Novel form of sea urchin pigments, and uses thereof

Abstract

The present invention relates to a method for extracting natural sea urchin pigments in a novel form (referred to as pigment matrix), to this pigment matrix and to the use thereof as a natural dye.

Description

[0001] New form of sea urchin pigments and uses

[0002] The invention belongs to the technical field of pigments of natural origin.

[0003] More specifically, it relates to a process for extracting natural sea urchin pigments in a new form (called pigment matrix), to this pigment matrix and to its use as a natural colorant.

[0004] Nowadays, although synthetic dyes offer a wider range of colors and are less expensive than natural dyes, they remain highly polluting. Their petrochemical-based production, as well as their use in the coloring industry, have a considerable impact on the environment and our health. Therefore, replacing polluting synthetic dyes with bio-sourced dyes that are more respectful of the environment and our health, is now a priority in various industries, such as paint, textiles, cosmetics, and construction. Marine invertebrates such as mollusks, brachiopods, crustaceans, crinoids, and sea urchins have varied colors ranging from red to green, including yellow, blue, and purple.This wide range of natural colors is due to the presence of organic pigments (usually of low molecular weight, typically < lkDa) biosynthesized by animals (e.g. porphyrins, bilins, melanin and quinonoids such as anthraquinones, polycyclic quinones and naphthoquinones) or obtained from dietary sources (e.g. carotenoids such as canthaxanthin and astaxanthin).

[0005] Echinoderms (Phylum Echinodermata) synthesize a large number of secondary metabolites such as hydroxyanthraquinones identified in crinoids and polyhydroxy-1,4-naphthoquinones (PHNQs) found in sea urchins.

[0006] Biosynthesized by enzymatic pathways, PHNQs have antimicrobial, anti-inflammatory, antioxidant, and UV-absorbing properties (Lebedev 2001, Zhou 2011, Coates 2018, Powell 2014, Brasseur 2017), making them interesting bioactive compounds, particularly for pharmaceutical applications (Hou 2018, Vasileva 2021, Shikov 2018). Approximately 40 different PHNQ molecules have already been identified in various species of adult sea urchins (Anderson 1969, Hou 2018, Vasileva 2021, Ageenko 2022) once isolated from their gonads, digestive system, red spherule cells, and colored skeletal biominerals (radioles) with their surrounding soft tissues. When sea urchins are consumed, estimated at 75,000 tonnes worldwide per year (2017 Stefansson report), the skeletons (shells and radioles (the "spines")) which represent almost 30% of the total weight of the sea urchin, constitute waste which remains very little recovered to this day.

[0007] As part of its research on the study of pigmentation mechanisms of mineralized tissues in sea urchins, the Applicant has isolated a new form of pigments (called pigment matrix) extracted from radioles, in which the pigments are complexed with organic macromolecules (in particular, proteins and polysaccharides), this new form of pigments has advantageous properties compared to free pigments (solubility, stability of color at pH, better yield in terms of mass than free pigments). This pigment matrix can be advantageously prepared from food waste resulting from the consumption of marine organisms.

[0008] The present invention thus relates to an intracrystalline sea urchin pigment matrix composed of pigments - of the polyhydroxynaphthoquinone type - complexed with organic macromolecules.

[0009] More specifically, this matrix includes:

[0010] - pigments naturally present in radioles.

[0011] These pigments have been characterized and belong to the family of polyhydroxylated naphthoquinones. Their exact nature can vary depending on the species of sea urchin considered, for example:

[0012] - spinochrome A (C12O7H8, 2-acetyl-3,5,6,8-tetrahydroxy-l,4-naphthoquinone)

[0013] - spinochrome B (CwOeHe, 2,3,5,7-tetrahydroxy-l,4-naphthoquinone)

[0014] - spinochrome C (CizOsHs, 2-acetyl-3,5,6,7,8-pentahydroxy-l,4-naphthoquinone)

