process for obtaining an aqueous solution enriched in blue pigment

Abstract

This application relates to a process for obtaining an aqueous solution enriched in blue pigment, comprising the steps a) of supplying an aqueous solution comprising a blue pigment from at least one microorganism of the genus Haslea, b) of precipitating the blue pigment with sodium hydroxide, c) of separating the precipitated phase from the aqueous phase, d) of solubilizing the blue pigment included in the precipitated phase and e) of dialysis.

Description

Title of the invention: process for obtaining an aqueous solution enriched with blue pigment Field of the invention

[0001] The present invention lies in the field of methods for obtaining an aqueous solution enriched in blue pigment, for example the pigment "marennine". It relates more particularly to a method for obtaining an aqueous solution enriched in blue pigment comprising the steps a) of providing an aqueous solution comprising a blue pigment originating from at least one microorganism of the genus Haslea, b) of precipitation of the blue pigment with sodium hydroxide, c) of separation of the precipitated phase from the aqueous phase, d) of solubilization of the blue pigment included in the precipitated phase, and e) of dialysis. Prior art

[0002] Certain species of diatom of the genus Haslea are known for their ability to produce a water-soluble blue pigment, responsible for the phenomenon of greening of the gills and other pallial organs of oysters. This greening is based on the erratic proliferation of the diatom in oyster ponds (oyster beds) and is of great economic interest to oyster farmers, green oysters (i.e. "verte de claire" or "fine de claire verte" type) being rarer and more expensive than non-green oysters refined in beds. The interest in the blue pigment for its applications in aquaculture is growing, particularly given the production and marketing of green oysters at the international level.

[0003] Methods for mass cultivation of the diatom Haslea have been described in particular in patent applications FR 2837833 and FR 2654743. Methods for extracting the blue pigment, in particular the blue pigment called "marennine" produced by the species H. ostrearia, from a culture medium have also been described (cf. Pouvreau et al., 2006a, Pouvreau et al., 2006b, Pouvreau et al., 2007, Turcotte et al., 2016). However, these methods have numerous limitations, in particular given the steps of ultrafiltration and anion exchange chromatography on a column. Furthermore, because these steps increase the cost and time of the process, the ultrafiltration step is not suitable for large volumes (e.g. around twenty liters or more, for example of supernatant) which are necessary in order to obtain aqueous solutions enriched in blue pigment on an industrial scale.Furthermore, a significant portion of the blue pigment also appears to be lost in the fraction discarded by ultrafiltration in the process described by Pouvreau et al., 2006a, reducing the final yield of the pigment.

[0004] There is therefore currently a need for new processes for obtaining aqueous solutions enriched with blue pigment, in particular from large volumes. There is also a need for new processes that are simpler and faster than those used to date. In particular, there is a need for new processes that do not include an ultrafiltration or column chromatography step, preferably without any adverse impact on the yield and / or quality of the blue pigment. There is also a need for new processes that make it possible to reduce the cost of enrichment. Finally, there is a need for new processes from which an aqueous solution comprising the blue pigment can be directly used in industrial applications, such as the greening of oysters. Summary of the invention

[0005] The subject of the present invention is a process for obtaining an aqueous solution enriched in blue pigment, comprising in particular a step of precipitation of the blue pigment with sodium hydroxide, a step of separation of the precipitated phase from the aqueous phase, a step of solubilization of the precipitated phase and a dialysis step.

[0006] Indeed, in the context of the present invention, the inventors have demonstrated that a process for obtaining an aqueous solution enriched in blue pigment comprising these steps makes it possible to avoid any ultrafiltration and column chromatography step. The process of the invention is advantageous because an aqueous solution enriched in blue pigment can be obtained, with a final concentration of blue pigment equal to or greater than that obtained in the prior art, while limiting the costs of the process. Indeed, it is estimated that the ultrafiltration steps according to the method of Pouvreau et al., 2006a results in a loss of the blue pigment of approximately 30 to 40% of the starting mass. In addition, the process is very easy to implement and can be used on large volumes of aqueous solution. This process is also advantageous because it makes it possible to reduce the total implementation time of the process necessary to obtain an aqueous solution enriched in blue pigment.

[0007] The process of the invention also makes it possible to obtain an aqueous solution enriched with blue pigment suitable for use in greening oysters.

[0008] In a first aspect, the present invention therefore relates to a process for obtaining an aqueous solution enriched in blue pigment comprising:

[0009] a) providing an aqueous solution comprising a blue pigment derived from at least one microorganism of the genus Haslea, b) a step of precipitation of the blue pigment with sodium hydroxide, c) a step of separation of the precipitated phase from the aqueous phase, d) a step of solubilization of the blue pigment included in the precipitated phase, and e) a dialysis step.

[0010] Steps b) and c) make it possible to obtain a precipitate of the blue pigment which can then be solubilized, in order to obtain a solution enriched in blue pigment without an intermediate step of ultrafiltration or column chromatography. The separation step (step c)) advantageously makes it possible to eliminate the soluble impurities not precipitated with sodium hydroxide. Advantageously, the dialysis step e) makes it possible to obtain an aqueous solution of blue pigment while reducing the impurities (e.g. elimination of salts).

[0011] Preferably, separation step c) comprises decantation and / or centrifugation, preferably decantation followed by centrifugation. This makes it possible to eliminate the majority, or even all, of the aqueous phase.

[0012] Preferably, the blue pigment is precipitated by the addition of at least 0.3 to 0.4 volumes of 1 M sodium hydroxide. The blue pigment is thus included in the precipitated phase while unwanted water-soluble compounds are retained in the aqueous phase.

