WO2020115439A1

WO2020115439A1 - Method for obtaining an aqueous solution enriched with blue pigment

Info

Publication number: WO2020115439A1
Application number: PCT/FR2019/052933
Authority: WO
Inventors: Charlotte FALAISE; Lila ZEBIRI; Boris JACQUETTE; Pamela PASETTO; Jean-Luc Mouget
Original assignee: Universite Du Mans; Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2018-12-04
Filing date: 2019-12-04
Publication date: 2020-06-11
Also published as: EP3924462A1; FR3089231A1

Abstract

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

Description

PROCESS FOR OBTAINING AN AQUEOUS SOLUTION ENRICHED IN BLUE PIGMENT

FIELD OF THE INVENTION

The present invention relates to the field of processes for obtaining an aqueous solution enriched with blue pigment, for example the "marennine" pigment. It relates more particularly 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 derived from at least one microorganism of the genus Haslea, b) of precipitation of the blue pigment with sodium hydroxide, c) separation of the precipitated phase from the aqueous phase, d) solubilization of the blue pigment included in the precipitated phase, and e) dialysis.

PRIOR ART

Certain species of diatom from the genus Haslea are known for their capacity to produce a water-soluble blue pigment, responsible for the greening phenomenon of the gills and other pallial organs of oysters. This greening is based on the erratic proliferation of diatoms in oyster basins (clear to oysters) and is of great economic interest for oyster farmers, green oysters (ie "clear green" or "clear green fines") being rarer and more expensive than non-green oysters ripened in the clears. The interest of the blue pigment for its applications in aquaculture is increasing in particular taking into account the production and the marketing of green oysters on an international level.

Methods for cultivating the Haslea diatom in mass 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 many limitations, in particular taking into account the stages of ultrafiltration and anion exchange chromatography on a column. In addition, because these steps increase the cost and the time for carrying out the process, the ultrafiltration step is not suitable for large volumes (eg around twenty liters or more, for example of supernatant) which are necessary in order to obtain aqueous solutions enriched with blue pigment on an industrial scale. In addition, a significant part of the blue pigment also seems 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.

There is therefore, at present, a need for new methods of obtaining aqueous solutions enriched with blue pigment, in particular from large volumes. There is also a need for new methods that are simpler and faster than those used to date. In particular, there is a need for new processes which do not include an ultrafiltration step or column chromatography, preferably without any harmful impact on the yield and / or on the quality of the blue pigment. There is also a need for new methods to reduce the cost of enrichment. Finally, there is a need for new methods 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

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 step of dialysis.

In fact, in the context of the present invention, the inventors have demonstrated that a process for obtaining an aqueous solution enriched with blue pigment comprising these steps makes it possible to avoid any step of ultrafiltration and column chromatography . The process of the invention is advantageous because an aqueous solution enriched with 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 leads to 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 since it makes it possible to reduce the total time for implementing the process necessary for obtaining an aqueous solution enriched with blue pigment.

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

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

a) the supply of an aqueous solution comprising a blue pigment originating from at least one microorganism of the genus Haslea, b) a step of precipitating the blue pigment with sodium hydroxide, c) a step of separating the precipitated phase from the aqueous phase, d) a step of solubilizing the blue pigment included in the precipitated phase, and

e) a dialysis step.

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

Preferably, step c) of separation comprises decantation and / or centrifugation, preferably decantation followed by centrifugation. This removes most, if not all, of the aqueous phase.

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

Preferably, the blue pigment included in the precipitated phase is dissolved in step d) with carboxylic acid, preferably formic acid.

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

Preferably, the method further comprises at least one step of filtration 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.

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

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

Preferably, during step e) of dialysis, the dialysis membrane has a cutoff 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 diatomaceous Haslea ostrearia NCC 497 deposited with the BEA ¹ October 2018 under the number BEA_IDA_0065B.

According to a second aspect, the present invention relates to an aqueous solution enriched with blue pigment capable of being obtained by the process as described here.

DESCRIPTION OF THE FIGURES

Fig. 1 Diagram illustrating the process according to the invention. The dotted frames represent optional steps.

