Red aqueous extract of tetraselmis and uses thereof

Abstract

The present invention relates to a red aqueous extract obtained from a green alga belonging to the genus Tetraselmis , a composition comprising same, and the uses thereof, in particular as a dye.

Description

Description Title of the invention: Red aqueous extract of Tetraselmis and its uses

[0001] Technical field

[0002] The invention relates to a red aqueous extract obtained from a green algae belonging to the genus Tetraselmis, a composition comprising it, and its uses, in particular as a colorant.

[0003] State of the art

[0004] In our current societies, the use of colorants is widespread given the strong consumer appetite for colors and consequently for colored products. For example, food colorings, mainly used as food additives, make it possible to enhance the attractiveness of food products, in order to restore or strengthen the color of certain foods. In the textile industry, the use of colorants, mainly synthetic, polluting and harmful to health, is, again, widespread in order to produce, for example, products with more vivid colors. In the cosmetics industry, colorants can be used as a coloring base for certain products, for example makeup products, or to enhance visual appeal, for example perfumes, shower gels, creams etc.

[0005] Dyes can be of natural origin, for example, plant, animal, or mineral; or synthetic origin. The chemical industry has enabled the development of a large number of new molecules, including synthetic dyes with improved stability compared to their natural alternatives. However, the absorption of certain synthetic dyes is not always without consequences for the health of consumers. Some are responsible for intolerances, irritation, and even allergies. Others are mutagenic and can cause cancer.

[0006] Furthermore, consumers are increasingly sensitive to the use of materials or products of natural origin, respectful of the environment and healthy. This interest is further increased when colorants are used in the food or cosmetics industry, reinforcing the need for new food or cosmetic ingredients of natural origin.

[0007] Thus, there is a need for new dyes of natural origin with increased stability properties comparable to synthetic dyes, as well as a lack of toxicity to humans or animals. Among the natural organisms that are recognized as an important resource, algae remain underexploited compared to other plant-based resources.

[0008] Natural colorants, i.e. a water-soluble molecule, are already known from the prior art. For example, mention may be made of natural red colorants derived from plants and soluble in water, such as anthocyanins, polyphenols (particularly in vines), as well as betalains (particularly in beets). In microalgae, mention may be made of red pigments, in particular carotenoids, of a liposoluble nature, such as antheraxanthin, astaxanthin and its esters, and canthaxanthin. However, no red colorant derived from green microalgae is described.

[0009] Summary of the invention

[0010] However, in the course of their research, the inventors unexpectedly discovered a green algae belonging to the genus Tetraselmis capable of producing a red dye that is particularly stable over time and therefore of particular interest for use as a dye, for example in the food industry but also in the cosmetics sector.

[0011] This biological resource, namely a green algae of the genus Tetraselmis, was isolated in New Caledonia, particularly in the Caledonian lagoons. Tetraselmis is a marine microalgae belonging to the Prasinophyte family, green in color and unicellular with 4 flagella. Several species have already been listed, the best known of which are Tetraselmis chuii and Tetraselmis suecica. The microalga Tetraselmis is known to be used as a food supplement in aquaculture, and recently there has been interest in the development of biofuels. However, there has never been any report of the use of the microalga Tetraselmis as a red dye, as it is naturally green in color. This is the subject of the present invention.

[0012] Thus, the invention relates to a red-colored aqueous extract obtained from a green algae of the genus Tetraselmis, in particular of the species Tetraselmis rubens, characterized in that the extract comprises at least one macromolecule with a molecular weight greater than 1000 kDa and / or two chromophores having individually a molecular weight less than 5 kDa.

[0013] Preferably, the molecular weight of the macromolecule contained in the extract is between 1000 kDa and 10,000 kDa.

[0014] According to a particularly preferred subject of the invention, the macromolecule greater than or equal to 1000 kDa is a glycoprotein, and this advantageously comprises between 60g and 75g of carbohydrates and between 25g and 35g of proteins per 100g of the glycoprotein.