[0013] Preferably, the blue pigment included in the precipitated phase is solubilized in step d) by carboxylic acid, preferably formic acid.

[0014] Preferably, the method further comprises a sterilizing filtration step, preferably on a filter having a porosity of 0.22 pmn before step e).

[0015] Preferably, the method further comprises at least one filtration step through a filter having an average pore size of at least 1.2 μm before step b), preferably a first filtration through a filter having an average pore size of between 12 and 15 μm followed by a second filtration through a filter having an average pore size of 1.2 μm. Advantageously, this first filtration makes it possible to avoid clogging during the second filtration.

[0016] Preferably, the aqueous solution enriched with blue pigment obtained in step e) comprises a blue pigment concentration of at least 90 mg / L.

[0017] Preferably, the aqueous phase obtained in step c) is subjected to a further precipitation step according to step b) followed by a separation step according to step c).

[0018] Preferably, during dialysis step e), the dialysis membrane has a cut-off threshold of between 1 and 3.5 kDa. Preferably, the aqueous solution provided in step a) is a supernatant of a culture of at least one diatom selected from: Haslea ostrearia, Haslea karadagensis, Haslea provincialis, Haslea nusantara, Haslea silbo sp nov, and Haslea acoran sp nov, preferably from a culture of diatom Haslea ostrearia NCC 497 deposited with the BEA on October 1, 2018 under number BEA_IDA_0065B.

[0020] According to a second aspect, the present invention relates to an aqueous solution enriched in blue pigment capable of being obtained by the process as described herein. Description of the figures

[0021] [Fig-1] Diagram illustrating the method according to the invention. The dotted frames represent optional steps.

[0022] Figure 2 shows the characterization of the aqueous solution enriched with blue pigment of the “marennine” type called “MCPN” obtained according to the present invention by:

[0023] [Fig.2a] Ultraviolet (UV)-visible absorption spectrum of concentrated marennine obtained according to the present invention (diluted 6 times), with comparison to the spectrum obtained with a solution of marennine purified according to the method of Pouvreau et al 2006a (“pure marennine”) at 0.2 mg / mL

[0024] [Fig.2b] Nuclear magnetic resonance (NMR) spectrum with comparison to the spectrum obtained with a solution of “pure marennine”

[0025] [Fig.2c] is a size exclusion chromatography (SEC-HPLC) analysis.

[0026] AU stands for "absorbance units", ppm for "parts per million".

[0027] Figure 3 is a comparison of the characteristics of the blue pigment of the “marennine” type prepared according to the process of the present invention (called “MCPN”) or according to the process of Turcotte et al 2016 (“blue water” or “BW”) or according to Pouvreau et al 2006a (“pure marennine”)

[0028] [Fig.3a] is an ultraviolet (UV)-visible absorption spectrum

[0029] [Fig.3b] is a nuclear magnetic resonance (NMR) spectrum

[0030] [Fig.3c] is a size exclusion chromatography (SEC-HPLC) analysis.

[0031] AU stands for “absorbance units”, ppm for “parts per million”. Detailed description of the invention

[0032] As indicated previously, in the context of the present invention, the inventors have demonstrated a new process for obtaining an aqueous solution enriched in blue pigment comprising the steps of precipitation of the blue pigment with sodium hydroxide, separation of the precipitated phase from the aqueous phase, solubilization of the precipitated phase, and dialysis. Indeed, the inventors have demonstrated that, surprisingly, a process comprising these steps advantageously makes it possible to avoid any ultrafiltration and column chromatography step.

[0033] The process according to the invention has the advantage of obtaining an aqueous solution enriched in blue pigment with a final concentration of blue pigment at least equal to that obtained using existing processes, while limiting the costs of the process. In addition, the process is very easy to implement, and can be used on large volumes of aqueous solution. The yield of blue pigment obtained by the process of the invention is, interestingly, higher than that obtained by the processes according to the prior art. This process also makes it possible to reduce the time required to obtain an aqueous solution enriched with blue pigment. Process of obtaining

[0034] A first aspect of the invention therefore relates to a process for obtaining an aqueous solution enriched in blue pigment comprising:

[0035] a) providing an aqueous solution comprising a blue pigment derived from at least one microorganism of the genus Haslea, b) a step of precipitation of the blue pigment with sodium hydroxide, c) separation of the precipitated phase from the aqueous phase, d) a step of solubilization of the precipitated phase, and e) a dialysis step.

[0036] Steps a) to e) are carried out in the order a), then b), then c), then d), then e). In certain cases (see below), additional steps (excluding any ultrafiltration and / or column chromatography step) may be interposed: • Before step a) For example, step a) may be preceded by a step of culturing diatoms of the genus Haslea and, optionally, by a step of lysing the diatoms of the genus Haslea present in the aqueous solution. • Between steps a) and b) For example, a filtration step can be added between steps a) and b). • Between steps c) and d) For example, the aqueous phase obtained in step c) can be resubmitted to steps b) and c) and the precipitate obtained grouped with that obtained previously. • Between steps d) and e) For example, a filtration step can be added between steps d) and e). • After step e) For example, a concentration or formulation step may be added after step e).

[0037] These various optional additional steps are detailed below.