[0015] - spinochrome E (CWOSHÊ, hexahydroxy-l,4-naphthoquinone)

[0016] - echinochrome A (C12O7H10, 7-ethyl-2,3,5,6,8-pentahydroxy-l,4-naphthoquinone)

[0017] For example, the pigment matrix according to the invention obtained with Paracentrotus lividus sea urchins contains mainly spinochrome A and in lesser quantities spinochromes B and C. and

[0018] - organic macromolecules also naturally present in radioles comprising proteins and polysaccharides to which the aforementioned pigments are complexed. These macromolecules were characterized by FT-IR and SDS-PAGE (see Example 2); in particular, these macromolecules are characterized by the amide functions of proteins and the characteristic functions of polysaccharides in the region 1200-950 cm 1 , in particular CO bonds, detected by FT-IR; these macromolecules are also characterized by a molecular weight between 17 and 72 KDa.

[0019] More specifically, these macromolecules exhibit FT-IR bands characteristic of proteins (amide A (NH stretching) at 3290 cm 1, amide B (NH stretching) at 3090 cm 1 , amide I (C=O stretching) at 1627 cm -1 , amide II (CN stretching / NH bending) at 1537 cm 1 and amide III (CN stretching / NH bending) at 1226 cm 1 ) and polysaccharides (vC-0 at 1093 cm 4 ). Furthermore, electrophoresis on 12% silver-stained Tris-tricine gels shows that these macromolecules have a molecular weight between 17 and 72 kDa, with in particular, a discrete band observed between 48 kDa and 63 kDa corresponding to a well-defined protein, probably the already identified SM50 (Killian 1989).

[0020] The pigment matrix according to the invention is such that the mass ratio between pigments and organic macromolecules is between 0.5:100 and 5:100, generally of the order of 1:100.

[0021] The pigment matrix according to the invention comprises a fraction of pigments complexed with organic macromolecules which is soluble in an acid medium, i.e. at a pH less than or equal to 7 (called the acid-soluble fraction of the pigment matrix) and a fraction of pigments complexed with organic macromolecules which is insoluble in an acid medium (called the acid-insoluble fraction of the pigment matrix).

[0022] Each of these two fractions is also the subject of the present invention.

[0023] The pigment matrix according to the invention can be obtained by the following process:

[0024] 1) Washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or in an oven at 37°C;

[0025] This step allows the destruction and elimination of residual external organic tissues.

[0026] 2) Reduction of skeletons into powder;

[0027] This step can be carried out by any grinding means; preferably, the powder is sieved to select particles with a size less than or equal to 200 pm, particles with a larger size are ground again.

[0028] 3) Total cold dissolution (4°C - 12°C) of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v;

[0029] The solution obtained at this stage contains free PHNQ pigments and the pigment matrix according to the invention.

[0030] 4) Separation of free pigments (< lkDa) and pigment matrix (> lkDa) by ultrafiltration on a membrane with a cut-off threshold of 1 kDa and recovery of the pigment matrix.

[0031] The acid-soluble and acid-insoluble fractions of the pigment matrix can be separated by centrifugation after step 3). Then, the free pigments (< lkDa) and the acid-soluble fraction of the pigment matrix (> lkDa) can be separated by ultrafiltration on a membrane with a cut-off threshold of 1 kDa.

[0032] This process can be applied to any type of sea urchin regardless of color (see the article by Vasileva et al. 2021). Without limitation, in the context of food waste recycling, we can particularly mention edible sea urchin species such as Paracentrotus lividus and Strongylocentrotus nudus.

[0033] According to a preferred embodiment, the fractions are in powder form, in particular lyophilisates; for this, they can be lyophilized according to conventional methods known to those skilled in the art. For the implementation of this embodiment, it is preferable to carry out an additional step of removing the salts prior to lyophilization; this can be done by dialysis with a cut-off threshold of 0.5-1 kDa (see example 2).