[0015] In particular, the glycosylated portion of the glycoprotein of 1000 kDa or more is composed of carbohydrates, preferably said carbohydrates being selected from ribose, arabinose, rhamnose, mannose, galacturonic acid, N-acetyl-glucosamine and combinations thereof. Their combinations may vary in ratio and their structure may be linear or branched.

[0016] The extract according to the invention may also comprise at least two chromophores. When the extract according to the invention comprises two chromophores, the extract preferably comprises at least one magenta red chromophore of a size less than 5 kDa and / or one red-orange chromophore of a size less than 5 kDa.

[0017] Advantageously, the extract according to the invention comprises at least one macromolecule comprising at least one and / or two chromophores, these being preferably polar and hydrophilic.

[0018] The inventors sought to identify the origin of this red coloration present in the extract according to the invention. Thus, in order to characterize this red-colored extract more specifically, the inventors measured the absorption peak of the extract, advantageously this is between 200 and 280nm, preferably the peak is 212nm (±2nm).

[0019] The inventors have also determined that the macromolecule and / or the two chromophores present in the extract according to the invention is / are not carotenoids, preferably chosen from canthaxanthin and / or astaxanthin, the extract being soluble in water. Also, the red coloration is not due to the presence of a possible carotenoid. In other words, preferably, the aqueous extract according to the present invention does not comprise carotenoids. More preferably, the aqueous extract does not comprise either canthaxanthin or astaxanthin.

[0020] On the other hand, the macromolecule and / or the two chromophores present in the extract are not one of the molecules chosen from phycobiliproteins, preferably phycoerythrin; betalains; anthocyanins; polyphenols; and combinations thereof. Indeed, the inventors have not observed fluorescence, characteristic of phycoerythrins, nor a change in color depending on the pH, characteristic of polyphenols. Finally, the inventors have shown by HPLC analysis that it is not betalain. In other words, preferably, the extract according to the present invention does not comprise phycobiliproteins (such as phycoerythrin), nor betalains, nor anthocyanins, nor polyphenols.

[0021] The green algae belongs to the species Tetraselmis rubens.

[0022] According to another preferred subject, said red aqueous extract is obtained by microwave extraction, this making it possible to extract a large quantity of the extract comprising at least the macromolecule and / or the two chromophores. More preferably, when the extract is obtained by microwave extraction, the extraction temperature is less than 55°C, even more preferably less than 40°C.

[0023] For the purposes of the invention, the term "extraction temperature" means the temperature of the sample, i.e., in the context of the invention, the microalga and / or the extract derived therefrom. In this case, the temperature is less than 55°C, preferably less than 40°C so as not to denature the molecules present in the microalga or the Tetraselmis rubens extract, in particular the osidic composition of the glycoprotein.

[0024] Also, in the context of the invention, the extract is likely to be obtained by any suitable extraction process, preferably this comprises the following steps: a. culture of the biomass of green algae of the species Tetraselmis rubens, b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. extraction of the biomass of Tetraselmis rubens by microwave and / or by enzymatic digestion and / or by high pressure homogenization, in particular in water or in an aqueous solvent, for example a phosphate buffer, PBS, Tris-HCl. d. possibly, purification by size exclusion chromatography.

[0025] According to a variant, the extract is also capable of being obtained by an extraction process comprising the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens, preferably in batch, harvested in the stationary phase of growth, b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. desalination of the culture supernatant of Tetraselmis rubens by diafiltration, d. optionally, purification by size exclusion chromatography.

[0026] Thus, according to another aspect, the invention also relates to such extraction methods.

[0027] Preferably, the extract obtained from the biomass and the extract obtained from the supernatant can be mixed in order to constitute a single extract or unique extract according to the invention.

[0028] According to another aspect, the extract according to the invention can also be integrated into a composition, advantageously a cosmetic or food composition. Also, the present invention also relates to a cosmetic composition comprising at least the extract according to the invention and a physiologically acceptable medium, preferably a cosmetically acceptable medium; or a food composition comprising at least the extract according to the invention and an acceptable food excipient, for example preservatives or flavorings.

