cells. These active substances may designate molecules of various nature: enzymes, peptides, amino acids, vitamins, polysaccharides, etc., and possess different biological properties capable of playing a role in the field of cosmetics, pharmaceuticals or cosmetics. nutrition.

  The Applicant has shown that, depending on the nature of the culture medium, the choice of the microorganism (s) grown and the cultivation protocol developed, new and very interesting active substances could be produced for cosmetic, dermatological, pharmaceutical and food applications.

  The composition of the invention is remarkable in that it has particularly advantageous cosmetic properties, especially depigmenting and antioxidant properties, thanks to the active substances present in the clarified medium.

  According to an advantageous embodiment of the invention, the marine microorganism and / or photosynthetic fresh water is a cyanobacterium and / or a microalga resulting from marine environments such as sea, ocean, brackish water, saline water or water soft.

  Saline water refers to water with varying degrees of salinity ranging from 35 g / L to 90 g / L.

  According to a preferred embodiment of the invention, the marine microorganism and / or photosynthetic freshwater is a cyanobacterium and / or a microalga belonging to the division of chlorophytes chosen from the group consisting of cyanophytes, chlorophytes , chrysophytes, chlorarachniophytes, dinophytes, cryptophytes, euglenophytes or rhodophytes.

  More particularly, the cyanobacterium and / or microalgae belongs to the class of Prasinophyceae chosen from the group consisting of cyanophyceae, chlorophyceae, prasinophyceae, pedinophyceae, prymnesiophyceae, eustigmatophyceae, raphidophyceae, chrysophyceae, diatomophyceae , tribophyceae, chlorarachniophyceae, dinophyceae, cryptophyceae, euglenophyceae and rhodophyceae.

  Among the microalgae that can be used, there may be mentioned, for example.

  - Nostoc commune, Synechococcus sp. and Synechocystis sp, cyanophycea class; - Scenedesmus dimorphus, Scenedesmus acutus, Dunaliella primolecta, Dunaliella tertiolecta, Dunaliella bardawil, Neochloris oleobundans, Clamydomonas reinhardtii, Botryococcus braunii, Chlorococcum oleofaciens, Dysmorphococcus globosus, Hormotilopsis gelatinosa, Ulothrix acuminata and Ourococcus sp, class chlorophyceae; - Tetraselmis suecica, from the class of prasinophyceae; - Pavlova lutheri and Isochrysis galbana, of the class of prymnesiophyceae; - Nannochloropsis oculata, of the class of eustigmatophyceae; - Phaeodactylum tricornutum, Skeletonema costatum, Chaetoceros calcitrans, Cyclotella sp, Thalassiosira pseudonana and Navicula pelliculosa, of the class of diatomophyceae; - Porphyridium cruentum and Rhodosorus marinas, of the rhodophycea class.

  The composition of the invention is also characterized in that the amount of the clarified culture medium is from 0.01% to 100% by weight relative to the total weight of the composition.

  The composition of the invention is further characterized in that said clarified culture medium is obtained by a process comprising the following steps: a) the selection of at least one marine microorganism and / or fresh photosynthetic water as defined herein above, b) the introduction, into a culture medium, of the microorganism (s) selected (s) during the previous step a), c) the amplification of said microorganism (s) in said culture medium, d) the elimination of all the microorganisms obtained at the end of the amplification step c), in order to obtain a clarified culture medium of said photosynthetic marine microorganism (s) and / or freshwater.

  The set of microorganisms obtained at the end of step c) of amplification constitutes the biomass.

  The clarified culture medium, obtained at the end of step d) of elimination, can undergo certain modifications before its use. Thus, it can for example be desalted by electrodialysis or nanofiltration to reduce its concentration of sodium chloride and facilitate its use in certain formulations. It can be concentrated by eliminating part of the water constituting it, or on the contrary it can be diluted. It can also be spray-dried or lyophilized to be in concentrated form more usable in certain applications.

  When at least two microorganisms are selected in step a) described above, these two microorganisms may be identical or different. Two different microorganisms denote two microorganisms, and in particular two microalgae, belonging to a species different from each other. In addition, they may belong to a different class and / or genre or order.

  The culture medium of step b) (in which at least one microorganism is introduced) is selected from the group consisting of Marine Source Water from Ile Grande, sea water, water from ocean, brackish water, saline water, fresh water, a clarified culture medium of at least one microorganism or mixtures thereof.

  According to an advantageous embodiment of the invention, the culture medium of step b) (in which at least one microorganism is introduced) is a clarified culture medium of at least one marine microorganism and / or water sweet photosynthetic as defined above or as obtained at the end of the process as described above.

  According to an advantageous embodiment, the culture medium of step b) can be enriched in nutrients selected from the group consisting of minerals, trace elements and vitamins.

  The culture (step c) amplification) can be carried out at the appropriate temperature appropriate to the species of the microorganism (s) cultivated (s), including the microalgae (s) cultivated (s). Generally, this temperature is between 15 and 25 C depending on the nature of the crop species.

  The pH of the culture medium is between 2 and 12, and preferably between 7 and 8 depending on the nature of the cultivated species.

  Each day, different parameters are measured (optical density, cell concentration, temperature, oximetry, etc.) in order to follow the evolution of cell growth in the culture medium.

  Step d) elimination of all the microorganisms obtained at the end of step c) amplification can be obtained by any conventional technique, such as centrifugation or filtration, and preferably centrifugation . This step d) makes it possible to separate the biomass from the culture medium and to obtain the clarified culture medium.

