Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/02—Algae
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
  • A61J3/00—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms
  • A61J3/07—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms into the form of capsules or similar small containers for oral use
  • A61J3/071—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms into the form of capsules or similar small containers for oral use into the form of telescopically engaged two-piece capsules
  • A61J3/077—Manufacturing capsule shells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/11—Encapsulated compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/73—Polysaccharides
  • A61K8/733—Alginic acid; Salts thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
  • A61K8/9706—Algae
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
  • B01J13/02—Making microcapsules or microballoons
  • B01J13/20—After-treatment of capsule walls, e.g. hardening
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/12—Unicellular algae; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6409—Fatty acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form

Definitions

• Gelled capsule comprising a plant cell
• the present invention relates to a gelled capsule comprising a plant cell, and a method of culturing said plant cell.
• macroscopic algae or macro-algae are either harvested in the wild or farmed at sea.
• Micro-algae are mainly grown in open-pond (open ponds). ) or bioreactor.
• the open-lay process has the advantage of being inexpensive, but does not produce algae in high concentration. On the other hand, it requires cultivation of extremophilic organisms in order to avoid contamination (by the external environment, since, by definition, no physical barrier exists between the culture medium and the outside). Among these contaminations, mention may be made of the result of animal droppings or the death of insects and / or animals.
• the bio-reactor process makes it possible to isolate the algae culture from the outside world and to concentrate the algae.
• the plant cells are in free suspension in a culture medium, it is also called bulk process.
• This process induces a significant production cost, mainly related to the need to continuously brew the crop and bring in the gases and light necessary for the growth of organisms.
• harvesting methods are not easy and expensive (centrifugation, tangential filtration, etc.).
• the mixing of the culture medium makes it difficult to cultivate fragile algae, because of the large cell death caused by this mixing.
• the object of the present invention is therefore to provide a means for the proliferation (here also referred to as growth) and, optionally, the elicitation of at least one plant cell, preferably at least one algal cell.
• the present invention relates to a capsule comprising:
• an internal phase comprising at least one plant cell
• a gelled external phase totally encapsulating said internal phase at its periphery, said external phase comprising at least one surfactant and at least one polyelectrolyte in the gelled state.
• the subject of the present invention is also a method for culturing plant cells, as well as a method for producing compounds of interest produced by said plant cells, if necessary after elicitation.
• the internal phase also refers to the heart of a capsule
• the gelled outer phase also refers to its gelled envelope, also called membrane.
• the capsules of the invention are also known as gelled capsules.
• the capsule of the invention is a so-called capsule
• a “simple” capsule is for example a capsule as described in the international application WO 2010/063937.
• the present invention also relates to a method for preparing the capsules according to the invention.
• the capsules of the invention are typically prepared by a process comprising the following steps:
• double drop a drop consisting of an internal phase and a liquid external phase, completely encapsulating said inner phase at its periphery.
• the production of this type of drop is generally carried out by concentric coextrusion of two solutions, according to a hydrodynamic mode of dripping or jetting, as described in applications WO 2010/063937 and FR2964017.
• the reagent capable of gelling the polyelectrolyte present in the gelling solution then forms bonds between the various polyelectrolyte chains present in the liquid external phase.
• the polyelectrolyte in the liquid state then passes to the gelled state, thus causing the gelation of the liquid external phase.
• the individual polyelectrolyte chains present in the liquid external phase are connected to each other to form a crosslinked network, also called a hydrogel, which traps the water contained in the external phase.
• a gelled external phase suitable for retaining the internal phase of the first solution, is thus formed.
• This gelled outer phase has a clean mechanical strength, that is to say that it is able to completely surround the internal phase and retain the plant cell or cells present in this internal phase to retain them in the heart of the capsule gelled.
• the capsules according to the invention remain in the gelling solution until the outer phase is completely gelled. They are then collected and optionally immersed in an aqueous rinsing solution, generally consisting essentially of water and / or culture medium.
• the size of the different phases initially forming the double drops, and ultimately the capsules is generally controlled by the use of two independent syringe pumps (at the laboratory scale) or two pumps (on an industrial scale), which respectively provide the first solution and the second liquid solution mentioned above.
• the flow rate Q of the syringe pump associated with the first solution controls the diameter of the internal phase of the final capsule obtained.
• the flow rate Q 0 of the syringe pump associated with the second liquid solution controls the thickness of the gelled external phase of the final capsule obtained.
• the relative and independent adjustment of the flow rates Q 1 and Q 0 makes it possible to control the thickness of the gelled external phase independently of the outer diameter of the capsule, and to modulate the volume ratio between the internal phase and the external phase.
• the capsules of the invention generally have an average size of less than 5 mm, and more generally from 50 ⁇ to 3 mm, advantageously from 100 ⁇ to 1 mm.
• the gelled outer phase has an average thickness of from 5 ⁇ to 500 ⁇ , preferably from 7 ⁇ to 100 ⁇ .