[0029] The invention also relates to several uses. Thus, the red aqueous extract according to the invention is particularly suitable for use as a colorant, preferably as a cosmetic or food colorant.

[0030] Surprisingly, the extract according to the invention has also demonstrated anti-UV properties and can thus be used as an anti-UV agent, either alone or as an additive to enhance the effectiveness of an anti-UV ingredient or as an anti-UV active ingredient. Antioxidant properties have also been reported.

[0031] Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow.

[0032] Brief description of the Figures

[0033] [Fig. 1] is a spectrum representing the absorption peak of the extract according to the invention from 200 to 400 nm.

[0034] [Fig. 2] is a diagram showing the steps of the extraction process from the culture of Tetraselmis rubens.

[0035] [Fig. 3] represents a spectrum of the extract obtained after elution on an S100 HR column. The Fl band corresponds to the red macromolecule.

[0036] Detailed description of the invention

[0037] Definition

[0038] For the purposes of the invention, the term "extract" means a set of molecules extracted from a raw material X, for example an algae; said molecules may be native or derived from processing, for example by extraction. Thus, for the purposes of the invention, the term "red aqueous extract obtained from a green algae of the species Tetraselmis rubens" means a set of several molecules obtained by an action on the plant raw material, in this case the green algae of the species Tetraselmis rubens. Preferably, the action used to obtain this set of molecules is an extraction, more particularly a microwave extraction and / or by high-pressure homogenization and / or an enzymatic digestion.

[0039] For the purposes of the invention, the term "dye" means a natural or synthetic substance that colors a medium and is soluble in water. The dye may be a water-soluble colored molecule comprising one or more functional groups, for example a chromophore, responsible for the color of the molecule. Unlike a pigment, which is a colored natural substance produced by animal or plant organisms, insoluble in water and in most common suspended media, with a high coloring and opacifying power, intended to give a surface coloring to the support on which it is applied.

[0040] Extract according to the invention

[0041] The present invention therefore relates to a red aqueous extract obtained from a green algae of the species Tetraselmis rubens, characterized in that the extract comprises at least one macromolecule with a molecular weight greater than 1000 kDa and / or two chromophores individually having a molecular weight less than 5 kDa.

[0042] According to a particular object of the invention, the macromolecule present in the extract preferentially has a molecular weight between 1000 kDa and 10000 kDa.

[0043] Particularly preferably, the macromolecule greater than or equal to 1000 kDa is a glycoprotein, and this advantageously comprises between 60g and 75g of carbohydrates and between 25g and 35g of proteins, per 100g of the glycoprotein. Also, the glycoprotein is essentially made up of carbohydrates and proteins.

[0044] Preferably, the carbohydrates present in the glycoprotein of at least 1000 kDa are chosen from ribose, arabinose, rhamnose, mannose, galacturonic acid, N-acetyl-glucosamine, and combinations thereof. Their combinations can vary in rate and their structure can be linear or branched.

[0045] Carbohydrates can be determined according to the method described in Kamerling et al., modified by Montreuil, et al. Briefly, samples were hydrolyzed for 4 hours at 100°C with 3 M MeOH / HCl, with myo-inositol used as an internal standard. After re-N-acetylation with acetic acid overnight at room temperature, methyl glycosides were converted to their corresponding trimethylsilyl derivatives using N,O-bis(trimethylsilyl)-trifluoroacetamide and trimethylchlorosilane (BSTFA:TMCS) 99:1. Separation and quantification of per-O-trimethylsilyl methyl glycosides were performed by gas chromatography (GC-FID, Agilent Technologies 6890N).

[0046] According to a very particular object of the invention, the glycoprotein comprises carbohydrates in the quantities indicated in Table 1 below.

[0047] [Table 1]

[0048] According to another subject of the invention, the extract according to the invention comprises at least one chromophore, preferably at least two chromophores. More preferably the two chromophores are linked to the macromolecule.

[0049] When the extract according to the invention comprises two chromophores, the extract preferably comprises at least one magenta red chromophore of a size less than 5 kDa and one red-orange chromophore of a size less than 5 kDa.