  The clarified culture medium can be obtained at different physiological stages of the culture, depending on the active substances that are sought (beginning or end of exponential growth phase, stationary phase).

  According to one embodiment of the invention, the clarified culture medium, obtained at the end of the process as described above, comprises for example: - Marine Source Water of the Big Island (possibly enriched in nutrients), - primary and secondary metabolites synthesized and released by the microalgual cells themselves (active substances).

  However, according to the microalgae grown, the Marine Source Water of the Big Island can be replaced by sea water, fresh water or brackish water (mixture of Marine Water Source of Great Island and fresh water). The Marine Source Water of the Big Island can also be replaced by the clarified culture medium of another microalga, see by its own clarified culture medium.

  According to an advantageous embodiment of the invention, a microalgae can therefore be cultured in its own clarified culture medium or else in the clarified culture medium of another microalgae, with or without the addition of a nutrient medium (concept of Clarificulture ", term invented by the Applicant). Similarly, as has already been mentioned, two or more microalgae, identical or different, can be grown in the same culture medium (concept of co-culture).

  Similarly, each nutrient medium (including minerals, trace elements, vitamins ...) is specific to each microalga and its growth, and can vary from one microalga to another.

  The type and concentration of active substances synthesized by the microalgual cells are functions of the cultivated microalgae species, the physiological phase of growth and the environmental conditions.

  Each microalgae culture, necessary for obtaining clarified culture media, is carried out in photobioreactors or basins. A small volume of a sterile microalgae concentrate solution serves as an inoculum to start the culture.

  The composition according to the invention is further characterized in that it contains at least one other active ingredient. This ingredient can be any asset useful to the properties of the composition.

  The active ingredient that can supplement the compositions of the invention may also come from the biomass as obtained at the end of the elimination step d) as defined above.

  According to an advantageous embodiment, the clarified culture medium used as an active agent in the compositions of the invention is vectorized in liposomes or other vectors in order to promote their pharmaceutical, cosmetic, dermatological or food activity.

  The present invention also relates to a method for preparing a clarified culture medium of at least one marine microorganism and / or photosynthetic freshwater, characterized in that it comprises the following steps: a) the selection of at least one marine microorganism and / or fresh photosynthetic water as defined above, b) introducing, in a culture medium, the microorganism (s) selected (s) during step a ) preceding, c) the amplification of said one or more microorganisms in said culture medium, d) the elimination of all the microorganisms obtained at the end of the amplification step c), in order to obtain a medium clarified culture of said one or more photosynthetic marine microorganisms and / or freshwater.

  The preparation process according to the invention is more particularly characterized in that it comprises the steps as described above.

  According to one embodiment of the invention, the preparation process of the invention is characterized in that a further step of screening the clarified culture medium as defined above, or such as obtained according to the process as defined above, in order to evaluate the pharmaceutical, cosmetic, dermatological or food properties of said clarified culture medium.

  The present invention also relates to the use of a clarified culture medium as defined above for the preparation of a cosmetic composition with lightening and / or depigmenting activity.

  The invention also relates to the use of a clarified culture medium as defined above for the preparation of a protective cosmetic composition against free radicals, to fight against aging.

  The invention also relates to the use of a clarified culture medium as defined above for the preparation of a cosmetic or pharmaceutical composition for combating cutaneous sensitivity and inflammatory reactions.

  The invention also relates to the use of a clarified culture medium as defined above for the preparation of a cosmetic composition with pigmenting activity on the skin and the hair.

  The invention also relates to the use of a clarified culture medium as defined above for the preparation of a cosmetic composition with slimming activity.

  The invention also relates to the use of a clarified culture medium as defined above for the preparation of a cosmetic composition with bronzing activity.

  The invention also relates to the use of a clarified culture medium as defined above for the preparation of a cosmetic composition promoting the growth and regrowth of the hair.

  The invention also relates to the use of a clarified culture medium as defined above for the preparation of a cosmetic composition with firming, toning, stimulating, balancing activity.

  Figure 1 illustrates the reaction to Dopa-Oxidase on separate epidermis. The top left square represents the DOPA control, the upper right square represents kojic acid, the bottom left represents the clarified culture medium of Phaeodactylum tricornutum and the bottom right square represents the culture medium. clarified Tetraselmis suecica.

  Other advantages and features of the invention will appear on reading the examples which follow. These examples illustrate the invention without limiting it in any way.

  Example 1. Preparation of a clarified culture medium from a culture of Tetraselmis suecica, and its cosmetic use as a depigmenting agent.

  A pool located under a greenhouse, after Deptil PA 5 disinfection, is filled with 20 m3 of Marine Source Water from Ile Grande.

  A sterile nutrient medium (CONWAY medium) is added to the Marine Source Water of Ile Grande.

  The composition of the nutrient medium is Na2 EDTA: 0.045 g / L Na2 NO3: 0.1 g / L H3BO3: 0.0336 g / L NaH2PO4, 2H2O: 0.026 g / L MnCl2, 4H2O: 3.6.10-4 g / L FeCl3: 6H2O: 1.3.10-3 g / L ZnCl2: 2.1.10-5 g / L CoCl2, 6H2O: 2.10-5 g / L (NH4) 6Mo7O24, 4H2O: 9.106 g / L CuSO4, 5H2O: 2.10- 5 g / L Vitamin B1: 2.104 g / L Vitamin B12: 1.10-5 g / L The pool is inoculated with 2500L of a concentrated culture of the microalga Tetraselmis suecica.