FIG. 2 shows the characterization of the aqueous solution enriched with blue pigment of the “marermine” type called “MCPN” obtained according to the present invention by:

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

Fig. 2B Nuclear magnetic resonance (NMR) spectrum with comparison to the spectrum obtained with a solution of "pure marennine"

Fig. 2C is a size exclusion chromatography analysis (SEC-HPLC). AU stands for "absorbance units", ppm for "parts per million".

FIG. 3 is a comparison of the characteristics of the blue pigment of “marennine” type prepared according to the method of the present invention (called “MCPN”) or according to the method of Turcotte et al 2016 (“blue water” or “BW”) or again according to Pouvreau et al 2006a ("pure marennine") Fig. 3A is an ultraviolet (UV) -visible absorption spectrum

Fig. 3B is a nuclear magnetic resonance (NMR) spectrum

Fig. 3C is a size exclusion chromatography analysis (SEC-HPLC). AU stands for "absorbance units", ppm for "parts per million". DETAILED DESCRIPTION OF THE INVENTION

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

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 the existing processes, while limiting the costs of the process. In addition, the process is very easy to carry out, and can be used on large volumes of aqueous solution. The yield of blue pigment obtained by the process of the invention is, advantageously, higher than that obtained by the processes according to the prior art. This process also makes it possible to reduce the time necessary to obtain an aqueous solution enriched with blue pigment.

Obtaining process

A first aspect of the invention therefore relates to a process for obtaining an aqueous solution enriched with blue pigment comprising: a) the supply of 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.

Steps a) to e) are implemented in order a), then b), then c), then d), then e). In some cases (see below), additional steps (excluding any ultrafiltration and / or column chromatography step) may be used:

• Before step a)

For example, step a) may be preceded by a step of culturing the diatoms of the genus Haslea and, optionally, by a step of lysis of 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 in steps b) and c) and the precipitate obtained grouped with that obtained previously.

• Between stages 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 can be added after step e).

These different optional additional steps are detailed below.

The purification process according to the invention is in particular implemented from an aqueous solution comprising a blue pigment derived from at least one microorganism of the genus Haslea, preferably from a supernatant from a culture medium d '' at least one microorganism of the genus Haslea.

Blue pigment

By “blue pigment” is meant, within the meaning of the present invention, a water-soluble blue pigment produced by a diatom of the genus Haslea. Said blue pigment can 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., 201 1). Said blue pigment can comprise or consists of several blue pigments produced by different diatoms. Preferably, the blue pigment according to the invention comprises marennine. According to a particular mode, the blue pigment according to the invention consists of marennine.

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 than the extracellular form (Pouvreau et al., 2006a).

In the context of the present invention, the blue pigment can comprise blue pigment in its intracellular form and / or its extracellular form. According to a first embodiment, the blue pigment essentially consists of its extracellular form. Of 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 essentially consists of its intracellular form. Preferably, the blue pigment consists of its intracellular form.

Step a)

Step a) of the process according to the invention is a step of supplying an aqueous solution comprising a blue pigment derived from at least one microorganism of the genus Haslea.

By "aqueous solution" is meant any solution comprising blue pigment derived from at least one microorganism of the genus Haslea. According to a preferred embodiment, said aqueous solution is a diatom culture. The diatom culture includes 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 diatoms from the culture medium in order to provide the supernatant are well known to those skilled in the art. Such a step can in particular be carried out by centrifugation with recovery of the supernatant or by filtration.

Preferably, said diatom is selected from the group consisting of: H. ostrearia, H. karadagensis, H. provincialis, H. nusantara, 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 Espanoi de Algas” (BEA), Universidad de Las Palmas de GC, Muelle de Taliarte s / n, 35214 Telde - Las Palmas, Spain on ¹ October 2018 under the BEA_IDA_0065B number.

According to a particular embodiment, the aqueous solution can 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 grown together, or grown individually. When the diatoms are cultivated individually, the culture, preferably the culture supernatants, of each diatom are added together in step a). 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. ostrearia NCC 497 deposited with the "Banco Espanoi de Algas" (BEA) on ¹ October 2018 under the number BEA_IDA_0065B.