• the volume ratio between the internal phase and the external phase is greater than 1, and preferably less than 50.
• the inner phase also called the heart of the capsules, generally consists of an aqueous composition, liquid or viscous, comprising at least one plant cell.
• At least one plant cell is meant at least one cell of a plant cell line.
• the internal phase may comprise several plant cells, of the same line or of different lines.
• Encapsulated plant cells are what is also called encapsulated biomass.
• the plant cells are differentiated from animal cells, among others, by the presence of a pectocellulosic wall and the presence of plastids, including chloroplasts that allow photosynthesis.
• the plant cells included in the internal phase are unicellular plant cells.
• the capsules of the invention comprise at least one algal cell (also called algal cell), preferably a microalgae cell.
• the capsules of the invention comprise several cells of algae, of the same species or of different species.
• Algae are living beings capable of photosynthesis whose life cycle generally takes place in the aquatic environment.
• prokaryotes cyanobacteria
• eukaryotes severe very diverse sets.
• micro-algae refers to microscopic algae. They are undifferentiated, photosynthetic, eukaryotic or prokaryotic unicellular or multicellular beings.
• algae that can be used in the capsules of the invention mention may be made of a green alga, a red alga or a brown alga. According to one embodiment, it is a prokaryotic algae.
• it is a eukaryotic algae.
• the alga is preferably of the genus Chlamydomonas, such as Chlamydomonas reinhardtii, or of the genus Peridinium, such as Peridinium cinctum.
• Suitable algae for the implementation of the invention may be chosen from the group consisting of Alexandrium minutum, Amphiprora hyalina, Anabaena cylindrica, Arthrospira platensis, Chattonella verruculosa, Chlorella vulgaris, Chlorella protothecoides, Chysochromulina breviturrita, Chrysochromulina kappa, Dunaliella Salina, Dunaliella minuta, Emiliania huxleyi (Haptophyta), Gymnodinium catenatum, Gymnodinium nagasakiense, Haematococcus pluvialis, Isochrysis galbana, Noctiluca scintillans, Odontella aurita, Oryza sativa, Ostreococcus lucimarinus, Pavlova utheri, Porphyridium cruentum, Spirodela oligorrhiza, Spirulina maxima,
• the internal phase comprises a buffer solution capable of survival of the plant cells.
• any buffer known per se can be used to be adapted to the survival of plant cells.
• the internal phase preferably has a pH of from 5 to 10, more preferably from 6 to 9.
• the internal phase comprises nutrients suitable for the proliferation of the plant cell or cells.
• the internal phase comprises a culture medium called MC1 in the context of the present invention.
• culture medium is meant a solution comprising nutrients suitable for the proliferation of the plant cell or cells and acting as a pH buffer.
• the osmolarity of the culture medium MC1 is preferably between 10 mOsm and 1000 mOsm.
• the MC1 culture medium is for example selected from Erdschreiber culture medium, F / 2 medium, TAP medium, reconstituted seawater, DM (diatom) medium, Minimum medium, and any of their mixtures.
• the Erdschreiber culture medium is a solution comprising NaCl (11.4 g / L),
• a soil extract refers to the filtrate obtained by filtration of a mixture of soil and water.
• F / 2 medium commercially available (in particular at the School of Biological Sciences of the University of Texas, or at Varicon Aqua), is a solution comprising NaNO 3 (8.82 ⁇ 10 -4 mol / L), NaH 2 PO 4 4 OH 2 O (3.62 ⁇ 10 -5 mol / L), Na 2 SiO 3 .9H 2 O (1.06 ⁇ 10 -4 mol / L), FeCl 3 .6H 2 O (1, 17.1 ⁇ -5 mol / L ), Na 2 EDTA.2H 2 0 (1, 17.10 "5 mol / l), CuS0 4 .5H 2 0 (3,93.10" 8 mol / L), Na 2 Mo0 4 .2H 2 0 (2,60.10 - 8 mol / L), ZnS0 4 .7H 2 0 (7,65.10 -8 mol / L), COCl 2 .6H 2 0 (4,20.10 "8 mol / L), MnCl
• TAP medium commercially available, (including LifeTech), is a mixture of Beijerincks buffer (2x) (50 mL), 1 M phosphate buffer pH 7 (1 mL), trace solution (1 mL), acid acetic acid (1 mL) and water (QSP 1 L).
• the compositions of the Beijerincks (2x) and 1 M phosphate pH 7 buffers and of the trace solution are described in the examples below.
• the pH of the TAP medium is 7.3.
• the osmolarity of the TAP medium is 60 mOsm.
• the reconstituted seawater is a solution comprising NaCl (11.7 g / L), Tris (6.05 g / L), NH 4 CI (3.00 g / L), KCl (0.75 g / L), L), K 2 HPO 4 .2H 2 O (74.4 mg / L), FeSO 4 H 2 O (2 mg / L), H 2 SO 4 (0.05 mg / L), MgSO 4 (7). 85 g / L), CaCl 2 (1.47 g / L) and water (QSP 1 L).