[0038] The purification method according to the invention is in particular carried out from an aqueous solution comprising a blue pigment derived from at least one microorganism of the genus Haslea, preferably from a supernatant of a culture medium of at least one microorganism of the genus Haslea. Blue pigment

[0039] For the purposes of the present invention, the term "blue pigment" means a water-soluble blue pigment produced by a diatom of the genus Haslea. Said blue pigment may in particular be the blue pigment called "marennine" produced by the diatom H. ostrearia or any other blue pigment produced by a diatom of the genus Haslea, such as H. karadagensis (Gastineau et al., 2012), H. provincialis (Gastineau et al., 2016), H. nusantara (Prasetiya et al., 2018), H. silbo sp nov (Gastineau et al., 2011), or H. acoran sp nov (Gastineau et al., 2011). Said blue pigment may comprise or consist of several blue pigments produced by different diatoms. Preferably, the blue pigment according to the invention comprises marennine. According to a particular embodiment, the blue pigment according to the invention consists of marennine.

[0040] A distinction can generally be made between the intracellular form (that present in the diatom) and the extracellular form of the blue pigment which is released into the marine environment. For example, the intracellular form of marennine has a different molar mass from the extracellular form (Pouvreau et al., 2006a).

[0041] In the context of the present invention, the blue pigment may comprise blue pigment in its intracellular form and / or its extracellular form. According to a first embodiment, the blue pigment consists essentially of its extracellular form. Preferably, the blue pigment consists of its extracellular form. According to another embodiment, the blue pigment comprises both the intracellular form and the extracellular form. According to another embodiment, the blue pigment consists essentially of its intracellular form. Preferably, the blue pigment consists of its intracellular form. Step a)

[0042] Step a) of the method according to the invention is a step of providing an aqueous solution comprising a blue pigment derived from at least one microorganism of the genus Haslea.

[0043] By "aqueous solution" is meant any solution comprising blue pigment from at least one microorganism of the genus Haslea. According to a preferred embodiment, said aqueous solution is a diatom culture. The diatom culture comprises the diatoms themselves and the supernatant (i.e. the culture medium without the diatoms). Preferably, said aqueous solution comprises the supernatant. This would be the case, for example, if the supernatant is mixed with another solution before step a), for example water. According to another preferred embodiment, said aqueous solution consists essentially of the supernatant. Preferably, said aqueous solution is the supernatant. Methods suitable for separating the diatoms from the culture medium, in order to provide the supernatant, are well known to those skilled in the art. Such a step may in particular be carried out by centrifugation with recovery of the supernatant or by filtration.

[0044] Preferably, said diatom is selected from the group consisting of: H. ostrearia, H. karadagensis, H. provincialis, H. n usantara, H. silbo sp nov, H. acoran sp nov. Preferably, said diatom is H. ostrearia, more preferably H. ostrearia NCC 497, even more preferably H. ostrearia NCC 497 deposited with the “Banco Espanol de Algas” (BEA), Universidad de Las Palmas de GC, Muelle de Taliarte s / n, 35214 Telde - Las Palmas, Spain on October 1, 2018 under number BEA_IDA_0065B.

[0045] According to a particular embodiment, the aqueous solution may comprise water-soluble blue pigment produced by at least two species of diatoms, or by at least two strains of a given species (eg two strains of H. ostrearia). Said diatoms may have been cultured together, or cultured individually. When the diatoms are cultured individually, the culture, preferably the culture supernatants, of each diatom are added together in step a).

[0046] According to a preferred embodiment, when at least two species are cultivated together, one of the two species is H. ostrearia. According to a preferred embodiment, when at least two strains of H. ostrearia are cultivated together, one of the two strains is H. ostrearia NCC 497, preferably H. ostreariaNCC 497 deposited with the “Banco Espanol de Algas” (BEA) on October 1, 2018 under the number BEA_IDA_0065B.

[0047] In some cases, the aqueous solution provided in step a) comprises lysed diatoms. A lysis step may therefore precede step a). A lysis step is particularly advantageous when a solution comprising both extracellular and intracellular forms of the blue pigment is desired. A lysis step in fact makes it possible to release intracellular blue pigment into the supernatant or into any other aqueous solution. For example, the lysis of diatoms may be carried out according to the method described in Pouvreau et al., 2006a as described on page 770.

[0048] According to a particular embodiment, the blue pigment concentration of the aqueous solution in step a) is between 2 and 7 mg / L, more advantageously between 3 and 5 mg / L. According to a particular embodiment, the blue pigment concentration of the aqueous solution in step a) is at least 2 mg / L, preferably at least 3 mg / L. The blue pigment concentration in the aqueous solution can be determined by the person skilled in the art in view of his general knowledge. By way of non-limiting example, the blue pigment concentration can be determined by spectrophotometry, by the Lowry method (Pouvreau, et al., 2006b), or by HPLC by gel filtration connected to a photodiode detector (Pouvreau et al., 2007). Preferably, the blue pigment concentration is determined by spectrophotometry using the extinction coefficient published in Pouvreau, et al. (2006b).

[0049] Preferably, the pH of the aqueous solution comprising blue pigment is between 7 and 9, more preferably approximately 8 (+ / - 0.2).

[0050] According to a particular embodiment, the aqueous solution comprising the blue pigment provided in step a) has not undergone any prior filtration step. Optionally, the aqueous solution provided in step a) is however filtered before step b). This additional step is particularly preferred when the aqueous solution comprises aggregates or any other non-water-soluble particle because the filtration step makes it possible to separate these impurities upstream of the precipitation, thus improving the purity of the precipitate obtained in step b). According to a preferred embodiment, a filtration step is interposed between steps a) and b) when the aqueous solution is a diatom culture, the filtration step making it possible to separate the supernatant from the diatoms before any other step. The porosity of the filter preferably makes it possible to retain the diatoms, without however retaining other compounds (e.g. the blue pigment).The methods and filters suitable for such a filtration step are well known to those skilled in the art. Such a step can in particular be carried out using glass microfibre filters (marketed by Whatman, for example type GF / C).