[0050] According to another preferred subject, the macromolecule and / or the two chromophores present in the extract are aqueous, polar and hydrophilic. Also, the extract according to the invention is only soluble in an aqueous solvent such as fresh water, sea water or aqueous buffer, for example a phosphate buffer or an acetate buffer. Consequently, the extract according to the invention is not soluble in organic solvents such as EtOH, MeOH, acetone, hexane, cyclohexane, MTBE, petroleum ether, or DMSO.

[0051] The inventors also determined the absorption peak of the extract, this is advantageously between 200 and 280nm, more preferably the peak is 212nm (±2nm), [Fig. 1], Such an absorption peak makes it possible to exclude that molecules such as neoxanthin, zeaxanthin, violaxanthin, lutein, beta-carotene, chlorophylls a and b are responsible for the red color of the extract according to the invention.

[0052] Thus, the particularity and originality of this extract obtained from the green algae Tetraselmis rubens is its capacity to include a red colorant, said red colorant not being molecules such as neoxanthin, zeaxanthin, violaxanthin, lutein, beta-carotene, chlorophylls a and b.

[0053] The inventors have thus continued their work in order to identify the origin of this red coloration. Thus, the macromolecule and / or the two chromophores present in the extract according to the invention is / are not a red-colored carotenoid, preferably the macromolecule and / or the two chromophores present in the extract is / are not canthaxanthin and / or astaxanthin, the extract being soluble in water. Also, the red coloration is not due to the presence of a possible red-colored carotenoid.

[0054] On the other hand, the macromolecule and / or the two chromophores present in the extract is / are not one of the molecules chosen from phycobiliproteins, preferentially phycoerythrin, betalains, anthocyanins, polyphenols, and their combinations. Indeed, the inventors did not observe fluorescence, characteristic of phycoerythrins, nor a change in color depending on the pH, characteristic of polyphenols.

[0055] According to another particularly preferred embodiment, the extract according to the invention comes from a green algae belonging to the species Tetraselmis rubens.

[0056] The extract according to the invention is preferably obtained or capable of being obtained by an extraction process comprising at least one microwave extraction step, this making it possible to extract a large quantity of the extract comprising at least the macromolecule and / or two chromophores, inextricably linked to the presence of this particular red color and stable over time, advantageously at least 5 years.

[0057] According to a preferred subject, when the extract is obtained by microwave extraction, the extraction temperature is lower than 55°C, even more preferably lower than 40°C. Thus, the molecules present in the microalgae of the species Tetraselmis rubens are not denatured, in particular the osidic composition. Thus, during microwave extraction, the temperature of the sample, i.e. of the algal raw material, is lower than 55°C, preferably lower than 40°C.

[0058] In a particular embodiment of the invention, the extract is capable of being obtained by an extraction process comprising the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens, advantageously in batch in stationary growth phase, b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. recovery and extraction of the biomass of Tetraselmis rubens by microwave and / or by enzymatic digestion by high pressure homogenization, with an aqueous solvent. d. optionally, purification by size exclusion chromatography.

[0059] According to one variant, the extract is capable of being obtained by an extraction process comprising the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens, advantageously in batch in the stationary growth phase, b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant. c. recovery and desalination of the Tetraselmis rubens culture supernatant by diafiltration, d. possibly, purification by size exclusion chromatography.

[0060] Preferably, the extract obtained from the biomass and the extract obtained from the supernatant are mixed in order to obtain a single extract according to the invention.

[0061] Also, according to another particular embodiment of the invention, the extract is capable of being obtained by an extraction process comprising the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens, advantageously in batch, in stationary phase of growth, b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. extraction of the biomass of Tetraselmis rubens by microwave and / or by enzymatic digestion by high pressure homogenization, with an aqueous solvent and / or desalination of the culture supernatant of Tetraselmis rubens by diafiltration, d. optionally, purification by size exclusion chromatography, and e. optionally mixing of the extract obtained from the biomass and the extract obtained from the supernatant.