  The bubbling, provided by 2 bubbler ramps located on each side of the basin, brings to the culture of air and CO2, and thus allows a permanent mixing of the cells.

  Tetraselmis suecica is cultivated at a pH of 7 thanks to an automatic regulation in CO2 and a temperature of 20 C.

  Weekly monitoring of the culture is performed to evaluate cell growth. The measured parameters are: pH, T, 02, DO.

  The culture is carried out at a pH of between 7 and 8, and at a temperature of between 20 and 23 ° C. The culture is conducted until a cell concentration corresponding to an optical density of 0.300 to the length of d is obtained. 435 nm wave.

  After ten days, the culture is centrifuged (16500 revolutions / min) and the clarified culture medium obtained is conditioned and stored in a cold room.

  The clarified culture medium is then concentrated and desalted by nanofiltration + diafiltration (cutoff threshold of the membrane = 1000 daltons) in order to achieve isotonicity and can be incorporated into cosmetic compositions with depigmenting activity.

  Example 2 Obtaining a clarified culture medium from a culture of Scenedesmus acutus, and its cosmetic use as an antioxidant.

  A photobioreactor, after disinfection with Deptil PA 5, is filled with 300 Liters of fresh water.

  A sterile nutrient medium (MLA medium) is added to the fresh water.

  The composition of the nutrient medium is as follows: Na 2 EDTA: 4.36 × 10 -3 g / L FeCl 3: 6H 2 O: 1.58 × 10 -3 g / L NaHCO 3: 0.6 × 10 -3 g / L MnCl 2, 4H 2 O: 0.36 × 10 3 g / L CuSO 4, 5H 2 O: 1.10-5 g / L ZnSO 4, 7H 2 O: 2.2 × 10 -5 g / L CoCl 2, 6H 2 O: 1.105 g / L MgSO 4: 4.95. 10-2 g / L NaNO3: 0.17 g / L H3BO3: 2.47. 10-3 g / L NaHCO 3: 4.23. 10-3 g / L K 2 HPO 4: 3.475. 10-2 g / L CaC12: 2, 94. 10-2 g / L Vitamin B1: 1. 10-4 g / L Vitamin B12: 5. 10-8 g / L Vitamin H: 5. 10-8 g / L The photobioreactor is inoculated with 25 liters of a sterile and concentrated culture of Scenedesmus acutus. A bubbling in air and CO2 ensures the culture a permanent mixing of the cells and a maintenance of the pH around the neutrality. The culture of Scenedesmus is carried out at a temperature of 20 ° C.

  24 hours after inoculation, neon ramps are lit around the crop to provide the essential light for photosynthesis.

  Weekly monitoring of the culture is performed to evaluate cell growth. The measured parameters are: pH, T, 02, DO.

  The culture is carried out at a pH between 6.5 and 8, and at a temperature between 17 and 21 C. The culture is conducted until a cell concentration corresponding to an optical density of 1.4 is obtained. at the wavelength of 679 nm.

  After 15 days, the culture is centrifuged (16500 rev / min) and the clarified product is conditioned and stored in a cold room.

  The clarified culture medium is then concentrated by thermal evaporation to be incorporated in cosmetic compositions with antioxidant activity.

  Example 3 Culture of Phaeodactylum tricornutum in the clarified culture medium of Porphyridium cruentum.

  Beforehand a Porphyridium cruentum culture is carried out as follows: A photobioreactor, after Deptil PA 5 disinfection, is filled with 300 Liters of Marine Water from the Grande Ile.

  A sterile nutrient medium (CONWAY medium) is added to Seawater.

  The composition of the nutrient medium is as follows: Na2 EDTA: 0.045 g / L Na2 NO3: 0.1 g / L H3BO3: 0.0336 g / L NaH2PO4, 2H2O: 0.026 g / L MnCl2, 4H2O: 3.6.10-4 g / L FeCl3: 6H2O: 1.3.10-3 g / L ZnCl2: 2.1.10-5 g / L CoCl2, 6H2O: 2. 10-5 g / L (NH4) 6Mo, O24, 4H2O: 9.10-6 g / L CuSO4, 5H2O: 2.10-5 g / L Vitamin B1: 2.10-4 g / L Vitamin B12: 1.10-5 g / L The photobioreactor is inoculated with 10 liters of a sterile and concentrated culture of Porphyridium cruentum. A bubbling in air and CO2 ensures the culture a permanent mixing of the cells and a maintenance of the pH around the neutrality. The culture of Porphyridium cruentum is carried out at a temperature of 20 C.

  24 hours after inoculation, neon ramps are lit around the crop to provide the essential light for photosynthesis.

  Weekly monitoring of the culture is performed to evaluate cell growth. The measured parameters are: pH, T, 02, DO.

  The culture is carried out at a pH of between 7.5 and 8 and at a temperature of between 19 and 23 ° C. The culture is conducted until a cell concentration corresponding to an optical density of 0.112 is obtained. 440 nm wavelength.

  After 15 days, the culture is centrifuged (16,500 rpm) and the clarified culture medium obtained is filtered on lpm in order to eliminate the residual cells.

  A new photobioreactor disinfected with Deptil PA 5 is filled with the 300 liters of the clarified filtered Porphyridium Cruent obtained previously.

  Liters from a sterile and concentrated culture of Phaeodactylum tricornutum serve as inoculants for culture. A bubbling in air and CO2 ensures the culture a permanent mixing of the cells and a maintenance of the pH around the neutrality. The culture of Phaeodactylum tricornutum is carried out at a temperature of 20 C.

  24 hours after inoculation, neon ramps are lit around the crop to provide the essential light for photosynthesis.

