EP3601318A2 - Purification of phycobiliproteins - Google Patents

Purification of phycobiliproteins

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Publication number
EP3601318A2
EP3601318A2 EP18714772.3A EP18714772A EP3601318A2 EP 3601318 A2 EP3601318 A2 EP 3601318A2 EP 18714772 A EP18714772 A EP 18714772A EP 3601318 A2 EP3601318 A2 EP 3601318A2
Authority
EP
European Patent Office
Prior art keywords
phycocyanin
apc
acidic
resistant
phycobiliproteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18714772.3A
Other languages
German (de)
French (fr)
Inventor
Olivier CAGNAC
Axel ATHANE
Julien DEMOL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fermentalg SA
Original Assignee
Fermentalg SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fermentalg SA filed Critical Fermentalg SA
Publication of EP3601318A2 publication Critical patent/EP3601318A2/en
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/46—Addition of dyes or pigments, e.g. in combination with optical brighteners using dyes or pigments of microbial or algal origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

Definitions

  • the present invention relates to a novel process for the purification of phycobiliproteins, in particular resistant to acidic pH, the phycobiliproteins obtained and their uses.
  • Galdieria sulphuraria phycobiliproteins particularly C-Phycocyanin (C-PC)
  • C-PC C-Phycocyanin
  • the presence of chlorophyll a and dissolved carotenoids in these micelles helps to increase the absorbance values at 280 nm (protein-specific UV absorbance) which may explain the low purity levels of the crude C-PC extracts compared to those of Spirulina (Sorensen et al., 2013).
  • the purity level of the crude extract can therefore be increased by eliminating micelles and soluble proteins other than phycobiliproteins.
  • the invention therefore relates to a method for purifying phycobiliproteins produced by bioreactor culture of phycobiliprotein-producing microorganisms, easy to implement and economically adapted for implementation on an industrial scale.
  • phycobiliproteins in particular phycocyanins
  • phycocyanins are mixtures of ⁇ -phycocyanin and allophycocyanin.
  • Ammonium sulfate precipitation purification causes the two proteins uncontrollably, so that it may be difficult to obtain a pigment with stable properties. This method of precipitation also generates losses of important extraction yield (Cruz de Jesus et al., 2006).
  • the invention therefore also relates to the preparation of purified phycobiliproteins, in particular purified phycocyanin essentially comprising cypycocyanin or essentially allophycocyanin, in particular phycobiliproteins resistant to acidic pHs of controlled phycocyanin composition, pH resistance acids that do not require the addition of stabilizing agents such as ascorbic acid (WO 2005/065697) or polyphenols (WO 2015/090697).
  • stabilizing agents such as ascorbic acid (WO 2005/065697) or polyphenols (WO 2015/090697).
  • the invention therefore relates to a method for purifying phycobiliproteins resistant to acidic pH from a crude extract of phycobiliproteins resistant to acidic pH, characterized in that it comprises the steps of
  • the invention also relates to phycobiliproteins resistant to acidic pHs obtained by the process and in particular phycocyanines resistant to acidic pH comprising a mixture of c-phycocyanin and allophycocyanin, more particularly whose molar ratio c-phycocyanin / allophycocyanin is at least 2.
  • Figure 1 Increasing the purity index of the raw extract as a function of pH from a fresh cell lysate.
  • Figure 2. Measurement of the concentration of C-PC and APC in different crude extracts obtained by centrifugation of a lysate of fresh cells at different pH. APC concentrations in mg / ml are represented in gray, and concentrations of C-PC in mg / ml are black.
  • FIG. 3 Measurement of the concentration of C-PC and APC in pellets obtained by centrifugation of a lysate of fresh cells at different pH. APC concentrations in mg / g of MS are shown in gray, and C-PC concentrations in mg / g of MS in black.
  • Figure 4 Increase in the purity index of the crude extract as a function of pH from a lysate of lyophilized and rehydrated cells.
  • FIG. 5 Measurement of the concentration of C-PC and APC in different crude extracts obtained by centrifugation of a cell lysate, lyophilized and rehydrated, at different pH. APC concentrations in mg / ml are represented in gray, and concentrations of C-PC in mg / ml are black.
  • the crude extract previously purified by precipitation at acidic pH, is filtered using a hollow fiber system.
  • the invention therefore relates to a method for purifying phycobiliproteins resistant to acidic pH from a crude extract of phycobiliproteins resistant to acidic pH.
  • the crude extract of phycobiliproteins is generally obtained from industrially grown microorganism cells in high capacity bioreactors, preferably to obtain fermentation musts comprising high densities of phycobiliprotein-producing microorganisms (in large densities).