[0034] These new forms of pigments, whether the pigment matrix, the acid-soluble fraction or the acid-insoluble fraction, have advantageous properties:

[0035] - their color remains stable whatever the pH;

[0036] - each has different solubility properties which allows a wide range of applications to be considered; while pigments alone are poorly soluble in water (at acid and neutral pH), the acid-soluble fraction of the pigment matrix is, which facilitates application in the field of dyeing, for example. The acid-insoluble fraction is, for example, interesting in the form of a paste for making prints.

[0037] Finally, these forms of pigments represent a greater quantity of material than the free pigments; in fact, the quantity of pigments in the radioles is quite low (approximately 0.01% by weight), the new forms of pigment matrix according to the invention can be extracted up to 1% by weight, which is very advantageous.

[0038] Depending on the desired applications and the solubility of the pigment matrix required, it may be advantageous to combine acid-soluble and acid-insoluble fractions of the pigment matrix in particular ratios.

[0039] The present invention thus also relates to the combination of the acid-soluble fraction of the pigment matrix and the acid-insoluble fraction of the pigment matrix; the fractions may be combined in a range of ratios between 1:100 and 100:1, or between 1:50 and 50:1. It should be noted that the pigment matrix is ​​a particular case of the combination of the acid-soluble fraction and the acid-insoluble fraction; in this particular case, the ratio acid-soluble fraction: acid-insoluble fraction is between 1:10 and 1:15.

[0040] Optionally, the acid-soluble fraction of the pigment matrix, the acid-insoluble fraction of the pigment matrix and / or the combination of the acid-soluble fraction of the pigment matrix and the acid-insoluble fraction are mixed with free pigments; due to the sensitivity of free pigments to pH, involving the change of color of the solutions in which they are dissolved depending on the pH, the color of such a mixture can be modified by varying the pH, while having a greater amount of material thanks to the pigment matrix or its fractions. By way of illustration, the ratio [free pigments]: [pigment matrix or its fractions] can be in a range from 1:1 to 1:100, preferably from 1:1 to 1:50.

[0041] Free pigments can be isolated as indicated above (by ultrafiltration at a cut-off of 1 kDa from the pigment matrix or acid-soluble fraction). Optionally, this separation can be followed by a purification step by extraction with an organic solvent (e.g. diethyl ether, chloroform or ethyl acetate) to remove dissolved salts as illustrated in Example 2.

[0042] For example, for dyeing applications with dark violet shades, the acid-soluble fraction should be greater than the free pigment fraction; for pinker shades, a larger portion of the free pigment fraction should be added; for printing applications, a larger portion of the insoluble portion is preferred for violet shades and a significant portion of free pigment may also be added for pinker shades.

[0043] The present invention also relates to the process for preparing the pigment matrix which comprises the following steps:

[0044] 1) washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or gentle steaming at 37°C;

[0045] 2) reduction of the skeletons into powder; 3) total dissolution in the cold (4°C - 12°C) of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v;

[0046] 4) separation of free pigments and pigment matrix by ultrafiltration on a membrane with a cut-off threshold of 1 kDa and recovery of the pigment matrix;

[0047] 5) optionally, removal of dissolved salts and acetic acid by dialysis;

[0048] 6) optionally, freeze-drying; if this step is implemented, step 5) will preferably also be carried out.

[0049] Alternatively, the pigment matrix may be obtained by separating the acid-soluble and acid-insoluble fractions, removing the free pigments from the acid-soluble fraction and then pooling the acid-soluble and acid-insoluble fractions; according to this embodiment, step 4) of the above method is replaced by the following steps:

[0050] 4bis) separation of the acid-soluble fraction and the acid-insoluble fraction by centrifugation;

[0051] 4ter) separation of free pigments and the acid-soluble fraction by ultrafiltration on a membrane with a cut-off threshold of 1 kDa;

[0052] 4quater) mixing the acid-insoluble fraction obtained in step 4bis) with the acid-soluble fraction obtained in step 4ter) to reconstitute the pigment matrix.