  Weekly monitoring of the culture is performed to evaluate cell growth. The measured parameters are: pH, T, O2, DO.

  The culture is carried out at a pH between 6.5 and 8, and at a temperature between 19 and 23 C. The culture is conducted until the stationary phase is obtained.

  After 10 days, the culture is centrifuged (16500 rev / min) and the clarified culture medium obtained is conditioned and stored in a cold room.

  EXAMPLE 4 Co-cultivation of Phaeodactylum tricornutum and Skeletonema costatum in the same medium

  A photobioreactor, after Deptil PA 5 disinfection, is filled with 300 Liters of Marine Water from Grande Ile.

  A sterile nutrient medium (CONWAY medium) is added to Seawater.

  The composition of the nutrient medium is as follows: Na2 EDTA: 0.045 g / L Na2 NO3: 0.1 g / L H3BO3: 0.0336 g / L NaH2PO4, 2H2O: 0.026 g / L MnCl2, 4H2O: 3.6.10-4 g / L FeCl3: 6H2O: 1.3.10-3 g / L ZnCl2: 2.1.10-5 g / L CoCl2, 6H2O: 2. 10-5 g / L (NH4) 6Mo, O24, 4H2O: 9.10-6 g / L CuSO4, 5H2O: 2.10-5 g / L Vitamin B1: 2.10-4 g / L Vitamin B12: 1.10-5 g / L Silica: 0.04 g / L The photobioreactor is inoculated with 10 liters of a sterile culture and concentrated Phaeodactylum tricornutum and then with 10 liters of a sterile and concentrated Skeletonema costatum culture. A bubbling in air and CO2 ensures the culture a permanent mixing of the cells and a maintenance of the pH around the neutrality. The co-culture of Phaeodactylum tricornutum and Skeletonema costatum is carried out at a temperature of 20 C.

  24 hours after inoculation, neon ramps are lit around the co-culture to provide the essential light for photosynthesis.

  Weekly monitoring of co-culture is performed to evaluate cell growth. The measured parameters are: pH, T, 02, DO.

  The culture is carried out at a pH of between 7 and 8 and at a temperature of between 21 and 23 ° C. The culture is carried out until a cell concentration corresponding to an optical density of 0.250 to the length of d wave of 440 nm. 10

  After 10 days, the co-culture is centrifuged (16500 rev / min) and the eclammate obtained is conditioned and stored in a cold room.

  Example 5. Preparation of a clarified culture medium from a culture of Skeletonema costatum.

  1) Cultivation of Skeletonema costatum in Marine Source Water of the Big Island.

  A pool located under a greenhouse, after Deptil PA 5 disinfection, is filled with 20 m3 of Marine Source Water from Ile Grande.

  A sterile nutrient medium (CONWAY medium) is added to the Marine Source Water of Ile Grande.

  The composition of the nutrient medium is as follows: Na2 EDTA: 0.045 g / L Na2 NO3: 0.1 g / L H3303: 0.0336 g / L NaH2PO4, 2H2O: 0.026 g / L MnCl2, 4H2O: 3.6.10-4 g / L FeCl3: 6H2O: 1.3.10-3 g / L ZnCl2: 2.1.10-5 g / L CoCl2, 6H2O: 2. 10-5 g / L (NH4) 6Mo, O24, 4H2O: 9.10-6 g / L CuSO4, 5H2O: 2.10-5 g / L Vitamin B1: 2.10-4 g / L Vitamin B12: 1.10-5 g / L Silica: 0.04 g / L The pool is inoculated with 2500L of a concentrated culture of the microalga Skeletonema costatum. 25 30

  The bubbling, provided by 2 bubbler ramps located on each side of the basin, brings to the culture of air and CO2, and thus allows a permanent mixing of the cells.

  Skeletonema costatum is cultivated at a pH of 7 thanks to an automatic regulation in CO2 and at a temperature of 20 C.

  Weekly monitoring of the culture is performed to evaluate cell growth. The measured parameters are: pH, T, O2, DO.

  The culture is carried out at a pH of between 7 and 8, and at a temperature of between 20 and 23 ° C. The culture is conducted until a cell concentration corresponding to an optical density of about 0.760 is obtained. 440 nm wavelength.

  After ten days, the culture is centrifuged (16500 revolutions / min) and the clarified culture medium obtained is conditioned and stored in a cold room.

  The clarified culture medium is then concentrated and desalted by nanofiltration + diafiltration (cut-off point of the membrane = 1000 daltons) in order to reach isotonicity and can be incorporated into cosmetic compositions.

  2) Cultivation of Skeletonema costatum in water from groundwater.

  An outdoor pool is filled with water from an underground aquifer.

  The pool is inoculated with a concentrated culture of the microalga Skeletonema costatum.

  The agitation of the culture, allowing a permanent mixing of the cells, is ensured by bubbler ramps located at the bottom of the basin.

  The culture is conducted until a cell concentration corresponding to an optical density of at least 0.600 at the wavelength of 440 nm is obtained.

  When the desired cell concentration is reached, the culture is centrifuged (16500 rpm) and the clarified culture medium obtained is conditioned and stored in a cold room.

  The clarified culture medium is then concentrated and desalted by nanofiltration + diafiltration to achieve isotonicity and can be incorporated into cosmetic compositions.

  EXAMPLE 6 Method for Screening Depigmented Clarified Culture Media by the Dopa-Oxidase Reaction on Separated Epidermis (BIO-EC Laboratory) The method consists in visualizing on human melanocytes the depigmenting potential of a composition tested by inhibition of L-DOPA oxidase (one of the enzymes responsible for the synthesis of melanins).