In some cases, the aqueous solution supplied in step a) comprises lysed diatoms. A lysis step can 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 in the supernatant or in any other aqueous solution. For example, the lysis of diatoms can be carried out according to the method described in Pouvreau et al., 2006a as described on page 770.

According to a particular embodiment, the concentration of blue pigment in 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 concentration of blue pigment in the aqueous solution in step a) is at least 2 mg / L, preferably at least 3 mg / L. The concentration of blue pigment in the aqueous solution can be determined by the skilled person in the light of his general knowledge. By way of nonlimiting example, the concentration of blue pigment 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 concentration of blue pigment is determined by spectrophotometry using the extinction coefficient published in Pouvreau, et al. (2006b).

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

According to a particular embodiment, the aqueous solution comprising the blue pigment supplied in step a) has not undergone any prior filtration step. Optionally, the aqueous solution supplied 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 inserted 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 (eg blue pigment). The methods and filters suitable for such a filtration step are well known to the person skilled in the art. Such a step can in particular be carried out using filters of the glass microfiber type (sold by Whatman, for example of the GF / C type).

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, preferably 1.4 μm. According to a second embodiment, the filtration step is carried out through two filters of different size, the second filter having an average pore size smaller than the first filter and at least 1.2 μm (for example . of 1, 4 pm). Advantageously, the first filtration makes it possible to avoid clogging during the second filtration.

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 1, 2 pm or 1, 4 pm.

Step b)

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

During step b), the majority of the blue pigment is precipitated and is therefore found in solid form, in the precipitated phase. Advantageously, sodium hydroxide is added until the blue pigment has completely precipitated. The amount of sodium hydroxide to be added varies depending on the concentration of the blue pigment in the aqueous solution of step a) as well as the volume of the aqueous solution. Complete precipitation leads to discoloration of the aqueous phase. Also, the precipitation step by adding sodium hydroxide corresponds to a step easily achievable by the person skilled in the art, taking into account its visual appearance. Advantageously, the unwanted water-soluble compounds present in the aqueous solution, such as proteins and carbohydrates, or even other compounds having no absorption in the visible spectrum, remain in the aqueous phase.

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%. Of 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 more than 1 M or 2 M. The 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 supplied in step a ).

For 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.

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

According to one embodiment, the sodium hydroxide is added in several stages, advantageously drop by drop. During the time of adding sodium hydroxide, the mixture may or may not be stirred. Preferably, the mixture is stirred. During the time of adding sodium hydroxide, the aqueous solution can 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 ° vs.

Step c)

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” here is meant 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 the person skilled in the art. Such a step can in particular be carried out by decantation and / or by centrifugation. By way of nonlimiting 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.

Preferably, the separation according to step c) comprises decantation. By “decantation” is meant, within the meaning of the present invention, an operation which consists in allowing the mixture to stand for a given period of time, so that the precipitate is deposited by difference in gravity, in order to remove the aqueous phase . Preferably, the decantation therefore comprises a step of resting the mixture before the physical separation of the precipitated phase from the aqueous phase. Preferably, while the mixture is left to stand, it is not stirred.

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 15 hours or more. According to another preferred embodiment, the mixture is left to stand for a period of time equal to 5 hours. Advantageously, resting 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 deposition of the precipitate at the bottom of the container. In some cases (eg when the amount of precipitate is small), it would be advantageous to allow the mixture to stand for a period of 16 hours or more.

The mixture can be left to stand at 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.

Preferably, part of the aqueous phase (for example, 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 part is removed from the mixture. The aqueous phase thus removed can be stored for later use or eliminated. 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.

As indicated above, by removing part of the aqueous phase, the volume of the mixture to be treated is further reduced. 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)).

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.

The person skilled in the art will know how to determine the appropriate centrifugation conditions to obtain the separation of the precipitated phase comprising the precipitate of the blue pigment from the aqueous phase. By way of nonlimiting example, the mixture can be centrifuged at an acceleration of at least 4000 xg, preferably at least 12000 x g. According to a preferred embodiment, the mixture is centrifuged at an acceleration of at least 4000 xg for at least 5 minutes, preferably at an acceleration of 4000 xg for 5 minutes. 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 20 min, preferably at least 30 min, preferably for 20 to 35 min. The mixture can undergo one or more centrifugations, which can be the same or different condition (ie with increasing duration or acceleration). Preferably, the mixture undergoes a single centrifugation step. Preferably, the mixture is centrifuged at an acceleration to 4000 xg for 5 minutes or between 12,000 and 13,000 xg for 30 minutes.