[0051] According to a particular embodiment, at least one filtration step is carried out, more preferably, at least two filtration steps are carried out. According to a first embodiment, the filtration is carried out through a filter having an average pore size of at least 1.2 μm. According to a second embodiment, the filtration step is carried out through two filters of different sizes, the second filter having an average pore size smaller than the first filter and of at least 1.2 μm. Advantageously, the first filtration makes it possible to avoid clogging during the second filtration.

[0052] Also, according to a particularly preferred embodiment, the filtration step comprises a first filtration through a filter having an average pore size of between 12 and 15 μm followed by a second filtration through a filter having an average pore size of 1.2 μm. Step b)

[0053] After step a) of providing an aqueous solution comprising the blue pigment, said blue pigment is precipitated with sodium hydroxide. From the moment the sodium hydroxide is added to the aqueous solution according to step b), the solution is called a "mixture", because it comprises both a precipitated phase and an aqueous phase.

[0054] During step b), the majority of the blue pigment is precipitated and is therefore found in solid form, in the precipitated phase. Advantageously, the sodium hydroxide is added until the blue pigment has completely precipitated. The quantity of sodium hydroxide to be added varies depending on the concentration of the blue pigment in the solution. aqueous solution from step a) as well as the volume of the aqueous solution. Complete precipitation leads to the discoloration of the aqueous phase. Also, the precipitation step by adding sodium hydroxide corresponds to a step easily carried out by the person skilled in the art, given its visual appearance. Advantageously, the unwanted water-soluble compounds present in the aqueous solution such as proteins and carbohydrates, or even other compounds not having absorption in the visible spectrum, remain in the aqueous phase.

[0055] According to a particular embodiment, the final percentage (mass / mass) of sodium hydroxide relative to the aqueous solution (mass of sodium hydroxide / mass of the aqueous solution x 100) used in step b) is between 0.02 and 0.04%. Preferably, the concentration of the sodium hydroxide solution added to the aqueous solution is less than or equal to 10 M, preferably less than or equal to 2 M, more preferably between 1 M and 2 M, even more preferably equal to 1 M or 2 M. A person skilled in the art will recognize that said final percentage of sodium hydroxide relative to the aqueous solution may vary depending on the initial concentration or quantity of blue pigment in the aqueous solution provided in step a).

[0056] As an example, complete precipitation can be observed by adding 5 to 10 mL of 1 M sodium hydroxide to an aqueous solution having a volume of 1 L and a blue pigment concentration of a few mg / L.

[0057] Preferably, the pH of the mixture obtained following step b) is at least 12.

[0058] According to one embodiment, the sodium hydroxide is added in several stages, advantageously drop by drop. During the time of addition of the sodium hydroxide, the mixture may or may not be stirred. Preferably, the mixture is stirred. During the time of addition of the sodium hydroxide, the aqueous solution may be maintained between 4°C and 25°C, for example between 4°C and 8°C or at room temperature, for example between 20 and 25°C. Step c)

[0059] After step b) precipitation of the blue pigment with sodium hydroxide, the precipitated phase and the aqueous phase of the mixture are separated. By "separation" is meant here the physical separation of the two phases. Advantageously, in addition to separating the two phases, step c) makes it possible to limit the volume of the mixture to be treated downstream. The methods suitable for such a separation step are well known to those skilled in the art. Such a step can in particular be carried out by decantation and / or by centrifugation. By way of non-limiting example, the separation step can be carried out between 4°C and 25°C, for example between 4°C and 8°C or at room temperature, for example between 20 and 25°C.

[0060] Preferably, the separation according to step c) comprises decantation. For the purposes of the present invention, the term "decantation" means an operation which consists of allowing the mixture to stand for a given period of time, so that the precipitate settles by gravity difference, in order to remove the aqueous phase. Preferably, the decantation therefore comprises a step of allowing the mixture to stand before the physical separation of the precipitated phase from the aqueous phase. Preferably, while the mixture is allowed to stand, it is not stirred.

[0061] According to a preferred embodiment, the mixture is left to stand for a period of time equal to or greater than 5 hours, more preferably equal to or greater than 10 hours, even more preferably equal to or greater than 15 hours. According to another preferred embodiment, the mixture is left to stand for a period of time equal to 5 hours. Advantageously, standing the mixture for a period of time equal to or greater than 5 hours makes it possible to optimize the formation of the precipitate in addition to allowing the precipitate to settle at the bottom of the container. In certain cases (e.g. when the quantity of precipitate is small), it would be advantageous to let the mixture stand for a period of time equal to or greater than 16 hours.

[0062] The mixture may be left to stand between 4°C and 25°C, for example between 4°C and 8°C or at room temperature, for example between 20 and 25°C.

[0063] Preferably, a portion of the aqueous phase (e.g., 80% to 90% of the aqueous phase) is removed from the mixture. Preferably, at least 80%, at least 85%, at least 90% of the aqueous portion is removed from the mixture. The aqueous phase thus removed may be stored for later use or discarded. The aqueous phase is preferably stored at a temperature between 4°C and 25°C, for example between 4°C and 8°C, or at room temperature, for example between 20 and 25°C.

[0064] As indicated above, by removing a portion of the aqueous phase, the volume of the mixture to be treated is reduced accordingly. This is particularly advantageous when large volumes (e.g., greater than 25, 50 or 100 L) are supplied as the starting aqueous solution (i.e., in step a)).