  The reaction is carried out by mixing the depigmenting composition and L-DOPA. The reaction is visualized by the intensity of melanocyte staining on separate epidermis.

  The 100% activity control (maximum staining) is carried out in the presence of L-DOPA (substrate of L-DOPA oxidase).

  The positive control (minimum staining) is carried out in the presence of LDOPA supplemented with 0.015% kojic acid. Kojic acid is known for its depigmenting activity.

  The clarified culture media tested are respectively: the clarified culture medium of Tetraselmis suecica as obtained in Example 1; the clarified culture medium of Phaeodactylum tricornutum as obtained in Example 3.

  The epidermals used are those from a frozen mammoplasty. The epidermis are separated by incubation in 2N NaBr (1:45) at 37 ° C.

  The epidermides are fixed in a buffered formaldehyde fixative, are rinsed and brought into contact with the volume / volume mixture: L-DOPA solutions (substrate) / clarified culture medium (50 μg of freeze-dried extract / ml of assay medium) .

  After incubation, the epidermis are rinsed, mounted between slide and coverslip with Mounting Medium mounting liquid (aqueous mixture containing glycerol and allowing perfect preservation of the sample). The observation is performed by optical microscopy (objective x 10).

  The results of the Dopa- Oxidase reaction on the separated epidermis are illustrated in FIG. 1. Conclusion: the clarified culture medium of Tetraselmis suecica has a depigmenting activity similar to that obtained with kojic acid on the epidermis model separated, while the clarified culture medium of Phaedactylum tricornatum does not exhibit depigmenting activity.

  Example 7: Effects of Clarified Culture Media on IL8 Production by IL-15-stimulated KHNs (SEPhRA Laboratory) Test Principle: Normal human keratinocytes are cultured in a microplate until 80% confluence.

  KHN primocultures are obtained from isolated cells of cutaneous samples from plastic surgery. The KHNs are seeded at the density of 20000 cells per well (KSFM specific culture medium, Life Technologies) in a 96-well microplate. The cells are incubated in a humid incubator at 37 ° C. under 5% CO 2 for 24 hours. The cells are then treated for the next 24 hours with the different clarified culture media to be tested at the same time as they are stimulated with IL-1 at 1 ng / ml.

  4 wells are treated by dilutions (n = 4), the dilutions being carried out in the culture medium. Cells are left untreated and others are treated only with IL-15. Culture supernatants containing IL-8 are stored at -20 ° C until the assay.

  Human IL-8 is quantified using the Quantikine ELISA microplate assay (Quantikine human kit IL-8 D8050, R & D systems). The test uses the enzyme immunoassay method of the sandwich. A monoclonal antibody specific for IL-8 was precoated on a microplate.

  The following are deposited in the wells of the titration microplate: the standards (recombinant human IL-8) (n = 2) at concentrations ranging from 0 to 1000 μg / ml, the culture supernatants to be assayed containing the IL 8 released by the cells treated with the clarified culture medium (= 4), - the anti-IL-8 polyclonal antibody conjugated to the Horse Radisch Peroxidase enzyme.

  If IL-8 is present, it is trapped between the immobilized antibody and the conjugated polyclonal antibody. After washing removing all non-adhered substances, a substrate solution (hydrogen peroxide and tetramethylbenzidine) is added to the wells. Staining develops in proportion to the amount of IL-8 trapped. The color reaction is stopped and the staining intensity (OD) measured at 450 nm.

  The quick summary of the protocol is as follows.

  1. Add 100 μl / well of assay buffer (provided by the kit), 2. Add 50 μl / well of standard (known IL8 concentrations) or assay samples (culture supernatants containing IL8 released from the cells treated with the clarified culture medium). Shake the plate to mix the reagents in each well.

  3. Add 100 μl / well of conjugated IL-8.

  4. Incubate the whole 2h30 at room temperature.

  5. Empty the wells and rinse with 5 or 6 washes.

  6. Add 200 μl / well of substrate solution (hydrogen peroxide and tetramethyl benzidine) and incubate for 30 minutes, protected from light.

  7. Add 50 μl stop solution (acid solution).

  8. Read the absorbance at 450 nm within 30 minutes and calculate the concentration results in μg / ml IL-8.

  In parallel, a protein assay for each well of the microplate is performed. The method used is that of bicinchoninic acid (or BCA). After 24 hours of treatment, the supernatants are frozen and the wells rinsed with PBS without Ca "and Mg '(Gibco) The cell mat is dissolved with a solution of 0.1M NaOH. Bicinchoninic acid and copper sulfate (50V / 1V) is added.The plate is placed at 37C under 5% Cq in the dark.After 30 minutes, the absorbance at 570 nm is measured. standard is made from a stock solution of BSA (Bovine serum albumin) 1 mg / ml (P0914, SIGMA) to convert the OD amounts (pg of protein).

  The final result is expressed in μg / ml of IL-8 / g of proteins (obtained by the BCA assay).

  20% inhibition of the release of IL8 by the keratinocytes after treatment with the clarified culture medium at 25 μg / ml is observed for the clarified culture medium of Dunalliela primolecta.

  Conclusion: Interleukin 8 release reflects an inflammatory terrain; therefore, by inhibiting the release of IL-8, an inflammatory condition of the skin is combated.

  Dunalliela primolecta therefore has in vitro anti-inflammatory properties.