• the pH of reconstituted seawater varies from 7.5 to 8.4.
• the osmolarity of reconstituted seawater is 676 mOsm.
• DM (diatom) medium is a solution comprising Ca (NO 3 ) 2 (20 g / L), KH 2 PO 4 (12.4 g / L), MgSO 4 .7H 2 O (25 g / L), NaHCO 3. 3 (15.9 g / L), FeNaEDTA (2.25 g / L), Na 2 EDTA (2.25 g / L), H 3 B0 3 (2.48 g / L), MnCl 2 .4H 2 0 (1.39 g / L), (NH 4 ) 6 Mo 7 O 2 .4H 2 O (1 g / L), cyanocobalamin (0.04 g / L), thiamine HCl (0.04 g / L) , biotin (0.04 g / L), NaSiO 3 .9H 2 O (57 g / L) and water (QSP 1 L).
• the Minimum medium is a mixture of Beijerincks buffer (2x) (50 mL), phosphate buffer (2x) (50 mL), trace solution (1 mL) and water (QSP 1 L).
• the compositions of Beijerincks (2x) and phosphate (2x) buffers and Trace Solution are described in the examples below.
• the osmolarity of the Minimum medium is 37 mOsm.
• the internal phase When encapsulating the plant cells (ie before any plant cell culture method), the internal phase typically comprises from 10 3 to 10 9 , preferably from 10 4 to 10 8 , more preferably from 10 5 to 10 8 , for example from 10 6 to 5.10 6 plant cells per milliliter of internal phase.
• the internal phase typically comprises from 1 to 10 7 , preferably from 5 to 10 6 , from 30 to 5 ⁇ 10 5 , from 50 to 10 5 , from 75 to 5 ⁇ 10 4 , from 100 to 10 4 , from 150 to 10 4 , or even 200 to 10 3 plant cells, per capsule.
• the counting of the plant cells of the internal phase is preferably carried out before encapsulation, or after the opening of the capsules.
• the counting of plant cells can be done by the counting method Malassez.
• Malassez's cell is a glass slide that counts the number of cells in suspension in a solution. On this glass slide, a grid of 25 rectangles has been engraved, containing 20 small squares themselves. To count the plant cells, one deposits on the cell of Malassez between 10 ⁇ ⁇ - and 15 ⁇ ⁇ - of internal phase including plant cells in suspension. After sedimentation, we count the number of plant cells in 10 rectangles (squared). The volume of a grid rectangle being 0.01 ⁇ , this number is multiplied by 10,000 to obtain the number of plant cells per milliliter of internal phase.
• counting of plant cells can be done by absorbance measurement.
• absorbance measurement for a given wavelength ⁇ , the absorbance of a solution is proportional to its concentration and to the length of the optical path (distance over which the light passes through the solution).
• concentration of plant cells in the inner phase can therefore be measured on the basis of an absorbance measurement method (also known as optical density). It suffices to measure the optical densities of internal phases containing a known quantity of plant cells, which makes it possible to construct a standard curve as a function of the cell concentration.
• the internal phase comprises one million cells per milliliter of internal phase before any culture method, which corresponds to approximately 60 cells per capsule of 500 ⁇ in diameter.
• the internal phase typically comprises from 50 million to 150 million plant cells, per milliliter of internal phase.
• the plant cells present in the inner phase of the capsules are suspended in the internal phase.
• the plant cells do not adhere to the gelled membrane of the capsules, and are not in prolonged contact with said membrane. The plant cells are thus completely immersed in the medium constituting the internal phase and are free to move in three dimensions.
• Those skilled in the art are able to verify that the plant cells are effectively suspended in the capsules, typically by observing the capsules by microscopy and demonstrating a differentiated movement between the capsule and the plant cells it contains. For example, in the particular case of flagellated microalgae, we can observe their swimming, due to the action of their flagella.
• the internal phase comprises at least one viscosity agent, preferably biocompatible, typically chosen from cellulose ethers.
• a viscosity agent makes it possible to facilitate the preparation of the capsules by reducing the difference in viscosity between the internal phase and the external phase, which comprises a polyelectrolyte in solution.
• viscosity agent is meant a product soluble in the internal phase capable of modulating its viscosity. It may especially be a natural polymer, such as glycosaminoglycans (hyaluronic acid, chitosan, heparan sulfate, etc.), starch, plant proteins, welan gum, or any other natural gum; of a semi-synthetic polymer, such as decomposed starches and their derivatives, cellulose ethers, such as hydroxypropyl methyl cellulose (HPMC), hydroxy ethyl cellulose (HEC), carboxymethylcellulose (CMC) and 2 ethylcellulose; or a synthetic polymer, such as polyethers (polyethylene glycol), polyacrylamides, and polyvinyls.