[0065] According to another embodiment, the separation is carried out by centrifugation. By "centrifugation" is meant the separation of the precipitated phase from the aqueous phase by centrifugal force (by rapid rotation). The mixture can also be left to stand under the conditions described above before centrifugation.

[0066] The person skilled in the art will be able to determine the centrifugation conditions suitable for obtaining the separation of the precipitated phase comprising the precipitate of the blue pigment from the aqueous phase. By way of non-limiting example, the mixture may be centrifuged at an acceleration of at least 4000 xg, preferably at least 12,000 xg. When the mixture is centrifuged at an acceleration of at least 12,000 xg (eg between 12,000 and 13,000 xg), the duration of the centrifugation is advantageously at least less than 20 min, preferably at least 30 min, preferably for 20 to 35 min. The mixture may undergo one or more centrifugations, which may be under the same or different conditions (i.e. with increasing duration or acceleration). Preferably, the mixture undergoes a single centrifugation step. Preferably, the mixture is centrifuged at an acceleration of between 12,000 and 13,000 xg for 30 minutes.

[0067] During centrifugation, the precipitated phase comprising the blue pigment forms the pellet while the non-precipitated water-soluble components remain in the aqueous phase. As during decantation, a large part of the aqueous phase is removed following centrifugation. The aqueous phase thus removed may be retained for later use or discarded.

[0068] Preferably, the centrifugation takes place at a temperature between 4°C and 25°C, more preferably between 4°C and 8°C or at room temperature, for example between 20 and 25°C.

[0069] According to a preferred embodiment, the separation step comprises decantation and centrifugation, preferably according to the aforementioned conditions. Preferably, the mixture is decanted before being centrifuged. The combination of these two techniques is particularly advantageous because decantation makes it possible to reduce the volume of the mixture to be centrifuged, a large part of the aqueous phase can now be removed before centrifugation. When part of the aqueous phase is removed during decantation and another part of the aqueous phase is removed following centrifugation, the aqueous phases can then be combined together. Optional step

[0070] At the end of step c), two separate phases are obtained: the precipitated phase and the aqueous phase. In certain cases, it may be advantageous to carry out the precipitation and separation steps (corresponding to steps b) and c) above) on the aqueous phase thus obtained. This is particularly preferred when the aqueous phase comprises a residual amount of blue pigment and makes it possible to improve the final yield of blue pigment.

[0071] Preferably, the aqueous phase subjected to steps b) and c) comprises all of the aqueous phases obtained during the separation in step c).

[0072] Preferably, during step c), the mixture is left to stand for a period of time equal to or greater than 3 hours, more preferably for a period of time equal to or greater than 16 hours. This makes it possible in particular to improve the recovery yield of the blue pigment obtained by the process according to the invention. Step d)

[0073] After step c) of separating the precipitated phase from the aqueous phase, the precipitated phase comprising the blue pigment is solubilized. The person skilled in the art will be able to identify a suitable solution for solubilizing the precipitated blue pigment.

[0074] Preferably, the blue pigment of the precipitated phase is solubilized by the addition of a weak acid, such as a carboxylic acid. By "carboxylic acid" is meant any acid comprising a carboxyl group, such as acetic acid, formic acid, carbonic acid, propenoic acid, etc. Preferably, the blue pigment is solubilized by the addition of formic acid (CAS no. 64-18-6). Preferably, the blue pigment is solubilized by the addition of a 5% (m / m) carboxylic acid solution, more preferably by the addition of a 5% (m / m) formic acid solution.

[0075] Preferably, the volume ratio of the aqueous solution provided in step a) and the aqueous solution enriched with blue pigment in step d) (solution provided in step a) / solution obtained in step d)) is at least 29 / 1, more preferably at least 30 / 1, even more preferably at least 40 / 1. According to a preferred embodiment, the ratio between the volume of the aqueous solution provided in step a) and the volume of the aqueous solution enriched with blue pigment obtained in step d) is between 29 / 1 and 40 / 1. The person skilled in the art will, however, recognize that said ratio between the volume of aqueous solution provided in step a) and the volume of the solution enriched with blue pigment in step d) may vary depending on the initial concentration of blue pigment in the aqueous solution provided in step a). Also, a higher concentration of the blue pigment in the aqueous solution provided in step a) (eggreater than 7 mg / mL) may require the addition of a larger volume of the solubilizing solution (e.g., formic acid), and thus a ratio lower than 29:1.

[0076] Preferably, the blue pigment-enriched solution obtained in step d) has a pH of between 3.0 and 4.0, preferably between 3.0 and 3.5.

[0077] Preferably, at the end of step d), the blue pigment is present in solution at a concentration equal to or greater than 90 mg / L. Also, the solution enriched in blue pigment obtained at the end of step d) is advantageously enriched in blue pigment by at least 12 times, advantageously by at least 18 times, more advantageously by at least 30 times compared to the aqueous solution provided in step a). Optional step

[0078] At the end of step d), one or more optional steps may be present. For example, a filtration step, such as a sterilizing filtration step, is carried out. Preferably, at the end of step d), a filtration step is carried out, more preferably on a filter having a porosity of 0.22 μm. This filtration step advantageously makes it possible to remove any remaining cellular debris quickly and inexpensively. Step e)

[0079] After solubilization step d), the blue pigment-enriched solution is subjected to a dialysis step. The blue pigment-enriched solution obtained in step d) can be dialyzed against any suitable aqueous solution, such as, for example, distilled water. The methods and membranes suitable for dialysis are well known to those skilled in the art. Such a step can in particular be carried out using a dialysis membrane having a cut-off threshold of between 1 and 3.5 kDa.