  Example 8: Anti-Radical Protocol - DNA Damaged Detection (DNA Damaged Detection): Quantitative Analysis of the Protective Effects of a Clarified Culture Medium of the Invention.

  The clarified culture media tested are Scenedesmus dimorphus and Nostoc commune.

  The measurement of the anti-free radical power is carried out using a dosing kit (IDN No. 003) marketed by SFRI Laboratoire (FSaint Jean d'Illac) for the purpose of highlighting the protection of the skin. DNA by potentially anti-radical extracts against lesions caused by hydroxyl radicals (represented by OH), radicals generated by the Fenton reaction below: Fe2 + + H2O2 OH + OH- + Fei + The detailed protocol is as follows: The wells are prewashed: 300 μl of washing solution (phosphate buffer, pH 7), diluted to 1 / 10th with ultra-pure water containing no traces of metals are added, then the water is removed by suction.

We repeat the operation once.

  50 μl of previously damaged or uninjured DNA is dispensed into the well. The titration microplate is covered with an adhesive film and incubated for 30 minutes at 30 ° C. with gentle stirring. The wells are washed twice.

  The clarified culture media to be tested are dispensed: 50 μl of each diluted sample is dispensed per well (the clarified culture media are tested in a concentration range from pure to 1: 100 dilution). C with gentle stirring. The wells are washed 3 times. The DNA lesions are repaired by an enzymatic repair system (mixture supplied in the assay kit and patented by the supplier) at the rate of 50 μl per well of repair in each well. The plate is covered with an adhesive film and incubated for 3 hours at 30 ° C. without stirring. The wells are washed 3 times. 50 μl of revealing solution I (avidin-labeled nucleotide peroxidase) is dispensed into all wells.

  The plate is covered with an adhesive film and incubated for 15 minutes at 30 ° C. with gentle stirring. The wells are washed 5 times. 50 of the revealing solution II (avidin chemiluminescent substrate peroxidase) are dispensed into each well. The mixture is incubated for 5 minutes at 30 ° C. with gentle stirring in the dark. The plate is read with a luminometer.

  The results are expressed in arbitrary chemiluminescence unit (RLU).

  Pourcentage percentage of nonspecific inhibition: (RLU DNA damage) - (RLU DNA lesion + sample) / (RLU DNA damage) x 100; Pourcentage% inhibition in the presence of free radicals: (RLU H2O2) - (RLU H2O2 + sample) / (RLU H202) x 10 0; % Specific protection percentage = (% inhibition in the presence of ROS) - (% nonspecific inhibition); (ROS = reactive oxygen species) Table 1 below represents the values obtained for the clarified culture medium of Scenedesmus dimorphus.

Table 1:

  Concentration tested in mghnL 1 0.1 0.01 0.001% non-specific inhbition 14.55 18.19 18.19% inhbition in the presence ROS 81.89 79.89 69.35 35% protection 67.34 61.7 51.15 35 20 Conclusion: the clarified culture medium of Scenedesmus dimorphus has an IC50 (concentration allowing obtain 50% inhibition of the effect of free radicals) close to 0.01 mg / ml.

  During external stress such as prolonged exposure to ultraviolet radiation or pollution etc ..., the cells express a high level of free radicals, resulting in deleterious effects on the cell (degradation of proteins, lipids, DNA damage, cell death). An extract with an antiradical property such as Scenedesmus dimorphus extract will allow the cell to better defend itself.

  Table 2 below represents the values obtained for the clarified Nostoc culture medium.

Table 2.

  Concentration tested in mgfmL 1 0.1 0.01 0.001% nonspecific inhibition 18.3 Io inhibition in the presence ROS 66.74 55.12 26.28 16.393% protection 48.43 36.82 7.973 -1.9102 Conclusion: the clarified culture medium of Nostoc commune presents an IC50 (concentration allowing to obtain 50 % inhibition of the effect of free radicals) close to 1 mg / ml.

  An extract presenting an antiradical property like the Nostoc common extract will allow the cell to better defend itself.

  Example 9. DNA Damaged Detection Test 3D: Demonstration of the Repair Effects of a Clarified Culture Medium of the Invention on DNA.

  The clarified culture medium tested is Skeletonema costatum.

  The measurement of the repairing power of the DNA is carried out by means of a kit of assay (ref IDN 003) marketed by the company SFRI Laboratory (F-Saint Jean d'Illac) aiming at the highlighting of activation of DNA repair by extracts to repair lesions caused by hydroxyl radicals (represented by OH), radicals generated by the Fenton reaction below: Fe2 + + H2O2 p OH '+ OH- + The detailed protocol is as follows: The wells are prewashed: 300 μl of washing solution (phosphate buffer, pH 7), diluted 1/10 with ultra pure water containing no traces of metals, are added. then the water is removed by suction. We repeat the operation once.

  50 μl of previously damaged or uninjured DNA is dispensed into the well. The titration microplate is covered with an adhesive film and incubated for 30 minutes at 30 ° C. with gentle stirring. The wells are washed twice.

  The clarified culture media to be tested are dispensed: 50 μl of each diluted sample is dispensed per well (the clarified culture media are tested in a concentration range from pure to a 1/100 dilution). 30 C with gentle stirring. The wells are washed 3 times. The DNA lesions are repaired by an enzymatic repair system (mixture supplied in the assay kit and patented by the supplier) at the rate of 50 μl per well of repair in each well. The plate is covered with an adhesive film and incubated for 3 hours at 30 ° C. without stirring. The wells are washed 3 times. 50 μl of revealing solution I (avidin-labeled nucleotide peroxidase) is dispensed into all wells.