• a natural polymer such as glycosaminoglycans (hyaluronic acid, chitosan, heparan sulfate, etc.), starch, plant proteins, welan gum, or any other
• the inner phase comprises a cellulose ether such as 2-ethylcellulose.
• the viscosity agent is present in the internal phase in a mass concentration of 0.01% to 5%, preferably 0.1% to 1%, relative to the total mass of the internal phase. .
• the external phase comprises at least one gelled polyelectrolyte, also called gelled polyelectrolyte, and at least one surfactant.
• the polyelectrolyte is chosen from polyelectrolytes reactive with multivalent ions.
• polyelectrolyte reactive with multivalent ions a polyelectrolyte likely to pass from a liquid state in a aqueous solution in a gelled state under the effect of contact with a gelling solution containing multivalent ions, such as multivalent cations of calcium, barium, magnesium, aluminum or iron.
• the individual polyelectrolyte chains are substantially free to flow relative to one another.
• An aqueous solution of 2% by weight of polyelectrolyte then exhibits a purely viscous behavior at the shear gradients characteristic of the forming process.
• the viscosity of this zero shear solution is between 50 mPa.s and 10,000 mPa.s, preferably between 1000 mPa.s and 7000 mPa.s.
• the individual polyelectrolyte chains in the liquid state advantageously have a molar mass greater than 65,000 g / mol.
• the gelling solution is, for example, an aqueous solution of a salt of formula
• X is selected from the group consisting of halide ions (chloride, bromide, iodide and fluoride) and tartrate, lactate and carbonate ions,
• M is selected from the group consisting of Ca 2+ , Mg 2+ , Ba 2+ , Al 3+ and Fe 3+ cations, and
• n and m are greater than or equal to 1.
• the concentration of salt X n M m in the gelling solution is advantageously from 1% to 20% by weight, preferably from 5% to 20% by weight.
• the individual polyelectrolyte chains together with the multivalent ions form a coherent three-dimensional network which retains the internal phase and prevents its flow.
• the individual chains are held together and can not flow freely relative to each other.
• the polyelectrolyte is preferably a biocompatible polymer, it is for example produced biologically.
• polysaccharides synthetic polyelectrolytes based on acrylates (sodium, lithium, potassium or ammonium polyacrylate, or polyacrylamide), or synthetic polyelectrolytes based on sulfonates (poly (styrene) sulfonate), for example).
• the polyelectrolyte is chosen from alkaline alginates, such as sodium alginate or potassium alginate, gelans and pectins.
• the reaction that occurs during gelation is as follows:
• 2NaAlg + CaCI 2 Ca (Alg) 2 + 2NaCI Alginates are produced from brown algae called “laminar", referred to as "sea weed”.
• the polyelectrolyte is an alkali metal alginate advantageously having an ⁇ -L-guluronate block content of greater than 50%, especially greater than 55%, or even greater than 60%.
• the polyelectrolyte is, for example, sodium alginate.
• the polyelectrolyte in the gelled state is typically a calcium alginate.
• the total weight percentage of polyelectrolyte in the gelled external phase is from 0.5% to 5%, preferably less than 3%.
• the total weight percentage of polyelectrolyte in the gelled external phase is for example between 0.5% and 3%, preferably between 1% and 2%.
• the presence of a surfactant in the external phase makes it easier to prepare the capsules of the invention by increasing the resistance of the liquid external phase during the impact of the double drop with the gelling solution.
• the surfactant is preferably an anionic surfactant, a nonionic surfactant, a cationic surfactant, or any mixture thereof.
• the molecular weight of the surfactant is typically between 150 g / mol and 10,000 g / mol, preferably between 250 g / mol and 1500 g / mol.
• the mass content of surfactant in the external phase is typically less than or equal to 2%, preferably less than 1%, relative to the total weight of the capsule.
• the surfactant is an anionic surfactant
• it is, for example, chosen from alkyl sulphates, alkyl sulphonates, alkyl aryl sulphonates, alkaline alkyl phosphates, dialkyl sulphosuccinates and alkaline earth salts of saturated or unsaturated fatty acids.
• These surfactants advantageously have at least one hydrophobic hydrocarbon chain having a number of carbons greater than 5 or even 10 and at least one hydrophilic anionic group, such as a sulphate, a sulphonate or a carboxylate linked to one end of the hydrophobic chain.
• An anionic surfactant particularly suitable for the implementation of the invention is sodium dodecyl sulphate (SDS).
• SDS sodium dodecyl sulphate
• the mass content of surfactant in the external phase is typically from 0.001% to 0.5%, preferably from 0.001% to 0.05%, based on the total weight of the capsule.
• the mass content of surfactant in the external phase is preferably less than or equal to 0.025%, preferably less than or equal to 0.010%, or even less than or equal to 0.005%, relative to the total weight of the capsule.