[0080] According to a preferred embodiment, the dialysis membrane has a cut-off threshold of between 1 and 3.5 kDa, preferably a cut-off threshold of 1 kDa, 2 kDa, or 3.5 kDa.

[0081] Preferably, the dialysis step is carried out for a minimum duration of 48 hours, more advantageously 72 hours. Preferably, the appropriate aqueous solution used during the dialysis (e.g. water) is changed approximately every 12 hours. Preferably, the dialysis of the blue pigment-enriched solution is carried out against approximately 2 L of the appropriate aqueous solution (e.g. water).

[0082] The dialysis step makes it possible in particular to increase the pH of the solution. The dialysis step also makes it possible to eliminate the salts and precipitating agents present in this aqueous solution (eg Mg(OH)2).

[0083] Preferably, at the end of step e), the aqueous solution enriched with blue pigment comprises a concentration equal to or greater than 90 mg / L of blue pigment, more preferably equal to or greater than 100 mg / L, equal to or greater than 110 mg / L, equal to or greater than 120 mg / L, equal to or greater than 130 mg / L, equal to or greater than 140 mg / L, even more preferably equal to or greater than 150 mg / L.

[0084] Preferably, the solution enriched with blue pigment obtained in step e) has a pH of between 4.5 and 5.5.

[0085] Said aqueous solution enriched with blue pigment is preferably stored at approximately 4°C (4 ± 2°C) and in the dark at the end of dialysis step e) or at the end of any other step when an additional step is present after step e). Indeed, the inventors have demonstrated that the blue pigment contained in the solution is stable for several months at this temperature and in the dark. It would therefore not be obligatory to add any excipient, such as a stabilizing agent, at the end of the process of the invention. Other optional subsequent steps

[0086] Even if the process described above makes it possible to obtain an aqueous solution enriched in blue pigment having a high purity and yield, in certain cases, it may however be advantageous to carry out one or more additional steps, for example concentration or formulation. The process according to the invention can thus further comprising at least one additional step subsequent to step e) of concentration or formulation.

[0087] By way of non-limiting example, it may be advantageous to subsequently concentrate the blue pigment included in the composition, for example by an evaporation step, in particular in order to obtain a higher concentration of blue pigment. By way of non-limiting example, it may also be advantageous to formulate the blue pigment in a composition. In order to adapt the aqueous solution enriched with blue pigment resulting from the process according to the invention to a particular use, the composition may undergo a formulation step, for example by the addition of an excipient and / or a vehicle and / or by any other change in the composition of said aqueous solution enriched with blue pigment, such as the addition of a buffer solution. In other cases, the formulation step may be a drying step, for example, making it possible to obtain the blue pigment in powder form.

[0088] In a particular embodiment, the method according to the invention further comprises, after step e) of dialysis, one or more of the following steps aimed at adapting the composition to a particular formulation and / or increasing the purity of the product: • a concentration stage; and / or • a formulation stage.

[0089] Each optional step identified above may be present as a single additional step of the method, in combination with one or more other optional steps, or with all other optional steps.

[0090] Another subject of the present invention is the aqueous solution obtained by the process described here.

[0091] The enriched blue pigment solution obtained by the process of the invention can in particular be used as a source of natural blue pigment for the food and cosmetic industries, or as an antimicrobial substance for use in aquaculture. Another subject of the present invention is the use of the aqueous solution enriched with blue pigment obtained by the process described here as a source of natural blue pigment for the food and cosmetic industries, or as an antimicrobial substance for use in aquaculture. EXAMPLES

[0092] The invention is illustrated by the following non-limiting examples. These teachings include alternatives, modifications and equivalents, which will be appreciated by a person skilled in the art. Example 1#: Method according to the invention

[0093] A process developed to obtain an aqueous solution enriched in blue pigment, here marennine, without any ultrafiltration or column chromatography step is described below. Materials and methods Biological material

[0094] Haslea ostrearia strains were isolated from oyster beds in Bourneuf Bay (France) and identified by scanning electron microscopy (NCC 497 strain). Batch cultures were performed in 200 mL Erlenmeyer flasks using an artificial seawater medium adapted from Harrison et al. (Harrison et al. 1980) and sterilized by autoclaving (final medium salinity 31 + 1 ppm and pH 7.6 ± 0.2). 77. ostrearia cultures were maintained in a temperature-controlled culture chamber at 16°C under illumination of 200 pmol of photons m 2 s 1 delivered by white fluorescent tubes with a 14 / 10 hour day / night cycle.When the cultures reached the beginning of the stationary phase of growth, the culture supernatant containing extracellular marennin was collected after decanting the cells and coarsely filtered through a 15 μm porosity rapid filtration filter (150 mm Filter paper, Fisher Scientific®). The filtered supernatant was kept in the temperature-controlled room in the dark before proceeding to the steps described in the general procedures.

[0095] Process for enriching blue pigment in aqueous solution

[0096] Optionally, the Haslea ostrearia culture medium is filtered at 1.2 pm (GF / C filters).

[0097] The H. ostrearia culture medium, comprising marennine at a concentration of approximately 3 to 4 mg / L, is then placed under moderate stirring and subjected to precipitation by adding 5 mL of sodium hydroxide (1 M) per 1 liter of culture filtered dropwise and then left to settle until a greenish layer appears at the bottom of the bottle and the color of the aqueous phase disappears (approximately 5 hours) at room temperature without stirring.

[0098] After decantation, a large portion of the aqueous phase (eg 80 to 90%) is removed. Optionally, the aqueous phase is set aside for further processing.