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 can be stored for later use or eliminated.

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.

According to a preferred embodiment, the separation step comprises decantation and centrifugation, preferably according to the above 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 At the end of step c), two separate phases are obtained: the precipitated phase and the aqueous phase. In some 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 quantity of blue pigment and makes it possible to improve the final yield of blue pigment.

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

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 allows in particular to improve the recovery yield of the blue pigment obtained by the process according to the invention. Step d)

After step c) of separation of the precipitated phase from the aqueous phase, the precipitated phase comprising the blue pigment is dissolved. The person skilled in the art will be able to identify an appropriate solution making it possible to dissolve the precipitated blue pigment.

Preferably, the blue pigment of the precipitated phase is dissolved 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 dissolved by the addition of formic acid (CAS no. 64-18-6). Preferably, the blue pigment is dissolved by the addition of a 5% carboxylic acid solution (m / m), more preferably by the addition of a 5% formic acid solution (m / m).

Preferably, the volume ratio of the aqueous solution supplied 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 supplied in step a) and the volume of the aqueous solution enriched in 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 supplied in step a) and the volume of the solution enriched in 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 blue pigment in the aqueous solution provided in step a) (eg greater than 7 mg / mL) may require the addition of a larger volume of the solubilizing solution (eg acid formic), and thus a ratio lower than 29/1.

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

Preferably, at the end of step d), the blue pigment present in solution at a concentration equal to or greater than 90 mg / L. Also, the solution enriched with blue pigment obtained at the end of step d) is advantageously enriched with blue pigment at least 12-fold, advantageously at least 18-fold, more advantageously at least 30- times relative to the aqueous solution provided in step a).

Optional step

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 performed. Preferably, at the end of step d), a filtration step, preferably a sterilizing 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. At the end of step d) and before the sterilizing filtration step, additional filtration and / or centrifugation steps can be carried out. By way of example, at least one step of filtration through a filter having an average pore size of between 0.45 μm and 15 is carried out. At least one filtration step can be followed by at least one centrifugation step. In certain cases, at least one filtration step can be carried out before and / or after a centrifugation step.

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, preferably 1, 4 μm or 15 μm. When several filters are used, they preferably have different average porosities. 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 preferred embodiment, the filtration step is carried out through two filters of different size, the second filter having an average pore size smaller than the first filter and at least 1.2 µm (e.g. . of 1, 4 pm). 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 1, 2 pm or 1, 4 pm.

Optionally, a centrifugation step can be carried out. By way of nonlimiting example, the solution enriched in blue pigment can be centrifuged at an acceleration of at least 4000 x g, preferably at least 10,000 x g. When the solution is centrifuged at an acceleration of at least 10,000 x g, the duration of the centrifugation is preferably at least 10 min, preferably 10 min. The solution can undergo one or more centrifugations, which can be the same or different condition (i.e. with increasing duration or acceleration). Preferably, the solution undergoes a single centrifugation step. Preferably, the solution is centrifuged at an acceleration at 10,000 x g for 10 minutes.

After a centrifugation step, at least one filtration step can be carried out. 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, filtration is carried out through a filter having an average pore size of at least 0.45 µm, preferably 0.45 µm or 0.7 µm. When several filters are used, they preferably have different average porosities. According to a particular embodiment, at least one filtration step is carried out, more preferably, at least two filtration steps are carried out after a centrifugation step. According to a preferred embodiment, the filtration step is carried out through two filters of different size, the second filter having an average pore size smaller than the first filter and at least 0.45 μm. Also, according to a particularly preferred embodiment, the filtration step carried out after a centrifugation step comprises a first filtration through a filter having an average pore size of between 0.7 and 1.2 μm, preferably 0 , 7 µm followed by a second filtration through a filter having an average pore size of 0.45 µm.