[0062] Process

[0063] The cultivation of the green algal biomass of the species Tetraselmis rubens can be carried out according to any method known to those skilled in the art that makes it possible to obtain a large quantity of green algal biomass. Advantageously, this is carried out with a supply of nitrogen and phosphate according to an N / P molar ratio of 9.2, making it possible to obtain optimal algal growth. The culture is advantageously carried out in batch mode and harvested in the stationary phase, i.e. the moment when all the nitrogen supply has been consumed to produce the biomass, the optimum moment for producing the red dye.

[0064] Algal biomass can be in wet form, in frozen form (resuspended at a rate of 50 g / L), or in freeze-dried or atomized form (dry powder), resuspended at a rate of 15 g / L in water.

[0065] Once the biomass is produced to obtain the algal biomass, the mixture comprising the culture medium and the biomass is separated by centrifugation, preferably between 3000 and 5000 g.

[0066] Once centrifuged, the centrifugation pellet is recovered and the extract according to the invention is, according to a preferred object, obtained from said pellet by microwave extraction and / or by enzymatic digestion and / or by high pressure homogenization, more preferably by microwave extraction in aqueous medium. Microwave extraction makes it possible to facilitate the lysis of algal cells in aqueous medium and thus obtain an optimum extraction yield and consequently to increase the red dye content of the extract according to the invention.

[0067] More preferably, the microwave extraction may comprise several cycles, advantageously at least two cycles, in order to further improve the lysis of the algal cells comprising the extract according to the invention.

[0068] According to a particular object of the invention, the algal biomass is previously suspended in a suitable aqueous solution, preferably a pure water solution. After homogenization, the microwave extraction is preferably carried out according to the following procedure: a. A first lysis cycle at maximum power for 1 minute with a temperature below 40°C; then b. Maintain in the microwave for 10 min without exceeding 40°C, c. A second lysis cycle at a power of 1000W for 10 minutes with a temperature below 40°C, d. Centrifugation and recovery of the supernatant, e. Optionally, addition of an aqueous solution to the pellet and repeating the extraction according to steps a) to d), until a translucent supernatant is obtained, and f. elimination of the pellet after exhaustion, i.e. when it no longer contains red dye.

[0069] Thus, the extraction steps are repeated as many times as necessary until the red dye in the pellet is used up, thus making it possible to obtain a supernatant comprising said dye and constituting a first form of the extract according to the invention.

[0070] After steps a) to d), when the supernatant is translucent, the pellet no longer contains red dye, the pellet is said to be exhausted. Thus, as long as the supernatant obtained after steps a) to d) is red in color, the pellet still contains red dye and steps a) to d) are repeated as many times as necessary until a translucent supernatant is obtained.

[0071] Advantageously, the pellet / aqueous solution ratio is between 50 and 100 g / L.

[0072] According to a variant, an extraction from the culture supernatant resulting from the separation step, i.e. the centrifugation step, can be carried out instead of or in addition to the extraction of the pellet by microwave. This is carried out, after the step of centrifugation of the culture and obtaining the pellet comprising the Tetraselmis rubens biomass, and the culture supernatant. The supernatant can thus be desalted by means of diafiltration, preferably a 5 kDa diafiltration, i.e. having a cut-off threshold of 5 kDa, then the diafiltered supernatant is concentrated.

[0073] Also, the method may comprise a step of microwave extraction of the pellet and, in parallel, diafiltration from the supernatant in order to maximize the quantity of extract according to the invention obtained. Indeed, the algal cells naturally secrete the red aqueous extract according to the invention in small quantities into the supernatant of the culture, which can thus be added, after diafiltration, to the extract obtained by microwave extraction in order to increase the quantity of extract according to the invention obtained.

[0074] Also, the method may comprise after the step of separating the Tetraselmis rubens biomass and the culture supernatant: a. a step of recovering the biomass and extracting the Tetraselmis rubens biomass, and, simultaneously or not, b. a step of recovering the supernatant and desalinating said Tetraselmis rubens culture supernatant.