• the surfactant is a cationic surfactant
• it is for example chosen from alkylpyridium or alkylammonium halide salts such as n-ethyldodecylammonium chloride or bromide, cetylammonium chloride or bromide (CTAB) .
• CTLAB cetylammonium chloride or bromide
• These surfactants advantageously have at least one hydrophobic hydrocarbon chain having a number of carbon atoms greater than 5 or even 10 and at least one hydrophilic cationic group, such as a quaternary ammonium cation.
• the mass content of surfactant in the external phase is typically from 0.001% to 0.5%, preferably from 0.001% to 0.05%, based on the total weight of the capsule.
• the mass content of surfactant in the external phase is preferably less than or equal to 0.025%, preferably less than or equal to 0.010%, or even less than or equal to 0.005%, relative to the total weight of the capsule.
• the surfactant is a nonionic surfactant
• it is for example chosen from polyoxyethylenated and / or polyoxypropylenated derivatives of fatty alcohols, fatty acids, or alkylphenols, arylphenols, or from alkyiglucosides, polysorbates and cocamides .
• a nonionic surfactant particularly suitable for the implementation of the invention is polysorbate 20 (Tween 20).
• the mass content of surfactant in the external phase is typically from 0.01% to 2%, preferably from 0.1% to 1%, relative to the total weight of the capsule .
• the capsules according to the invention comprise an intermediate phase between the internal phase and the gelled external phase.
• This intermediate phase forms an aqueous or, where appropriate, oily, generally biocompatible, intermediate envelope which completely encapsulates the internal phase and is completely encapsulated by the gelled external phase.
• Such capsules are generally obtained by concentric coextrusion of three solutions, by means of a triple envelope: a first stream constitutes the internal phase, a second stream constitutes the intermediate phase and a third stream constitutes the external phase.
• the production of such capsules, called “complex”, is described in particular in the international application WO 2012/089820.
• the three flows come into contact and then form a multi-component drop, which is then gelled when immersed in a gelling solution, in the same way as in the process for preparing capsules "Simple" described above.
• the intermediate phase may comprise at least one plant cell, preferably an algal cell, which may be identical or different from the plant cells present in the internal phase.
• the intermediate phase may also comprise at least one viscosity agent as described above.
• the intermediate phase when present and comprising plant cells, is preferably suitable for the survival of said plant cells. It advantageously comprises a culture medium capable of culturing said cells, typically one of the MC1 culture media mentioned above for the internal phase.
• the intermediate phase when present and comprising plant cells, typically comprises from 1 to 10 7 , preferentially from 5 to 10 6 , from 30 to 5 ⁇ 10 5 , from 50 to 10 5 , from 75 to 5 ⁇ 10 4 from 100 to 10 4 , from 150 to 10 4 , even from 200 to 10 3 plant cells, per capsule.
• the capsule according to the invention consists of:
• an internal phase comprising at least one plant cell
• a gelled external phase totally encapsulating said internal phase at its periphery, said external phase comprising at least one surfactant and at least one polyelectrolyte in the gelled state.
• the capsule according to the invention does not comprise an envelope (or membrane) other than the internal phase and the gelled envelope (gelled external phase).
• the capsule according to the invention does not comprise a stiffened envelope such as that described in FR 2 986 165 or in WO 2013/1 13855.
• the stiffened envelope of FR 2 986 165 and WO 2013/1 13855 notably makes it possible to fix and develop mammalian cells.
• the capsule according to the invention is devoid of stiffened envelope, including stiffened intermediate envelope.
• the subject of the present invention is also a method for culturing plant cells comprising a step of culturing at least one capsule according to the invention.
• culturing is meant the action of placing the capsules of the invention in a culture medium called MC2 in the context of the present invention, typically suitable for the cultivation of encapsulated plant cells, under temperature conditions. and brightness adapted to the culture of said plant cells, for a time necessary to obtain the desired plant cell concentration within the capsules.
• the culture medium MC2 is, for example, chosen from Erdschreiber culture medium, F / 2 medium, TAP medium, reconstituted seawater, DM (diatom) medium, Minimum medium, and any of their mixtures.
• the osmolarity of the culture medium MC2 is preferably between 10 mOsm and 1000 mOsm.
• the ratio (osmolarity of MC1) / (osmolarity of MC2) is between 1/50 and 50, preferably between 1/10 and 10.
• the culture medium MC2 is identical to the culture medium MC1.
• the culture medium MC2 is different from the culture medium MC1.
• the capsules are typically cultured under light conditions ranging from total black to 500 ⁇ . ⁇ ⁇ 2 .5 ⁇ 1 .
• the capsules are typically cultured for a period of one hour to one month, usually 24 hours to a week.
• capsule harvesting is typically by removal of the MC2 culture medium by filtration of the capsules, or by any other capsule recovery technique.
• a sieve having an opening size smaller than the average diameter of the capsules of the invention, which are substantially spherical, is typically used.