[0053] The present invention also relates to the process for preparing the acid-soluble fraction of the pigment matrix which comprises the following steps:

[0054] 1) washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or gentle steaming at 37°C;

[0055] 2) reduction of the skeletons to powder;

[0056] 3) total cold dissolution (4°C - 12°C) of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v;

[0057] 4) separation of the acid-soluble fraction and the acid-insoluble fraction by centrifugation and recovery of the acid-soluble fraction (liquid phase); 5) separation of the free pigments and the acid-soluble fraction from the pigment matrix by ultrafiltration on a membrane with a cut-off threshold of 1 kDa and recovery of the pigment matrix;

[0058] 6) optionally, removal of salts and acetic acid by dialysis;

[0059] 7) optionally, freeze-drying; if this step is implemented, step 6) will preferably also be carried out.

[0060] The present invention also relates to the process for preparing the acid-insoluble fraction of the pigment matrix which comprises the following steps:

[0061] 1) washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or gentle steaming at 37°C;

[0062] 2) reduction of the skeletons to powder;

[0063] 3) total cold dissolution (4°C - 12°C) of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v;

[0064] 4) separation of the acid-soluble fraction and the acid-insoluble fraction by centrifugation and recovery of the acid-insoluble fraction (solid phase).

[0065] According to another of its objects, the present invention relates to a kit comprising:

[0066] - the acid-soluble fraction of the pigment matrix,

[0067] - the acid-insoluble fraction of the pigment matrix, and

[0068] - free PHNQ pigments.

[0069] Preferably, the acid-soluble and acid-insoluble fractions and the free pigments of this kit are in powder form, in particular lyophilisate.

[0070] Such a kit allows the preparation of dye with a solubility adapted to the need and being able to have the desired shade of color by varying the proportions of each of the ingredients and the pH.

[0071] The present invention also relates to an ink comprising:

[0072] - the pigment matrix, the acid-soluble fraction of the pigment matrix or the acid-insoluble fraction of the pigment matrix according to the invention;

[0073] - a solvent; and

[0074] - optionally, a binder. For example, in the case of an ink for printing on fabric, the solvent may be water and the binder a gum, particularly a vegetable one such as guar gum.

[0075] According to another of its objects, the present invention relates to the use of the acid-soluble fraction of the pigment matrix, the acid-insoluble fraction of the pigment matrix or their combination according to the invention as a coloring agent for various supports. In particular, mention may be made of textiles (synthetic or natural), cellulosic supports such as paper, food, cosmetic products, paint, construction materials, etc.

[0076] Due to the known properties of PHNQ pigments, the use of the acid-soluble fraction of the pigment matrix, the acid-insoluble fraction of the pigment matrix or their combination according to the invention can also be aimed at applications as an antibacterial agent, an antifungal agent, an anti-inflammatory agent, an antioxidant agent and a UV absorber.

[0077] According to a particular embodiment, the acid-soluble fraction of the pigment matrix, the acid-insoluble fraction of the pigment matrix or their combination according to the invention can be used as a biocidal agent in anti-fouling treatment products (which prevents the development of soiling due to the proliferation of microorganisms including algae on surfaces immersed in water).

[0078] Figures:

[0079] Figure 1: A) Photographs of color changes as a function of pH from 4.4 to 12 of the pigment matrix in the presence of free pigments from orange-pink to magenta-violet to violet-blue, of the pigment matrix without free pigments (no change in dark violet color) and of free pigments from orange to pink to blue and B) photographs of the acid-soluble fraction and the acid-insoluble fraction of the pigment matrix, after centrifugation and after resuspension for the latter, for extracts from purple sea urchins Paracentrotus lividus from Roscoff (Brittany, France).