Also, according to a particular embodiment, before the sterilizing filtration step, the solution enriched with blue pigment is:

- filtered 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 1.4 µm,

- centrifuged at 10,000 x g acceleration for 10 min, and then

- filtered through a filter having an average pore size of between 0.7 and 1.2 µm, followed by a second filtration through a filter having an average pore size of

0.45 pm.

Step e)

After step d) of solubilization, the solution enriched in blue pigment is subjected to a dialysis step. The solution enriched with blue pigment 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 the person skilled in the art. Such a step can in particular be carried out using a dialysis membrane having a cutoff threshold of between 1 and 3.5 kDa.

According to a preferred mode, the dialysis membrane has a cutoff threshold of between 1 and 3.5 kDa, preferably a cutoff threshold of 1 kDa, 2 kDa, or 3.5 kDa.

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 dialysis (eg water) is changed approximately every 12 hours. Preferably, dialysis of the solution enriched with blue pigment is carried out against approximately 1 L to 2 L of the appropriate aqueous solution (eg water), more preferably against about 1 L or 2 L of the appropriate aqueous solution (eg water). Preferably, the aqueous solution used for dialysis is ultra pure water. "Ultra pure" water meets the criteria established in at least one of the following specifications, describing water quality standards: ASTM ("American Society for Testing and Materials") D1193, ISO ("International Organization for Standardization") 3696, or CLSI-CLRW ("Clinical and Laboratory Standards Institute-Clinical Laboratory Reagent Water").

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

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 or greater than 100 mg / L, equal or greater than 1 10 mg / L, equal or greater than 120 mg / L, equal or greater than 130 mg / L, equal or greater than 140 mg / L, even more preferably equal or greater than 150 mg / L .

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

Said aqueous solution enriched in blue pigment is preferably stored at approximately 4 ° C (4 ± 2 ° C) and in the dark at the end of step e) of dialysis 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 mandatory to add any excipient, such as a stabilizing agent, at the end of the process of the invention. Other optional subsequent steps

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 of concentration or of formulation. The method according to the invention can thus also comprise at least one additional step and subsequent to step e) of concentration or formulation.

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. As a non-limiting example, it may also be advantageous to formulate the blue pigment. in a composition. In order to adapt the aqueous solution enriched in blue pigment resulting from the process according to the invention to a particular use, the composition can undergo a formulation step, for example by the addition of an excipient and / or of a vehicle and / or by any other change in composition of said aqueous solution enriched in blue pigment, such as adding a buffer solution. In other cases, the formulation step can be a drying step, for example, making it possible to obtain the blue pigment in powder form.

In a particular embodiment, the method according to the invention further comprises, after step e) of dialysis, one or more of the following steps aiming to adapt the composition to a particular formulation and / or to increase the purity of the product :

• a concentration stage; and or

• a formulation stage.

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

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

The enriched blue pigment solution obtained by the process of the invention can in particular be used as a source of natural blue pigment intended for the food industry and for cosmetics, 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 intended for the food industry and for cosmetics, or as an antimicrobial substance for use in aquaculture. EXAMPLES

The invention is illustrated by the following nonlimiting examples. These lessons include alternatives, modifications and equivalents, which may be appreciated by a person skilled in the art.

Example 1: Process according to the invention A process perfected making it possible to obtain an aqueous solution enriched in blue pigment, here marennine, without any step of ultrafiltration or column chromatography is described below. Materials and methods

Biological material

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

Method for enriching the blue pigment in aqueous solution

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

Culture medium ύΉ. ostrearia, 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 filtered culture drop by drop then allowed to settle until a greenish layer appears at the bottom of the bottle and the coloring of the aqueous phase disappears (about 5 hours) at room temperature without stirring.

After decantation, a large part of the aqueous phase (e.g. from 80 to 90%) is removed. Optionally, the aqueous phase is set aside for further processing.

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 allowed to settle as described above, decanting for at least 3 hours, or even overnight (eg 16 hours), to improve the recovery yield. of marennine and further reduce the volume of work. After centrifugation, the precipitated phase obtained is dissolved in a 5% solution of formic acid (HCOOH). The solubilization is carried out in a volume of 250 to 350 ml of formic acid.