• the inventors have discovered, surprisingly, that the cultivation of plant cells, in particular of algae, within the capsules according to the invention makes it possible to access higher plant cell concentrations than in the bulk processes mentioned in FIG. introduction. For example, cell concentrations of 50 million to 150 million cells per ml are obtained in the capsules, 5 to 15 times more than those obtained in bulk processes.
• the encapsulation of plant cells and the capsule culture of these plant cells, in particular algae such as microalgae, also has the following advantages over conventional methods:
• encapsulated plant cells are protected from mechanical stresses, such as shearing, thus decreasing cell death during the cell culture process;
• the encapsulated plant cells are partially protected from the external medium, because the membrane of the capsule has a selective permeability (in particular, the bacteria can not penetrate the capsule), which advantageously makes it possible to avoid possible contaminations and therefore to reduce cell death;
• the size of the capsules (several hundred micrometers) and their mechanical properties make them easier to handle than the algae, in particular for changes in MC2 culture medium or during harvesting;
• the hydrogel network confers indeed semisolid mechanical properties to the capsules of the invention, much higher than those of plant cells.
• the hydrogel membrane of the capsules of the invention also has a semi-permeability, based on the porous nature of the structure of the chain network of the polyelectrolyte.
• This network is characterized by an average pore size of between 5 nm and 25 nm, and therefore has a cut-off size of the order of 20 nm to 25 nm.
• This porosity allows the free passage of dissolved gases, minerals, nutrients necessary for the proliferation of plant cells, such as small biomolecules (amino acids and peptides), and small macromolecules with a molecular weight of less than 1 MDa.
• the membrane retains any element with a characteristic size greater than the cutting size, ie macromolecules or biomolecules with a molecular weight greater than 1 MDa, or cellular organisms such as plant cells, for example algae, or even bacteria. and mushrooms.
• cellular organisms such as plant cells, for example algae, or even bacteria. and mushrooms.
• this is of significant interest for the cultivation of plant cells, such as algae, by controlling the exchanges between the outside and the inside of the capsule, that is to say between the culture medium MC2 and encapsulated biomass.
• this semi-permeability also makes it possible to control the release of encapsulated element, such as compounds of interest produced by the plant cells.
• the production of plant cells, in particular of algae, in encapsulated form according to the invention also makes it possible to limit the quorum sensing effects.
• Washing is also useful for eliminating metabolic or catabolic waste, and thus orienting the activity of biomass. This approach proves to be a method of elicitation in itself.
• one of the limiting factors of algae culture is the contribution of light.
• the light flux is reduced by "fouling", that is to say the bonding of biomolecules and microorganisms on the walls of the bioreactor.
• This layer absorbs the light, which induces a decrease in the luminous flux captured by the plant cells.
• the accumulation of this deposit induces increasing pressure losses and increases production costs.
• this washing can also be envisaged to eliminate possible contaminants from the biomass culture, without direct manipulation thereof.
• contaminants can be of chemical origin (molecules of the type heavy metals or other chemical releases), physical (of particulate type), or biological (dead cells, exogenous bacteria ). This method can therefore limit operating losses.
• the present invention also relates to a process for producing a compound of interest, comprising:
• the step of culturing the above production method generally corresponds to the step of culturing the culture process of the invention.
• the culture medium MC2 may be identical or different from the culture medium MC1 of the heart of the capsules.
• elicitation is understood to mean the stimulation of the production of compounds of interest by a plant cell, said stimulation being caused by the setting in particular conditions, whether physicochemical, resulting from a modulation of temperature, pressure or illumination, or that they rely on the presence of a particular molecule, called “elicitant molecule”. Artificial production of compounds of interest by the encapsulated plant cells is thus artificially induced. According to one embodiment, the elicitation step takes place during the culturing step.
• This embodiment is for example implemented by carrying out the step of culturing under eliciting conditions, for example by carrying out the step of culturing in a culture medium MC2 different from the culture medium MC1, said medium MC2 culture containing an elicitant molecule.
• the elicitation step takes place at the end of the culturing step, that is to say once the plant cells have proliferated within the capsules of the invention.
• This embodiment is for example implemented by carrying out the step of culturing under conventional conditions, that is to say by choosing a culture medium MC2 identical to the culture medium MC1, and then, at the resulting from the culturing step, placing itself under eliciting conditions, for example by replacing the culture medium MC2 with another culture medium, different from the culture medium MC1.
• the elicitation step of the plant cells typically comprises:
• the elicitation stage of the plant cells consists, for example, in culturing the capsules of the invention in a culture medium MC2 different from the culture medium MC1, or in a culture medium comprising an eliciting molecule, or in the same MC1 medium with a change in culture conditions (temperature, light, etc.).
• the capsules according to the invention can be cultured under conditions that are well known to those skilled in the art, such as by adding them to the culture medium.
• MC2 salicylic acid, ethylene, jasmonate or chitosan see for example the international application WO 2003/077881).