  The plate is covered with an adhesive film and incubated for 15 minutes at 30 ° C. with gentle stirring. The wells are washed 5 times. 50 μl of revealing solution II (chemiluminescent substrate of avidin peroxidase) is dispensed into each well. The mixture is incubated for 5 minutes at 30 ° C. with gentle stirring in the dark. The plate is read with a luminometer.

  The results are expressed in arbitrary chemiluminescence unit (RLU).

  É Percentage increase of the repair rate: (RLU damaged DNA + sample) * 100 / (RLU DNA lesioned); Conclusion: The clarified culture medium of Skeletonema costatum induces an increase in the level of DNA repair from 40% to 1 μg / ml.

  During external stress, the cells express a high level of free radicals, resulting in deleterious effects on the cell, including lesions on the DNA.

  An extract showing the ability to increase the speed of repair of the damaged DNA, such as Skeletonema costatum extract, will allow the cell to recover its DNA capital more quickly.

  Example 10: Cosmetic emulsion depicting a clarified culture medium Tetraselmis suecica.

  The composition of a depigmenting cosmetic emulsion of the invention is for example: clarified culture medium of Tetraselmis suecica obtained according to Example 1: 20 g conventional cosmetic emulsion (fatty alcohols, polyoxyethylenated fatty alcohols, vegetable oil, palmitate, isopropyl, glycerin, carbopol type gelling agent, preservatives, perfumes, water). qsp 100 g Example 11: Dry skin protective powder for bathing clarified culture medium of Scenedesmus acutus.

  The clarified culture medium of Scenedesmus acutus is obtained according to Example 2, then is concentrated by thermal evaporation and lyophylized until a powder is obtained to homogenise before packaging in 20 g bags to be used in the bath.

  EXAMPLE 12 Moisturizing Lotion for Sensitive Skins of Clarified Culture Medium of Dunalliela Primolecta The clarified culture medium of Dunalliela primolecta is obtained according to the protocol described below.

  A photobioreactor, after disinfection with Deptil PA 5, is filled with 300 Liters of fresh water.

  A sterile nutrient medium (CONWAY medium) is added to the fresh water.

  The composition of the nutrient medium is as follows: Na, EDTA: 0.045 g / L Na, NO3: 0.1 g / L H3BO3: 0.0336 g / L NaH2PO4, 2H2O: 0.026 g / L MnCl2, 4H2O: 3.6 . 10-4 g / L FeCl3: 6H2O: 1.3.10-3 g / L ZnCl2: 2.1.10-6 g / L CoCl2, 6H2O: 2.106 g / L (NH4) 6Mo, O24, 4H2O: 9.10-6 g / L L CuSO4, 5H2O: 2.10-6 g / L Vitamin B1: 2.10-4 g / L Vitamin B12: 1.10-5 g / L The photobioreactor is inoculated with 10 liters of a sterile and concentrated Dunaliella primolecta culture. A bubbling in air and CO2 ensures the culture a permanent mixing of the cells and a maintenance of the pH around the neutrality. The culture of Dunaliella is carried out at a temperature around 20 C.

  24 hours after inoculation, neon ramps are lit around the crop to provide the essential light for photosynthesis.

  Weekly monitoring of the culture is performed to evaluate cell growth. The measured parameters are: pH, T, 02, DO.

  The culture is carried out at a pH between 6.5 and 8, and at a temperature between 19 and 27 C. The culture is conducted until a cell concentration corresponding to an optical density of 0 is obtained. , 6 at the wavelength of 438 nm.

  After 16 days, the culture is centrifuged (16500 rpm).

  The clarified culture medium is then concentrated by thermal evaporation and desalted by dialysis membrane (cutoff threshold = 500 Da). The composition of a moisturizing lotion of the invention is, for example: clarified culture medium of Dunalliela primolecta obtained such that described above: 50 g - clarified culture medium Porphyridium cruentum obtained according to the first phase of the protocol described in Example 3, but until a viscous clarified (marketed by the Applicant under the name hydrocomplex 2) 50 g Example 13: Bioprotective foundation.

  The clarified culture medium of Scenedesmus dimorphus is prepared according to the following protocol: A photobioreactor, after disinfection with Deptil PA 5, is filled with 300 Liters of fresh water.

  A sterile nutrient medium (MLA medium) is added to the fresh water.

  The composition of the nutrient medium is as follows.

  Na2 EDTA: 4.36 × 10 -3 g / L FeCl3: 6H2O: 1.58 × 10 -3 g / L NaHCO3: 0.6 × 10 -3 g / L MnCl2, 4H2O: 0.36 × 10 -3 g / L CuSO4, 5H2O 1.10- 5 g / L ZnSO4, 7H2O 2.2.10-5 g / L CoCl2, 6H2O: 1.10-5 g / L MgSO4: 4.95. 10-2 g / L NaNO3: 0.17 g / L H3BO3: 2.47 . 10-3 g / L NaHCO 3: 4.23. 10-3 g / L K 2 HPO 4: 3.475. 10-2 g / L CaC12: 2, 94. 10-2 g / L Vitamin B1: 1. 10-4 g / L Vitamin B12: 5. 10-8 g / L Vitamin H: 5. 10-8 g / L The photobioreactor is inoculated with 10 liters of a sterile and concentrated culture of Scenedesmus dimorphus. A bubbling in air and CO2 ensures the culture a permanent mixing of the cells and a maintenance of the pH around the neutrality. The culture of Scenedesmus is carried out at a temperature of 20 C.