  • DM dry matter
  • These culture methods are known to those skilled in the art, which can be carried out in autotrophy, heterotrophy or mixotrophy, in particular described in the applications WO 2017/050917, WO 2017/050918 and PCT / EP2016 / 079325 filed on November 30, 2016.
  • the Phycobiliproteins produced by the microorganisms grown must be released after cell lysis.
  • the microorganism cells contain large amounts of phycobiliproteins (Moon et al., 2015, Sorensen et al., 2013, Eriksen 2008). Therefore, the implementation of the process according to the invention first requires the preparation of an aqueous extract from the fermentation must.
  • the aqueous extract can be prepared directly from the fermentation broth as it is recovered from the reactor at the end of fermentation, optionally supplemented with a suitable amount of water.
  • It can be prepared from fresh cells separated from the fermentation must by any separation method well known to those skilled in the art. It can also be prepared from cells previously lyophilized or dried for preservation.
  • the aqueous extract is prepared from fresh cells separated from the fermentation must after culturing.
  • Cell lysis can be by any means of cell lysis known to those skilled in the art. It can be done while the cells are suspended in water, fermentation wort or reconstituted suspension.
  • the cell lysis is made on cells separated from the fermentation must, before resuspension.
  • the aqueous extract is obtained from the suspension comprising the lysed cells by separating the solids, by any means of separation known to those skilled in the art to remove solid residues of cell lysis, especially filtration.
  • aqueous extract is thus obtained called “phycobiliprotein crude extract” or “crude extract” which comprises, in addition to the phycobiliproteins sought, in particular resistant to acidic pH, other organic materials such as micelles and other water-soluble proteins.
  • the crude extract of phycobiliproteins can be prepared from fresh cells lysed (directly in the fermentation must or after separation of the fermentation must) or from freeze-dried or dried cells, the cell lysis taking place before or after lyophilization or drying.
  • the crude extract is prepared from fresh cells.
  • the purification process according to the invention consists in separating the phycobiliproteins, particularly resistant to acidic pH, sought from these other organic materials such as micelles and other water-soluble proteins.
  • microorganisms cultivated to produce phycobiliproteins are well known to those skilled in the art, in particular chosen from the group of Cyanophyceae such as Arthrospira platensis (Spirulina), Spirulina maxima, Synechococcus elongatus, or the group of cyanidiophyceae such as Galdieria sulphuraria, Cyanidium caldiarium, Cyanidioschyzon merolae.
  • Cyanophyceae such as Arthrospira platensis (Spirulina), Spirulina maxima, Synechococcus elongatus, or the group of cyanidiophyceae such as Galdieria sulphuraria, Cyanidium caldiarium, Cyanidioschyzon merolae.
  • phycobiliproteins are phycocyanins resistant to acidic pH.
  • Phycobiliproteins resistant to acidic pH or phycocyanin resistant to acidic pH are phycobiliproteins that are resistant to precipitation at acidic pH.
  • acid pH is meant according to the invention a pH of less than 7, preferably 6 or less.
  • phycobiliproteins resistant to acidic pH do not precipitate in an aqueous solution at pH lower than 6. They can also be described indifferently as resistant or stable at acidic pH.
  • the purified phycobiliproteins according to the invention will be more or less stable depending on the acid pH considered. Some will be stable in a range of pH values close to 6. Others will be stable at pH values well below 6. Therefore, phycobiliprotein resistant to acidic pH also means a mixture of phycobiliproteins, the majority of which not precipitate at a pH below 7, preferably below 6 or less.
  • the invention relates to stable phycobiliproteins at pHs of less than 5, preferably less than or equal to 4, more preferably ranging from 4 to 2, even more preferentially less than or equal to 3.5.
  • Such phycocyanines resistant to acidic pH are known to those skilled in the art, in particular described in the application WO 2016/099261 or the application WO 2017/050918.
  • these are phycocyanins produced by microalgae strains of the genera Cyanidioschyzon, Cyanidium or Galdieria, chosen in particular from the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, especially strains of Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae.
  • C-PC ⁇ -phycocyanin
  • APC allophycocyanin
  • the apoprotein of C-PC comprises the protein of SEQ ID NO 1 or SEQ ID NO 2 or a variant thereof.
  • the ⁇ -subunit apoprotein of C-PC comprises the protein of SEQ ID NO 1 and the apoprotein of the ⁇ subunit of C-PC comprises the protein of SEQ ID NO 2 or variants thereof.