[0075] After the extraction and / or desalination step, the process may optionally include a final purification step by size exclusion chromatography.

[0076] Thus, the present invention relates, according to a particular embodiment, to an extraction method comprising the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. extraction of the Tetraselmis rubens biomass by microwave and / or by enzymatic digestion and / or by high-pressure homogenization; and / or desalination of the Tetraselmis rubens culture supernatant by diafiltration, d. optionally, purification by size exclusion chromatography, e. optionally, mixing the extract obtained from the biomass according to step c) and the extract obtained from the supernatant according to step c).

[0077] Composition

[0078] The extract according to the invention can be used alone or integrated into a cosmetic or nutritional composition, at a rate of 0.1 to 100% by weight of dry matter of the composition. The subject of the present invention is therefore such a composition comprising as ingredient or active principle, a red aqueous extract according to the invention, according to any one of the preceding embodiments, comprising at least one macromolecule with a molecular weight greater than 1000 kDa and / or two chromophores each having a molecular weight less than 5 kDa.

[0079] The compositions comprising an extract according to the invention may contain other known suitable components, such as excipients, selected according to the form and intended use of the composition or other active ingredients or active molecules.

[0080] Preferably, the invention relates to a cosmetic composition comprising at least the extract according to the invention and a physiologically acceptable medium, preferably a cosmetically acceptable medium; or a food or nutritional composition comprising at least the extract according to the invention and an acceptable food excipient, for example preservatives or flavorings.

[0081] Use

[0082] The extract according to the invention and the compositions including it can be used in numerous applications, in particular as a colorant or as an anti-UV agent.

[0083] Also, the invention also relates to several uses. In particular, the red aqueous extract according to the invention is particularly suitable for use as a colorant, preferably as a cosmetic or food colorant.

[0084] Surprisingly, the extract according to the invention has also demonstrated anti-UV properties and can thus be used as an anti-UV agent, either alone or as an additive to enhance the effectiveness of an anti-UV ingredient or active ingredient.

[0085] Finally, according to another object, the extract according to the invention can be used as an antioxidant.

[0086] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results.

[0087] Examples

[0088] Example 1 - Extract according to the invention from biomass

[0089] A first example according to the invention is an extract obtained by implementing the method consisting of implementing the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens, carried out with a supply of nitrogen and phosphate according to the molar ratio N / P equal to 9.2 (Walne's medium and sea water), in batch, and harvested in stationary phase, in which the cell density can reach approximately 7.10 6cells / mL. b. separation by centrifugation of the Tetraselmis rubens biomass and the culture supernatant, c. extraction of the Tetraselmis rubens biomass by microwave comprising the following steps (possibly repeated): i. First lysis cycle at maximum power for 1 minute with a temperature below 40°C; then ii. Maintain in the microwave for 10 min without exceeding 40°C, iii. Second lysis cycle at a power of 1000W for 10 minutes with a temperature below 40°C; d. purification by size exclusion chromatography on a Sephacryl S100 HR column (GE healthcare or Cytiva) XK 26 / 100 (500 mL gel). The eluent used is filtered and degassed milli-Q water (resistivity of 18.2 MQ-cm).

[0090] The extract is characterized by: ■ A red macromolecule > lOOOkDa (comprising two chromophores), represented in [Fig. 3] fraction Fl, ■ A complex sugar representing 66% of the mass weight of the red macromolecule, represented in [Fig. 3] fraction F3, ■ A protein representing 29% of the mass weight of the red macromolecule, represented in [Fig. 3] fraction F2, ■ A magenta-red chromophore of size less than 5 kDa, represented in [Fig. 3] fraction F4, ■ A red-orange chromophore of size less than 5 kDa, represented in [Fig. 3] fraction F5.

[0091] Example 2 - Extract according to the invention from the culture supernatant

[0092] A second example according to the invention is an extract obtained by implementing the method consisting of implementing the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens, according to the protocol described in Example 1. b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. diafiltration of the culture supernatant of Tetraselmis rubens at a cut-off threshold of 5 kDa and using purified water with a resistivity of 18.2 MΩ cm, as solvent at a rate of 5 volumes of purified water for 1 volume of culture supernatant, d. lyophilization of the extract and storage in solid form.