[0099] The mixture comprising the remainder of the aqueous phase and the precipitated phase is centrifuged at 12,880 xg (10,000 rpm) for 30 minutes. The remaining aqueous phase is then separated from the precipitated phase. Optionally, the aqueous phases are combined and resubmitted to precipitation by adding sodium hydroxide and then left to settle as described above, by settling for at least 3 hours, or even overnight (eg 16 hours), to improve the recovery yield of marennine and further reduce the working volume.

[0100] After centrifugation, the precipitated phase obtained is solubilized in a 5% formic acid (HCOOH) solution. The solubilization is carried out in a volume of 250 to 350 mL of formic acid.

[0101] The solution obtained is filtered at 0.22 pm on a regenerated cellulose membrane. Finally, dialysis against 2 L of water is carried out in a 1 kDa dialysis sausage for 72 hours, changing the dialysis water twice a day. Results

[0102] An aqueous solution enriched in marennine (90 mg / L minimum) with a pH between 4.5 and 5.5 noted "MCPN" is obtained. Also, very advantageously, the marennine in the enriched aqueous solution is concentrated 26-fold.

[0103] Example 2: Characterization of the “MCPN” solution

[0104] The aqueous solution enriched in marennine was characterized by UV-Visible spectrometry, nuclear magnetic resonance (NMR) and size exclusion chromatography (HPLC-SEC). A solution of marennine considered pure and obtained according to the method of Pouvreau et al (2006a) was also analyzed for comparison. Materials and methods

[0105] UV-visible absorption spectra were performed on a UV-3100PC spectrophotometer (VWR). The analysis is carried out in scan mode from 190 to 800 nm.

[0106] The NMR analysis was carried out on the liquid NMR platform of the Institute of Molecules and Materials of Le Mans at the University of Le Mans. The device used is a Bruker AVANCE 400MHz Spectrometer with the BBFO+ 5mm broadband probes (1H, 19F and from 15N to 31P, z-gradient, automatic tuning) and QNP 5mm (1H, 19F, 31P, 13C, z-gradient) and a B-ACS 60 type sample changer.

[0107] The chromatographic analysis was carried out on a Waters Alliance 2690 HPLC system equipped with a PAD 996 pulsed amperometric detector and Empower software. An Xbridge® Protein BEH SEC 125 A°, 3.5 pm, 7.8 * 300 mm column (Waters) was used with isocratic elution (250 mM Na2CO3, 0.05% NaN3, pH 8) and a flow rate of 0.5 mL / min. Results

[0108] The absorption spectrum and NMR spectrum of MCPN solution are very similar to those of pure marennine ([Fig.2a] and 2b). Moreover, the chromatogram of MCPN solution, only represents another impurity (peak at 10.963 min) and a small shoulder on the marennine peak at 17.607 minutes ([Fig.2c]).

[0109] Advantageously, the aqueous solution enriched with blue pigment (here, marennine) obtained by the process of the invention has the same characteristics as those of pure marennine. Also, the process developed advantageously makes it possible to obtain a high purity product without an ultrafiltration or column chromatography step.

[0110] Example 3: Evaluation of variants of the process according to FE example 1

[0111] A series of tests was carried out by varying the conditions of the different stages, according to Table 1 below.

[0112] [Tables 1] STEP / MODIFICATION RESULTS l OBSERVATIONS b> / precipitation of the blue pigment with 2M sodium hydroxide No difference observed compared to the process described in Example 1 b) / temperature at 4: C No difference observed compared to the process described in Example 1, or even an improvement in the precipitation d) / re-solubilization by addition of strong acid (HCl) Differences observed in the spectrocolorimetric properties of Marennine e) / 2 kDa or 3.5 kDa dialysis sausage No difference observed compared to the process described in Example 1 Operating conditions of representative tests

[0113] It is noted that the different tested variants of the process do not modify the aqueous solution enriched with blue pigment, with the exception of the resolubilization phase by hydrochloric acid.

[0114] Example 4: Enrichment of the blue pigment produced by other species of H aslea

[0115] The process for obtaining an aqueous solution enriched in blue pigment as described herein was tested using the supernatant of different Haslea species producing the blue pigment as the starting aqueous solution (in step a)) (see Table 2).

[0116] [T ableaux2] Haslea species Origin H. ostrearia Polder de Bouin (Vendée, France) H. provinciabs Boulouris (Var, France) H. ostrearia San Juan Islands (Oregon, USA) H. sitbo sp nov Morehead City (North Carolina, USA) Haslea species tested

[0117] It is found that the culture of different species of Haslea producing blue pigment can be used as an aqueous solution in step a) of the process according to the invention, including the culture of the species described above. Example 5#: Comparative test

[0118] The method according to the invention was carried out in parallel with the method currently used as described by Pouvreau (Pouvreau et al., 2006a). Materials and methods

[0119] The process for enriching the blue pigment according to the invention was carried out as described in Example 1. The processes of Pouvreau et al., 2006a and Turcotte et al., 2016 were carried out in parallel. Briefly, these processes begin with a filtration step on a Whatman GF / F filter (0.45 μm) of the supernatant of a Haslea ostrearia culture, followed by an ultrafiltration step (recovery of the fraction between 3 kDa and 30 kDa) (Turcotte et al., 2016), followed for Pouvreau et al., 2006a by a semi-preparative anion exchange chromatography step (passage through a column containing a Sepharose resin). The pigment retained on the resin is eluted by a saline solution and then the solution is dialyzed.