  • the ⁇ subunit of said APC comprises SEQ ID NO 3 or variants thereof and the apoprotein of the ⁇ subunit of said APC comprises SEQ ID NO 4 or variants thereof .
  • the apoproteins of C-PC and APC from the same source of phycocyanins generally have different isoelectric points. By lowering the pH, it will be possible to separate at least part of the C-PCs from the APCs.
  • the inventors have found that the lower the pH of the crude extract was adjusted, the more C-PC obtained were pure.
  • lowering the pH below the isoelectric point of the APC makes it possible to obtain a phycocyanin comprising a C-PC / APC mixture whose molar ratio is at least 5, preferably at least 10, more preferably at least 15.
  • the pH of the crude extract in step a) is adjusted to a pH of less than 5.
  • Phycocyanin resistant to acidic pH comprising less than 5 mol% of APC, preferably less than 1, can be obtained.
  • % more preferably less than 0.1% APC, the percentages being expressed relative to the total sum of APC and C-PC.
  • step a) the pH adjustment is done by adding an acid, mineral or organic, strong or weak, in the form of solid or solution, the amount of acid added being determined by the pH the raw extract to be treated and the pH value that the skilled person will seek to obtain.
  • mineral acids well known to those skilled in the art, mention will be made more particularly of hydrochloric acid and phosphoric acid.
  • organic acids well known to those skilled in the art, mention will in particular be made of acetic acid, citric acid, tartaric acid, lactic acid, preferably citric acid.
  • Acidic polyphenols such as rosmarinic acid, tannic acid, digallic acid, quercitannic acid, gallotannic acid, acidic tannins such as quercitin, ellagitannines, castalagine, castaline, casuariticin, grandinine may also be mentioned. Punicalin, punicalin, roburin A, tellimagrandin II, terflavin B, vescalin, pendunculagin, casua ine, castline, vescaline, preferably tannic acid.
  • the acids used are acids authorized for food use, in particular phophoric acid, citric acid or tannic acid.
  • any separation method known to those skilled in the art may be employed, in particular by tangential filtration on ceramic membranes or organic membranes such as hollow fibers polyethersulfone .
  • the thresholds of these filters can be chosen to separate molecules of molecular weight higher or lower than targeted phycobiliproteins.
  • the separation in step b) is done by tangential filtration. This step helps to focus and eliminate a part of the proteins other than phycobiliproteins, thus increasing the degree of purity of the final product.
  • Step c) drying / dehydration phycobiliproteins resistant to acidic pH from the supernatant is by any method of removing the solvent, the water in this case, for example by evaporation at atmospheric pressure or under vacuum.
  • atomization, lyophilization, zeodration, infra-red drying, or refractive window drying are mentioned.
  • step b it is possible, after recovery of the supernatant in step b), to recycle the phycobiliproteins contained in the precipitate.
  • the residual phycobilliproteins are solubilized in an aqueous solution of acidic pH, about 6 or less, pH at which the impurities remain insoluble while the phycobiliproteins are soluble.
  • the residual phycobiliproteins are then separated from the impurities and isolated by repeating steps b) and c) of the process. It is an iterative process that can be repeated as many times as necessary.
  • the residual phycobiliproteins are an APC enriched C-PC / APC mixture.
  • phycobiliproteins comprising a C-PC / APC mixture whose molar ratio is less than 5, in particular less than 4, advantageously of the order of 3 to 0.1, are obtained.
  • a mixture of APC comprising less than 5 mol% of CPC, preferably less than 1%, more preferably less than 0.1% of CPC, can then be obtained, the percentages being expressed with respect to the total sum of APC and C-PC.
  • the invention also relates to phycobiliproteins resistant to acidic pH, in particular phycocyanin, which can be obtained by the purification process.
  • the invention also relates to purified phycocyanins resistant to acidic pH comprising a C-PC / APC mixture having a molar ratio of at least 2.
  • the invention relates to phycocyanin resistant to acidic pH which comprises at least 95 mol% of C-PC and less than 5 mol% of APC, preferably at least 99 mol% of C-PC and less than 1 mol% of APC, the percentages being expressed in relation to the total sum of APC and C-PC.
  • C-PCs are known to those skilled in the art and in particular defined above, in particular those whose ⁇ -subunit of C-PC comprises the protein of SEQ ID NO 1 and the apoprotein of the ⁇ subunit of C-PC comprises the protein of SEQ ID 2 or variants thereof.
  • the variants according to the invention have a sequence identity of at least 83% for the ⁇ -subunits of C-PC, and at least 82% for the ⁇ subunits of the C-PC. PC.
  • the variants according to the invention have an identity of at least 90% for the ⁇ (SEQ ID NO 1) and ⁇ (SEQ ID NO 2) subunits.