• the compounds of interest produced by the production method of the invention are typically subjected to one or more treatments, such as purification, concentration, drying, sterilization and / or extraction. These compounds are then intended to be incorporated into a cosmetic, agri-food or pharmaceutical composition.
• the capsules of the invention make it possible in particular to produce lipids of interest in cosmetics, such as, for example, fatty acids, such as linoleic acid, alpha-linoleic acid or gamma acid. linoleic, palmitic acid, stearic acid, eicosapentaenoic acid, docosahexanoic acid, arachidonic acid; fatty acid derivatives, such as ceramides; or sterols, such as brassicasterol, campesterol, stigmasterol and sitosterol.
• fatty acids such as linoleic acid, alpha-linoleic acid or gamma acid.
• fatty acid derivatives such as ceramides
• sterols such as brassicasterol, campesterol,
• the capsules of the invention also make it possible to produce organic selenium, an essential trace element.
• Selenium is typically incorporated into amino acids, peptides and proteins, especially in the form of selenomethionine.
• the recovery of the compound of interest is carried out by treatment of the culture medium (MC2) in which the capsules are immersed, by conventional purification methods, such as liquid / liquid extraction (phase separation organic / aqueous), acid-base washing, solvent concentration and / or purification by chromatography, among others.
• conventional purification methods such as liquid / liquid extraction (phase separation organic / aqueous), acid-base washing, solvent concentration and / or purification by chromatography, among others.
• This embodiment is particularly suitable for cases where the compound of interest is excreted by the plant cells and then diffuses out of the capsules.
• This embodiment has the advantage of keeping capsules and cells intact.
• the recovery of the compound of interest is effected by opening the capsules, then opening the membrane of the cells, and then treating the resulting mixture with conventional purification methods.
• the opening of the capsules can be of chemical or mechanical type.
• a mode of chemical opening is for example the depolymerization of the membrane, typically by contact with a solution of citrate ions.
• a mechanical opening mode is typically the grinding of the capsules.
• the opening of the cell membrane is typically performed by grinding the cells.
• This embodiment is particularly suitable for cases where the compound of interest remains confined within the plant cells.
• the present invention also relates to the use of a capsule according to the invention, for the production of plant cells and / or the production of molecules of interest.
• the production of molecules of interest is obtained by elicitation of plant cells, as described above.
• the present invention also relates to a composition comprising at least one capsule according to the invention.
• the capsule according to the invention has been treated by a process for producing a compound of interest according to the invention.
• the capsule according to the invention has, in addition, undergone a post-process treatment for the production of a compound of interest, consisting in forming a second membrane completely encapsulating at its periphery the gelled membrane of said capsule.
• a second membrane may be formed by gelling in the presence of a compound capable of forming electrostatic bonds with the constituents of the gelled membrane, typically in the presence of a polyelectrolyte.
• This variant has the advantage of protecting the heart of the capsules and to avoid the migration out of the capsules of the compounds of interest contained in said core.
• composition according to the invention is typically a cosmetic, pharmaceutical or agri-food composition.
• the subject of the present invention is also the use of a capsule according to the invention for the preparation of a cosmetic, pharmaceutical or agri-food composition.
• the present invention also relates to a composition comprising a capsule extract.
• capsule extract is meant a compound of interest produced by the plant cells, typically by elicitation, and recovered as described above.
• a plant cell is also meant, originating from a cultivation process of the invention, and possibly from a process for producing a compound of interest according to the invention.
• This filtration step avoids the presence of particles or solid aggregates leading to the clogging of the nozzles used for production, but is also used to sterilize the phases. It is also possible to heat these phases to a temperature above 60 ° C to sterilize them.
• a microalgae solution was prepared at a typical concentration of 1 million cells / ml in TAP (MC1) medium.
• 2-ethylcellulose (0.5% by weight) was added to facilitate the coextrusion of the phases and to stabilize the process by avoiding excessive viscosity differences between the internal and external phases.
• Chlamydomonas reinhardtii strains WTS24-, Sta6 and CW15.
• the method of manufacturing capsules is based on the concentric coextrusion of two solutions, in particular described in WO 2010/063937 and FR2964017, to form double drops.
• the size of the internal phase and the thickness of the external phase of the drops formed were controlled by the use of two independent syringe pumps (HA PHD-2000).
• the ratio r q between the flow rate of the fluid constituting the core and the flow rate of fluid constituting the membrane has been set at 1, 6. This made it possible to obtain capsules having a membrane thickness-to-radius ratio of less than 0.9, which maximizes the rate of encapsulation of the internal phase, and therefore micro-algae.
• the capsules obtained have a diameter of 300 ⁇ (+/- 50 ⁇ ).
• the drops formed were gelled with a gelling solution of sterile calcium chloride 200 mM (minimum concentration 50 mM), to which were added a few drops of a sterile solution of Tween 20 (Sigma AIdrich) to 10% (w / w).