[0080] Figure 2: A) HPLC chromatography of purified free pigments extracted from purple sea urchins Paracentrotus lividus from Roscoff (Brittany, France), B) chemical structure of spinochromes A, B and C, C) complete HR-MS spectrum and D) simulation of C12O7H7, E) HR-MS spectrum from 218 to 228 m / z and F) simulation of CwOeHs, G) HR-MS spectrum from 276 to 296 m / z and H) simulation of C12O8H7. Figure 3: A) NMR spectrum 1 H and B) 2D HMBC of purified free pigments from purple sea urchins P. lividus from Roscoff (Brittany, France)

[0081] Figure 4: A) FT-IR spectra of purified acid-soluble (ASM) (dotted curve) and acid-insoluble (AIM) (solid curve) fractions of the pigment matrix of purple sea urchins P. lividus and B) 12% Tris-glycine SDS-PAGE gels after silver staining of acid-insoluble (AIM), acid-soluble (ASM) and free pigment (Pig.) fractions of purple sea urchins P. lividus.

[0082] Figure 5: A) UV-visible spectra of free pigments (solid curve) and acid-soluble fraction of pigment matrix (dotted curve) and 15% Tris-tricine SDS-PAGE gels of acid-soluble (ASM), acid-insoluble (AIM) and free pigment (Pig.) fractions of purple P. lividus sea urchins B) before and C) after silver staining.

[0083] Figure 6: AB) 15% Tris-tricine SDS-PAGE gels of the acid-soluble (ASM) and acid-insoluble (AIM) fractions of extracts from green P. lividus sea urchins A) before silver fixation and B) after fixation. C) 15% Tris-tricine SDS-PAGE gels of the acid-soluble (ASM) and acid-insoluble (AIM) fractions of extracts from purple P. lividus sea urchins during the beginning of fixation at pH > 8 and at the end at pH < 7 showing color changes of the bands due to free pigments (< 11 KDa) from purple to red and no color change for the bands > 11 kDa due to proteins and polysaccharides. The migration front is indicated by an arrow on the gels before Ag staining. and fractions according to the invention

[0084] Biological material: Radioles of sea urchins Paracentrotus lividus from Brittany (Roscoff, France).

[0085] Method: sea urchin radioles:

[0086] Collect the sea urchin spines in a 50 mL centrifuge tube previously filled with a 5% NaCIO aqueous solution for rotary shaking for 3 h.

[0087] Then rinse with water 3 times for 10 minutes and let the spines air dry (or

[0088] 37°C).

[0089] Then, grind the thorns in an agate mortar until a fine powder with a maximum particle size of 200 pm is obtained by sieving it using a 200 pm sieve.

[0090] Total elimination of intercrystalline macromolecules by treatment Suspend the powder thus obtained in a 12% NaCIO solution (50 mL / gram of powder) in several centrifuge tubes by rotary shaking (15 rpm) for 48 hours.

[0091] Centrifuge the tubes (6000 rpm, 15 min) and pipette off the 12% NaClO solution. Rinse the powder with milli-Q® water under rotary agitation for 1.5 hours, then remove the water with a pipette after centrifugation. This step is repeated five times.

[0092] Rinse the powder with 5 mL of ethanol for 5 min under rotary agitation and finally let it air dry (or at 37°C) in glass Petri dishes.

[0093] Dissolution of the mineral phase:

[0094] Slowly dissolve the powder with a 10% (v / v) acetic acid solution =1.75 M, at 4°C at a slow addition rate of 1.2 mL / min using an electronic digital titration burette. For complete dissolution, approximately 25 mL of 10% acetic acid solution is required per gram of powder.

[0095] Separation of acid-soluble and acid-insoluble fractions:

[0096] Distribute the resulting solution into several 50 mL centrifuge tubes before centrifuging them. Pipette the colored supernatants (containing the acid-soluble fraction) from each tube and collect them in a 500 mL glass bottle.