[0120] The UV-visible absorption spectra, NMR spectra, and size exclusion chromatography (HPLC-SEC) of the aqueous solution obtained by the method described here (the “MCPN” solution) and that obtained by the methods of Pouvreau et al., 2006a (“pure marennine” and Turcotte et al. 2016 (solution called “blue water, BW”) were then determined and compared together. These techniques are detailed in Example 2, above. Results

[0121] As indicated in Table 3 above, it is noted that the duration of the process according to the present invention is shorter than that of the prior art, that the cost is greatly reduced. Furthermore, it is noted that the absorption spectra of “BW”, “pure marennine” and “MCPN” are very comparable. The shift observed at the level of the 640-680 m zone is due to the difference in pH between the solutions. In addition, the chromatograms obtained by HPLC-SEC show that “MCPN” is purer than “BW”.

[0122] [Tables3] Parameter / Result Process according to the invention Process according to Pouvreau, 2006a Process duration (in number of hours) 108 (for 10 L supernatant) 562 (for 10 L supernatant) Estimated cost of materials NaOH: 52€7kg Formic acid: 50€ / L Average price of nion exchange resin: W00€ / kg Average cost of disposable ultrafiltration column: 25Q€ Initial marennine concentration (in mg / L) 3.4 3.4 Final marennine concentration (in mg / L) 90 to i 10 (depending on the test) 110 Comparison of processes List of cited documents

[0123] Gastineau, 2011. Biodiversity, reproduction and phylogeny of blue diatoms of the genus Haslea and valorization of their marennine-type pigments. Agricultural Sciences, University of Maine.

[0124] Gastineau, etal., 2012. Haslea karadagensis (Bacillariophyta): a second blue diatom, recorded from the Black Sea and producing a novel blue pigment. Eur. J. Phycol., 47: 469-479

[0125] Gastineau, et al., 2016. A new blue-pigmented hasleoid diatom, Haslea provincialis, from the Mediterranean Sea. Eur. J. Phycol, 51: 156-170

[0126] Harrison, et al., 1980. A broad spectrum artificial sea water medium for Coastal and open océan phytoplankton. J. Phycol. 16, 28-35.

[0127] Pouvreau, et al., 2006a. Purification of the blue-green pigment - marennine from the marine tychopelagic diatom Haslea ostrearia (Gaillon / Bory) Simonsen. J. Appl. Phycol., 18,769-781.

[0128] Pouvreau, et al., 2006b. Preliminary characterization of the blue-green pigment -marennine from the marine tychopelagic diatom Haslea ostrearia (Gaillon / Bory) Simonsen. J. Appl. Phycol., 18, 757-767.

[0129] Pouvreau, et al., 2007. Method for the quantification of the blue-green pigment marennine synthesized by the marine diatom Haslea ostrearia (Gaillon / Bory) Simonsen using HPLC gel-filtration and photodiode-array détection. J. Appl. Phycol. 19, 263-270.

[0130] Prasetiya, et al., 2018. Haslea nusantara, a new blue diatom from the Java Sea, Indonesia: Morphology, biometry and molecular characterization. Crypt. & Algol, (sous presse)

[0131] Robert, et al., 2002. Extraction and quantitative analysis of the blue pigment "marennine" synthesized by the diatom Haslea ostrearia. Journal of Applied Phycology, 14: 299-305.

[0132] Turcotte, et al., 2016. Prophylactic effect of Hasl e a ostrearia supernatant culture containing the pigment marennine to stabilize bivalve hatchery production. Aquatic Living Resources, 29: 401 (9 pages).

Claims

Claims

1. Process for obtaining an aqueous solution enriched in blue pigment, comprising: a) providing an aqueous solution comprising a blue pigment originating from at least one microorganism of the genus Haslea, b) a step of precipitation of the blue pigment with sodium hydroxide, c) a step of separation of the precipitated phase from the aqueous phase, d) a step of solubilization of the blue pigment included in the precipitated phase, and e) a dialysis step.

2. Method according to claim 1, characterized in that the separation step c) comprises decantation and / or centrifugation, preferably decantation followed by centrifugation.

3. A method according to claim 1 or 2, characterized in that the blue pigment is precipitated in step b) by the addition of 0.02 and 0.04% (m / m) of sodium hydroxide.

4. Process according to any one of claims 1 to 3, characterized in that the blue pigment included in the precipitated phase obtained in step c) is solubilized in step d) by carboxylic acid, preferably formic acid.

5. Method according to any one of claims 1 to 4, characterized in that it further comprises a sterilizing filtration step, preferably on a filter having a porosity of 0.22 pm, before the step

6. ej. Method according to any one of claims 1 to 5, characterized in that it further comprises at least one filtration step through a filter having an average pore size of at least 1.2 pm before step b), preferably a first filtration through a filter having an average pore size of between 12 and 15 pm followed by a second filtration through a filter having an average pore size of 1.2 pm.

7. Process according to any one of claims 1 to 6, characterized in that the aqueous phase obtained in step c) is subjected to a further precipitation step according to step b) followed by separation according to step c).

8. Method according to any one of claims 1 to 7, characterized in that the dialysis membrane has a cut-off threshold of between 1 and 3.5 kDa.

9. Method according to any one of claims 1 to 8, characterized in that the aqueous solution provided in step a) is a supernatant of a culture of at least one diatom selected from: Haslea ostrearia, Haslea karadagensis, Haslea provincialis, Haslea nusantara, Haslea silbo sp nov, Haslea acoran sp nov, preferably from a culture of diatom Haslea ostrearia NCC 497 filed with the BEA on October 1, 2018 under number BEA_IDA_0065B.

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