The solution obtained is filtered at 0.22 μm on a regenerated cellulose membrane. Finally, dialysis against 2 L of water is carried out in a 1 kDa dialysis rod for 72 hours by changing the dialysis water 2 times a day.

Results

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

Example 2: Characterization of the “MCPN” solution

The aqueous solution enriched in marennin was characterized by UV-Visible spectrometry, nuclear magnetic resonance (NMR) and exclusion-diffusion 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

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

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

The chromatographic analysis was carried out on a Waters Alliance 2690 HPLC chain equipped with a PAD 996 pulsed amperometry detector and Empower software.

An Xbridge® Protein BEH SEC 125 A ° column, 3.5 pm, 7.8 ^* 300 mm (Waters) was used with isocratic elution (Na ₂ C0 ₃ 250 mM, NaN ₃ 0.05%, pH 8) and a flow rate of 0.5 mL / min.

Results

The absorption spectrum and the NMR spectrum of the MCPN solution are very similar to those of pure marennine (FIGS. 2A and 2B). In addition, the chromatogram of the solution MCPN, represents only another impurity (peak at 10.963 min) and a small shoulder on the peak of marennine at 17.607 minutes (Figure 2C).

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 product of high purity without an ultrafiltration or column chromatography step.

Example 3: Evaluation of variants of the process according to Example 1

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

Operating conditions for representative tests

It is noted that the different variants tested of the process do not modify the aqueous solution enriched with blue pigment, with the exception of the phase of re-solubilization by hydrochloric acid.

Example 4: Enrichment of the Blue Pigment Produced by Other Haslea Species

The process for obtaining an aqueous solution enriched with blue pigment as described here was tested using the supernatant of different Haslea species producing the blue pigment as the aqueous starting solution (in step a)) (cf. Table 2). Table 2

Species of astea tested

It is noted 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

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 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 T urcotte et al., 2016 were carried out in parallel . Briefly, these methods begin with a filtration step on a Whatman GF / F filter (0.45 μm) of the supernatant of a culture of Haslea ostrearia, 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 chromatography step by anion exchange (passage over a column containing a Sepharose resin). The pigment retained on the resin is eluted with a saline solution and then the solution is dialyzed.

The UV-visible absorption spectra, the NMR spectra, and the exclusion-diffusion chromatography (HPLC-SEC) of the aqueous solution obtained by the method described here (the “MCPN” solution) and that obtained by the 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

As indicated in Table 3 above, it can be seen 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. In addition, it can be seen that the absorption spectra of "BW", "pure marennine" and "MCPN" are very comparable. The difference observed in 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 the “MCPN” is purer than the “BW”.

Table 3

Process comparison

LIST OF CITED DOCUMENTS

Gastineau, 201 1. Biodiversity, reproduction and phylogeny of blue diatoms of the genus Haslea and development of their pigments of the marennine type. Agricultural Sciences, University of Maine.

Gastineau, et al., 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

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

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

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.

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.

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 detection. J. Appl. Phycol. 19, 263-270.

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

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.

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

Claims

1. A process for obtaining an aqueous solution enriched with blue pigment, comprising: a) the supply of an aqueous solution comprising a blue pigment derived from at least one microorganism of the genus Haslea, b) a step of precipitation of the pigment blue with sodium hydroxide, c) a step of separating the precipitated phase from the aqueous phase, d) a step of solubilizing 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. 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. Method 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 dissolved in step d) with 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 step e).

6. Method according to any one of claims 1 to 5, characterized in that it further comprises at least one step of filtration through a filter having an average pore size of at least 1, 2 pm before the 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.

7. Method according to any one of claims 1 to 6, characterized in that the aqueous phase obtained in step c) is subjected to a new 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 cutoff threshold between 1 and 3.5 kDa.

9. Method according to any one of claims 1 to 8, characterized in that the aqueous solution supplied in step a) is a supernatant from 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 diatom culture Haslea ostrearia NCC 497 deposited with the BEA on October 1, 2018 under number BEA_IDA_0065B.