  • the invention also relates to a purified phycocyanin enriched in APC that can be obtained by the process according to the invention.
  • the invention relates to purified phycocyanin which comprises a mixture enriched in APC whose Molar ratio C-PC / APC is less than 5, in particular 4, advantageously of the order 3 to 0.1.
  • the mixture enriched with APC has an APC / C-PC ratio of at least 5, preferably at least 10, more preferably at least 15.
  • the phycocyanin consists essentially of APC with at least 95% APC Molar and less than 5% C-PC Molar, preferably at least 99% APC Molar. and less 1 mol% of C-PC, the percentages being expressed with respect to the total sum of APC and C-PC.
  • APCs are known to those skilled in the art and in particular defined above, in particular those whose ⁇ subunit of said APC comprises SEQ ID NO 3 or variants thereof and the apoprotein of the ⁇ subunit. of said APC comprises SEQ ID NO 4 or variants thereof.
  • the variants according to the invention have a sequence identity of at least 83% for the ⁇ subunits of APC, and at least 82% for the S subunits of APC.
  • polypeptide may be modified by substitution, insertion and / or deletion of at least one amino acid without substantially modifying its function.
  • substitution of an amino acid at a given position by another chemically equivalent amino acid is a known example of sequence variation that does not substantially affect the properties of the protein.
  • the apoprotein variants of the phycocyanins and / or allophycocyanins according to the invention may comprise from 1 to 30 amino acids of difference in number with respect to the corresponding reference sequence, particularly with regard to the subunits a and / or or ⁇ of phycocyanin, to the extent that the variant obtained retains the properties of the reference protein and the percentages of homology / identity stated above.
  • the apoprotein variants of the phycocyanin subunit usable in the acid compositions according to the invention may comprise from 1 to 27 amino acids of difference with respect to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;
  • variants of the apoproteins of the ⁇ subunit of the phycocyanins that can be used in the acid compositions according to the invention, derived from substitutions, insertions and / or deletions, they may comprise from 1 to 30 amino acids of difference relative to to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;
  • the apoprotein variants of the ⁇ -subunit of allophycocyanines that may be used in the acid compositions according to the invention, derived from substitutions, insertions or deletions, they may comprise from 1 to 24 amino acids of difference relative to the sequence so-called reference reference, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;
  • the apoprotein variants of the ⁇ subunit of the allophycocyanines that can be used in the acid compositions according to the invention, resulting from substitutions, insertions and / or deletions, they may comprise from 1 to 20 amino acids of difference with respect to the so-called reference reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above.
  • the variants of said subunits may advantageously comprise from 1 to 15 amino acids of difference, preferably from 1 to 10 amino acids of difference, in particular 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids of difference relative to to the so-called reference sequence corresponding, to the extent that the obtained variant retains the properties of the reference protein and the percentages of identity stated above.
  • the invention relates to C-PC whose alpha subunit protein consists of the protein of SEQ ID 1 and the subunit beta protein consists of the protein of SEQ ID 2.
  • the invention relates to APC whose alpha subunit protein consists of the protein of SEQ ID 3 and the subunit beta protein consists of the protein of SEQ ID 4.
  • Phycobiliproteins are natural dyes essentially used for coloring foods.
  • the invention also relates to the use of phycobiliproteins resistant to acidic pH obtained by the method according to the invention and in particular phycocyanines resistant to acidic pH defined above as a colorant in a food product.
  • the invention also relates to a particular food composition
  • a particular food composition comprising phycobiliprotein resistant to acidic pH obtained by the process according to the invention and in particular phycocyanin resistant to acidic pH defined above.
  • the food product or the food composition is an acid composition, as defined in the application WO 2017/050918.
  • acid composition is meant according to the invention any composition comprising a mineral or organic acid and phycocyanin.
  • This composition may be liquid, fluid or viscous, pasty or solid which has an acidic pH and in which phycocyanin resistant to acidic pH is incorporated.
  • the pH is measured in the usual manner.
  • the pH is measured after dissolution of the composition in an amount of water sufficient to dissolve the soluble compounds it contains, including inorganic or organic acids and phycocyanin.
  • the composition according to the invention is an aqueous liquid composition, optionally in the form of a gel, or a pasty or solid composition intended to be dissolved in an aqueous solution or in a composition solid or pasty including water.
  • the pasty or solid acid composition composition intended to be used and / or stored in a humid environment.
  • the mineral or organic acids that can be used in the compositions according to the invention are well known to those skilled in the art.
  • mineral acids mention will in particular be made of carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulphonic acid and nitric acid.