[0097] Collect all pellets containing the acid-insoluble fraction in a tube, resuspend them in 10% acetic acid (approximately 10 mL) and pipette them. This extracts the remaining acid-soluble fraction and completes the dissolution of any residual carbonate fraction (mineral phase). The tube is then centrifuged (to re-pellet the acid-insoluble fraction) and the acid-soluble supernatant is pipetted. Repeat the step of resuspending / centrifuging the acid-insoluble fraction in acetic acid until the supernatant becomes colorless; add any additional colored supernatants to the vial containing the acid-soluble fraction.

[0098] Rinse the acid-insoluble fraction with milli-Q® water three times by performing three resuspension / centrifugation cycles, then transfer it to a 2 mL Eppendorf tube.

[0099] The acid-soluble and insoluble fractions can be stored at 4°C.

[0100] Separation of the acid-insoluble fraction and free pigments:

[0101] This separation is carried out by ultrafiltration on a membrane with a cut-off threshold of 1 kDa. Example 2: characterization of the fractions obtained For the purpose of characterizing the fractions obtained, the protocol described in example 1 is implemented by adding:

[0102] - a step of purification of the free pigments by extraction with an organic solvent (for example diethyl ether, chloroform or ethyl acetate) to remove the salts dissolved in the aqueous phase which must have a pH of 2 (addition of HCl if necessary) to allow the migration of the free pigments (insoluble at acid pH) into the organic phase. The organic solvent is finally left to evaporate under a hood;

[0103] - a dialysis step of the acid-soluble fraction: the acid-soluble fraction is distributed in dialysis tubes with a cut-off threshold of 0.5-1 kDa then the tubes are introduced into beakers under magnetic stirring filled with milli-Q® water, the water is renewed until the volume of liquid in the dialysis tubes remains constant;

[0104] Freeze-drying:

[0105] Freeze the obtained fractions with liquid nitrogen or place them in a freezer at -80°C for 3 hours before proceeding with lyophilization according to a standard protocol.

[0106] The organic fractions obtained according to the protocol of Example 1 are very colorful (Figure 1). In particular, the pigment matrix is ​​dark purple and the free pigments give a pinkish coloration to the aqueous solution (pH 4.4) (Figure 1), which suggests the presence of pigments with the organic macromolecules.

[0107] Elucidation of the structure of the PHNQ mixture and identification of macromolecules associated with PHNQ:

[0108] Solution nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS) data show that spinochrome A is the most abundant polyhydroxylated naphthoquinone (PHNQ) in the free pigment fraction (Figures 2 and 3) as well as in the pigment matrix extracted from the spines of purple sea urchins P. lividus. Spinochrome B and spinochrome C are also identified by HR-MS (Figure 2) but in smaller amounts than spinochrome A.

[0109] The organic macromolecules of the acid-soluble and acid-insoluble fractions were characterized by Fourier transform infrared spectrometry (FT-IR) and sodium dodecyl sulfate Tris-tricine polyacrylamide gel electrophoresis (SDS-PAGE). The obtained FT-IR spectra exhibit characteristic bands of protein and polysaccharide structures (Figure 4A). In particular, the spectrum of the purified acid-soluble fraction exhibits amide A bands (N-H stretching) at 3290 cm 1 , amide B (NH stretching) at 3090 cm' 1 , amide I (C=O stretching) at 1627 cm 1 , amide II (CN stretching / NH bending) at 1537 cm 1 and amide III (CN stretching / NH bending) at 1226 cm . Bands attributed to vCH and 5-CH (asymmetric and symmetric) are also observed.

[0110] These bands are also all detected in the acid-insoluble fraction with the addition of a significant band at 1093 cm 1indicating the presence of other bonds characteristic of polysaccharides (vC-0).

[0111] SDS-PAGE gels with 12% Tris-tricine after silver staining show discrete bands characteristic of proteins as well as more diffuse signals characteristic of the presence of polysaccharides. In agreement with the FT-IR results, the acid-insoluble fraction presents more polysaccharides than the acid-soluble one. The molecular weights of these macromolecules are between 17 and 72 KDa, with higher concentrations from 34 kDa. A defined band around 50 KDa is identified for the acid-soluble fraction, another around 34 kDa for the acid-insoluble fraction.