• the formed capsules were harvested using a sieve and then transferred to one of the culture media described below.
• Chlamydomonas reinhardtii (strain WTS 24-) according to an initial cell concentration adjusted to 1 million cells / ml, were cultured in TAP medium or Minimum medium (MC2) (ie about 200 capsules per flask of 20 ml, or 10,000 capsules for 1 L of medium MC2), in flasks allowing gaseous exchange, with moderate stirring, at 25 ° C.
• TAP medium or Minimum medium ie about 200 capsules per flask of 20 ml, or 10,000 capsules for 1 L of medium MC2
• a light source of the order of 3400 lumens was used.
• the monitoring of cell growth was done qualitatively, comparing the volume revolution occupied by microalgae in the capsules, and quantitatively, by classical cell biology experiments.
• the capsules were opened by contacting with a solution of citrate (sodium) at 10% by mass for a few seconds.
• citrate sodium
• the citrate anions make it possible to complex the calcium cations, which depolymerizes the alginate gel of the membrane.
• the contents of the capsules were analyzed by flow cytometry and Malassez counting.
• the microalgae proliferated to a mean concentration in the capsules of between 120 and 250 million cells / mL after one week, as measured by Malassez counting.
• the microalgae did not exceed a concentration of 10 million cells / mL, all conditions being equal.
• the stability of the capsules was evaluated in TAP culture medium, TAP culture medium supplemented with 10 mM CaCl 2 and TAP culture medium to which was added 0.1% EDTA by mass.
• Example 1 remained stable for at least 3 weeks in each of these 3 culture media (MC2).
• Microalgae Chlamydomonas reinhardtii strain WTS 24- were cultured at 250,000 cells / ml in TAP medium (ie about 10,000 capsules per 1 L of medium TAP), firstly in free form (in bulk), and secondly in encapsulated version according to the invention (capsules of Example 1).
• the two samples obtained were imaged in the presence of SYTOX Green, a fluorescence-visible cell death marker with the Nikon FITC filter, before and after strong stirring.
• the encapsulated microalgae showed a green marking equivalent to the marking observed before stirring. Few cells died, despite the strong shear imposed. As in the initial microalgae suspension, the presence of a small proportion of dead micro-algae is normal and stems simply from the cellular cycle of the microalgae considered.
• Capsules according to Example 1 were cultured in TAP medium (approximately 10,000 capsules per 1 L of TAP medium), to which were added Escherichia coli RFP bacteria, i.e. E. coli species genetically engineered to synthesize a fluorescent molecule, visible using the Nikon TRITC filter.
• capsules according to Example 1 were placed in the presence of a 1 mM solution of rhodamine (visible in fluorescence using the Nikon TRITC filter) for a few minutes, then were transferred to a bath of oil and imaged under the microscope. These results showed that, unlike E. coli RFP bacteria, rhodamine diffused through the alginate membrane to be found inside the capsules.
• the capsules according to the invention are therefore semi-permeable: they allow the passage of molecules, such as the nutrients of the culture medium MC2, and do not allow the bacteria to penetrate, which has the advantage of preventing any bacterial contamination during the microalgae cultivation process.
• EXAMPLE 3 Elicitation of encapsulated microalgae for the production of lipids and organic selenium
• Capsules according to Example 1, comprising microalgae Chlamydomonas reinhardtii (strain Sta6) were cultured in TAP medium for 48 hours (ie about 10,000 capsules per 1 L of TAP medium), then the latter was removed and replaced with N0 medium, for 48 h.
• microalgae then produced lipids, in the form of lipid bodies present inside said microalgae.
• Capsules were removed and placed in the presence of 25% of DMSO and 1 ⁇ l of Nile Red for 10 minutes. The capsules were then imaged under bright field microscopy and fluorescence (source: mercury lamp).
• the chloroplast of micro-algae was revealed in fluorescence thanks to the Nikon filter
• the Nile Red testifying to the presence of lipids produced by elicitation, was revealed thanks to the Nikon FITC filter.
• microalgae produce molecules containing selenium, such as Peridinium cinctum. Such microalgae can therefore produce selenium in organic form, from mineral selenium.
• Microalgae producing selenium-containing molecules have been encapsulated according to the present invention and then incubated in a medium enriched with mineral selenium, in order to induce bioaccumulation of organic selenium by these microalgae.

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• 2014
  • 2014-10-22 FR FR1460169A patent/FR3027608B1/fr active Active
• 2015
  • 2015-10-22 EP EP20174903.3A patent/EP3718530A1/de active Pending
  • 2015-10-22 EP EP15786905.8A patent/EP3209266A1/de not_active Ceased
  • 2015-10-22 WO PCT/EP2015/074549 patent/WO2016062836A1/fr active Application Filing
  • 2015-10-22 CN CN201580057823.0A patent/CN107105737A/zh active Pending
  • 2015-10-22 US US15/520,269 patent/US20170312323A1/en not_active Abandoned

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