  24 hours after inoculation, neon ramps are lit around the crop to provide the essential light for photosynthesis.

  Weekly monitoring of the culture is performed to evaluate cell growth. The measured parameters are: pH, T, 02, DO.

  The culture is carried out at a pH between 6.5 and 8, and at a temperature between 17 and 23 C. The culture is conducted until a cell concentration corresponding to an optical density of 1.8 is obtained. at the wavelength of 677 nm. 10

  After 22 days, the culture is centrifuged (16500 rev / min) and the clarified product is conditioned and stored in a cold room.

  The clarified culture medium is then concentrated by thermal evaporation. The composition of a bioprotective foundation of the invention is, for example: clarified culture medium of Scenedesmus dimorphus obtained as described above g conventional bottom formulation complexion (alcohols and fatty esters, silicones, phospholipids of soya, butylene glycol, xanthan gum, iron oxides, sunscreens, preservatives, perfumes, water) qs 100g Example 14. Tablet for oral absorption.

  The composition of a tablet for oral absorption is, for example: clarified culture medium obtained from a co-culture of Phaeodactylum tricornutum and Skeletonema costatum as described in Example 4 and then lyophilized 100 mg - starch 25 mg - lactose 80 mg - aroma 5 mg - tablet excipients (talc, magnesium stearate) qs 250 mg

Claims (16)

  1. Cosmetic, dermatological, pharmaceutical or food composition, characterized in that it comprises a culture medium of at least one marine microorganism and / or fresh photosynthetic water, said medium being clarified.   2. Composition according to claim 1, characterized in that the marine microorganism and / or photosynthetic fresh water is a cyanobacterium and / or a microalga from (s) marine environments such as sea, ocean, brackish water, salt water or pure water.   3. Composition according to any one of claims 1 or 2, characterized in that the marine microorganism and / or photosynthetic fresh water is a cyanobacterium and / or a microalga belonging to the division of chlorophytes chosen (s) in the group consisting of cyanophytes, chlorophytes, chrysophytes, chlorarachniophytes, dinophytes, cryptophytes, euglenophytes or rhodophytes 4. Composition according to claim 3, characterized in that the cyanobacterium and / or the microalgae belongs to the class Prasinophyceae selected from the group consisting of cyanophyceae, chlorophyceae, prasinophyceae, pedinophyceae, prymnesiophyceae, eustigmatophycées, raphidophycées, chrysophycées, diatomophycées, tribophycées, chlorarachniophycées, dinophycées, cryptophycées, euglenophycées and rhodophycées.   2873038 38 5. Composition according to one of claims 1 to 4, characterized in that the amount of the clarified culture medium is from 0.01% to 100% by weight relative to the total weight of the composition.   6. Composition according to any one of claims 1 to 5, characterized in that said clarified culture medium is obtained by a process comprising the following steps: a) the selection of at least one marine microorganism and / or water photosynthetic sweetness as defined in one of claims 2 to 4, b) the introduction into a culture medium of the microorganism (s) selected (s) in the previous step a), c) l amplification of said one or more microorganisms in said culture medium, d) elimination of all the microorganisms obtained at the end of the amplification step c), in order to obtain a clarified culture medium of said one or more photosynthetic marine and / or freshwater microorganisms.   7. Composition according to claim 6, characterized in that step a) consists in selecting at least two microorganisms, said microorganisms may be identical or different.   8. Composition according to any one of claims 6 or 7, characterized in that the clarified culture medium, obtained at the end of step d) of elimination, is subjected to an additional stage of 30 desaling , concentration, dilution and / or drying.   9. Composition according to any one of claims 6 to 8, characterized in that the culture medium of step b) (wherein is introduced at least one microorganism) is selected from the group consisting of Source Water Marine Grande Ile, sea water, ocean water, brackish water, saline water, fresh water, a clarified culture medium of at least one microorganism or mixtures thereof.   10. Composition according to one of claims 6 to 9, characterized in that the culture medium of step b) (wherein is introduced at least one microorganism) is a clarified culture medium as defined in one Claims 1 to 8.   11. Composition according to any one of claims 6 to 10, characterized in that the culture medium of step b) is enriched in nutrients selected from the group consisting of minerals, trace elements and vitamins.   12. Composition according to any one of claims 6 to 11, characterized in that it contains at least one other active ingredient from the biomass as obtained at the end of the elimination step d) described in claim 6.   13. Composition according to one of claims 1 to 12, characterized in that the clarified culture medium is vectorized in liposomes or 2873038 other vectors to promote their pharmaceutical, cosmetic, dermatological or food.   14. A method for preparing a clarified culture medium of at least one marine microorganism and / or fresh photosynthetic water, characterized in that it comprises the following steps: a) the selection of at least one marine microorganism and / or photosynthetic freshwater   as defined in one of claims 2 to 4,   b) introducing, in a culture medium, the microorganism (s) selected (s) during the previous step a), c) the amplification of said microorganism (s) in said culture medium, d) l) elimination of all the microorganisms obtained at the end of the amplification step c), in order to obtain a clarified culture medium of said one or more photosynthetic marine microorganisms and / or freshwater.   15. Preparation process according to claim 14, characterized in that it comprises the steps as described in any one of the Claims 7 to 11.   16. Method according to any one of claims 14 or 15, characterized in that one further proceeds to a step of screening the clarified culture medium as defined in any one of claims 1 to 13, or such obtained according to the method of any one of claims 14 or 15, to evaluate the pharmaceutical, cosmetic, dermatological or food properties of said clarified culture medium.