[0112] Characterization of PHIMQ-macromolecule complexes present in the pigment matrix: Comparison of the UV-visible spectra of the free pigments and the acid-soluble fraction first highlights the presence of PHNQ in the latter (Figure 5A). In particular, the free pigments present 3 UV bands at 232, 373, 315 nm and a broad band in the visible with two peaks at 450 and 510 nm characteristic of spinochrome A (Figure 4B).

[0113] These bands are also observed in the acid-soluble fraction, this confirms the presence of spinochrome A in this fraction.

[0114] 15% Tris-tricine SDS-PAGE gels were run (Figures 5B and C and 6).

[0115] The 15% Tris-tricine gels are special in that they allow the separation of low molecular weight macromolecules and therefore the detection of PHNQJibres ( <lKDa) sous le front de migration (indiqué par une flèche sur les gels) (Figure 5B et C et Figure 6).

[0116] After Ag staining, free PHNQ.S are no longer visible (Figure 5C, Figure 6B), their staining fades, which is why the gels were also photographed before this staining (Figure 5B and Figure 6A).

[0117] For purple sea urchins, before gel staining (Figure 5B), thin purple bands corresponding to PHNQ are observed at the bottom of the gels. In addition, long purple spots (diffuse bands) are observed throughout the gel above the migration front, very visible for the acid-soluble fraction in particular, indicating that a large amount of PHNQ interacts with proteins and polysaccharides.

[0118] After staining (Figure 5C), a band is detected at 50 kDa in all acid-soluble and acid-insoluble fractions, probably corresponding to the well-known SM50 protein. Other bands are observed around 20 kDa, indicating the presence of other proteins. Long spreads are also observed, suggesting the presence of polysaccharides.

[0119] Therefore, for the purple sea urchin specimen from Brittany, the PHNQ mixture is associated with proteins and polysaccharides.

[0120] Figure 6A and 6B show 15% gels obtained for the fractions extracted from the spines of green sea urchins from Brittany. Before Ag staining, a discrete green band is detected around 10 kDa for the acid-insoluble and acid-soluble fractions obtained on a 10 KDa ultrafiltration membrane. After staining, the diffuse signal obtained at this green band indicates that the highlighted chromoprotein does not stain very well with Ag. Note that the acid-soluble fraction between 1 and 10 kDa shows an intense and long purple band before staining in the same way as the acid-soluble fraction of purple sea urchins.

[0121] Finally, stability at pH values ​​from 4 to 12 was tested for the different fractions both in solution (pH mediated by HCl and NaOH) and during gel fixation (before staining) where the pH ranges from 8 to 4 (Figures 1 and 6C). Interestingly, while the free PHNQ mixture changes color as a function of pH, this is not the case for the acid-soluble fraction extracted from purple spines (Figures 1A). This is confirmed by monitoring the color changes during gel fixation (Figure 6C). Indeed, all free PHNQ bands change from purple to red at acidic pH for green and purple spines, which is not the case for PHNQ-macromolecule complexes (Figure 6C). Indeed, the acid-insoluble fraction extracted from purple spines remains dark purple and the acid-soluble fraction extracted from green spines remains dark green at pH <4.

Claims

CLAIMS 1. pH-stable colored sea urchin pigment matrix composed of intracrystalline polyhydroxynaphthoquinone pigments complexed with organic macromolecules including proteins and / or polysaccharides.

2. Pigment matrix according to claim 1, characterized in that the intracrystalline polyhydroxynaphthoquinone pigments are spinochrome A, spinochrome B and spinochrome C.

3. Pigment matrix according to claim 1 or 2, characterized in that the organic macromolecules are characterized by the amide functions of proteins and the characteristic functions of polysaccharides in the region 1200-950 cm 1 detected by FT-IR and by a molecular weight between 17 and 72 KDa.