[0093] The extract obtained is characterized by: ■ A red macromolecule > 1000kDa (comprising two chromophores), ■ A complex sugar representing 66% of the mass weight of the red macromolecule, ■ A protein representing 29% of the mass weight of the red macromolecule.

[0094] Example 3 - Extract according to the invention from biomass and supernatant

[0095] A third example according to the invention is an extract obtained by implementing the method according to examples 1 and 2, in which a final step of mixing the extract obtained from the supernatant and the extract obtained from the biomass is carried out in order to obtain a single extract according to the invention.

[0096] The extract obtained is characterized by: ■ A red macromolecule > 1000kDa (comprising two chromophores) ■ A complex sugar representing 66% of the mass weight of the red macromolecule ■ A protein representing 29% of the mass weight of the red macromolecule ■ A magenta-red chromophore less than 5 kDa in size ■ A red-orange chromophore less than 5 kDa in size.

Claims

Claims 1. Red aqueous extract obtained from a green algae of the species Tetraselmis ruben, characterized in that the extract comprises at least one macromolecule with a molecular weight greater than 1000 kDa and / or two chromophores with a molecular weight less than 5 kDa.

2. Red aqueous extract according to the preceding claim, characterized in that the macromolecule and / or the chromophores is / are polar and hydrophilic.

3. Red aqueous extract according to one of the preceding claims, characterized in that the absorption peak of the extract is between 200 and 280nm, preferably 212nm.

4. Red aqueous extract according to one of the preceding claims, characterized in that the macromolecule and / or the chromophores is / are not carotenoids, preferably canthaxanthin and / or astaxanthin.

5. Red aqueous extract according to the preceding claim, characterized in that the macromolecule and / or the chromophores is / are not molecules chosen from phycobiliproteins, betalains, anthocyanins, polyphenols, and combinations thereof.

6. Red aqueous extract according to one of the preceding claims, characterized in that the macromolecule greater than or equal to 1000 kDa is a glycoprotein.

7. Red aqueous extract according to the preceding claim, characterized in that the glycoprotein comprises between 60g and 75g of carbohydrates and between 25g and 35g of proteins per 100g of glycoprotein.

8. Red aqueous extract according to the preceding claim, characterized in that the carbohydrates are chosen from ribose, arabinose, rhamnose, mannose, galacturonic acid, N-acetyl-glucosamine, and combinations thereof.

9. Red aqueous extract according to one of claims 6 to 8, characterized in that the two chromophores are linked to the macromolecule.

10. Red aqueous extract according to one of the preceding claims, characterized in that a chromophore is a magenta red chromophore of a size less than 5 kDa and / or a chromophore is a red-orange chromophore of a size less than 5 kDa.

11. Red aqueous extract according to one of the preceding claims, characterized in that the extract is obtained by microwave extraction.

12. Red aqueous extract according to the preceding claim, characterized in that the extraction temperature is less than 55°C, preferably less than 40°C.

13. Red aqueous extract according to one of the preceding claims, characterized in that the extract is obtainable according to the extraction process comprising the following steps: a. culture of the biomass of green algae of the species Tetraselmis rubens, b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. extraction of the biomass of Tetraselmis rubens by microwave and / or by enzymatic digestion and / or by high pressure homogenization, d. optionally, purification by size exclusion chromatography.

14. Process for preparing a red aqueous extract according to one of claims 1 to 12 comprising the following steps: a. cultivation of the biomass of green algae of the species Tetraselmis rubens, b. separation by centrifugation of the biomass of Tetraselmis rubens and the culture supernatant, c. diafiltration of the culture supernatant of Tetraselmis rubens, d. optionally, purification by size exclusion chromatography.

15. Use of the extract according to one of claims 1 to 13, as a colorant, preferably as a cosmetic or food colorant.

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