4. Pigment matrix according to any one of the preceding claims, characterized in that it is composed of an acid-soluble fraction.

5. Pigment matrix according to any one of claims 1 to 3, characterized in that it is composed of an acid-insoluble fraction.

6. Pigment matrix according to any one of claims 1 to 3, characterized in that it is composed of a combination of the acid-soluble fraction and the acid-insoluble fraction, said fractions being in a range of ratios between 1:100 and 100:

1.

7. Pigment matrix according to any one of the preceding claims, characterized in that it is in powder form.

8. Pigment matrix according to any one of the preceding claims, characterized in that it also comprises free polyhydroxynaphthoquinone pigments.

9. Process for preparing the pigment matrix according to any one of claims 1 to 3 comprising the following steps: 1) washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or gentle steaming at 37°C; 2) reduction of the skeletons to powder; 3) total cold dissolution of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v; 4) separation of free pigments and pigment matrix by ultrafiltration on a membrane with a cut-off threshold of 1 kDa and recovery of the pigment matrix; 5) optionally, removal of dissolved salts by dialysis; 6) optionally, freeze-drying.

10. Process for preparing the pigment matrix according to any one of claims 1 to 3 comprising the following steps: 1) washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or gentle steaming at 37°C; 2) reduction of the skeletons to powder; 3) total cold dissolution of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v; 4bis) separation of the acid-soluble fraction and the acid-insoluble fraction by centrifugation; 4ter) separation of free pigments and the acid-soluble fraction by ultrafiltration on a membrane with a cut-off threshold of 1 kDa; 4quater) mixing the acid-insoluble fraction obtained in step 4bis) with the acid-soluble fraction obtained in step 4ter) to reconstitute the pigment matrix; 5) optionally, removal of dissolved salts by dialysis; 6) optionally, freeze-drying.

11. Process for preparing the acid-soluble fraction of the pigment matrix according to claim 4, characterized in that it further comprises the steps: 1) washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or gentle steaming at 37°C; 2) reduction of the skeletons to powder; 3) total cold dissolution of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v; 4) separation of the acid-soluble fraction and the acid-insoluble fraction by centrifugation and recovery of the acid-soluble fraction (liquid phase); 5) separation of free pigments and the acid-soluble fraction of the pigment matrix by ultrafiltration on a membrane with a cut-off threshold of 1 kDa and recovery of the pigment matrix; 6) optionally, removal of salts and acetic acid by dialysis; 7) optionally, freeze-drying; if this step is implemented, step 6) will preferably also be carried out.

12. Process for preparing the acid-insoluble fraction of the pigment matrix according to claim 5, characterized in that it further comprises the steps: 1) washing of sea urchin skeletons, preferably radioles, with an aqueous solution of NaCIO at a concentration between 5% v / v and 12% v / v, rinsing with water and drying, preferably at room temperature or gentle steaming at 37°C; 2) reduction of the skeletons to powder; 3) total cold dissolution of the mineral part of the powder obtained in an aqueous solution of acetic acid at a concentration of between 5 and 10% v / v, preferably of the order of 10% v / v; 4) separation of the acid-soluble fraction and the acid-insoluble fraction by centrifugation and recovery of the acid-insoluble fraction (solid phase).

13. Kit including: - the acid-soluble fraction of the pigment matrix, - the acid-insoluble fraction of the pigment matrix, and - free PHNQ pigments.

14. Ink comprising: - the pigment matrix according to any one of claims 1 to 8; - a solvent; and - optionally, a binder.

15. Use of the pigment matrix according to any one of claims 1 to 8 as a coloring agent.

16. Use of the pigment matrix according to any one of claims 1 to 8 as an antibacterial agent, antifungal agent, antioxidant agent, UV absorbing agent or biocidal agent.

17. Pigment matrix according to any one of claims 1 to 8 for its use in the treatment of inflammation.

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