  • organic acids mention will be made in particular of citric, lactic, malic, tartaric and succinic acids, advantageously citric acid.
  • acidic food composition any composition intended to be ingested by humans or animals which falls within the above definition.
  • Nutraceutical acid compositions must be considered as falling within the definition of acidic food compositions within the meaning of the invention.
  • the acidic food compositions according to the invention are well known to those skilled in the art. They may comprise a vehicle that may comprise structural constituents associated with active compounds identified with regard to their nutritive contributions or for their properties beneficial to the health of humans or animals.
  • the acidic food composition according to the invention may also comprise food additives such as texturizing agents, flavoring agents, preserving agents, all components well known to those skilled in the art.
  • the vehicle may comprise water and / or proteins and / or fats and / or fibers and / or sugars.
  • the constituents of the vehicle may have only structural properties but they are generally known for their nutrient inputs.
  • the acidic food composition according to the invention can be ready for use or in the form of a food additive that is added to a solid, pasty or liquid preparation to prepare the food that can be ingested.
  • the acid will preferably be selected from the list of acidifiers authorized in the diet, in particular carbonic acid, phosphoric acid, citric acid, malic acid, tartaric acid and lactic acid, more particularly citric acid.
  • non-food acidic compositions may be, for example, pharmaceutical, veterinary or cosmetic and further include any additives and / or active known and used in this kind of composition.
  • the phycocyanin may be incorporated, for example, in the form of a powder.
  • Said acid composition, particularly said acidic food composition may then be in any known known form such as creams, gels, foams, pastes, etc.
  • a solid food composition include cakes or biscuits, dry foods for cooking, powders for dilution, solid gelatinous compositions or "jelly", foams, etc.
  • said liquid acid composition may be an aqueous composition in which the phycocyanin is dissolved. It may be in the form of a ready-to-use composition or as a liquid concentrate to be diluted, especially for its ingestion or to be added to a solid food either for its preparation or for its ingestion, for example a liquid composition. concentrated coating or "topping" which will be deposited on a cake to bring its color. Among these concentrated compositions include syrups, alcoholic or not.
  • the liquid acid composition according to the invention may be of variable viscosity and may or may not comprise additives such as viscosity agents, gelling agents, and other structuring additives known to those skilled in the art and customary for the preparation of liquid food compositions.
  • the liquid food composition may be an acidic beverage, gaseous or not.
  • examples include sodas, juices, sports drinks, exercise drinks, salvage drinks, etc.
  • the compositions of these drinks are well known to those skilled in the art and may include, in particular, sugars, mineral salts, food additives, dissolved gas, etc.
  • the beverage according to the invention is a usual acidic beverage in which the dye usually employed has been replaced in whole or in part by phycocyanin resistant to acidic pH according to the invention.
  • the phycocyanin content in the compositions according to the invention may be in accordance with the practice of those skilled in the art.
  • the phycocyanin content in said composition may be in accordance with the practices of a person skilled in the art of coloring.
  • the content of phycocyanin may be between 2.5 mg / L and 2500 mg / L, preferably between 25 mg / L and 300 mg / L.
  • the phycocyanin content may generally be between 25 mg / l and 300 mg / l, preferably between 50 mg / l and 100 mg / l.
  • the phycocyanin content may generally be between 250 mg / l and 2500 mg / l, preferably between 500 mg / l and 1000 mg / l.
  • the phycocyanin content may generally be between 0.01 mg / g and 10 mg / g, preferentially between 0.1 mg / g and 5.0 mg / g, very preferably between 0.25 mg / g g and 2.5 mg / g.
  • Example 1 Purification by acid precipitation on fresh cell.
  • the ground material is acidified by adjusting the pH unit by 0.5. addition of citric acid.
  • a sample of the mixture is made and then centrifuged for 10 min at 1000 g.
  • the supernatant containing the phycobiliproteins is removed and the purity index measured by making the absorbance ratio at 618 nm on the absorbance at 280 nm with a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).
  • the acidification also makes it possible to obtain a better separation of the liquid and solid phases and to obtain a pellet of cellular debris and proteins which is more compact and easier to separate from the aqueous phase.
  • Example 2 Purification by acid precipitation on lyophilized cell and rehydrated.
  • the ground then freeze-dried.
  • the freeze-dried dry matter is suspended in a volume of water equivalent to the initial volume of the must and then acidified by adjusting 0.5 pH unit by addition of citric acid.
  • a sample of the mixture is made and then centrifuged for 10 min at 1000 g.
  • the supernatant containing the phycobiliproteins is removed and the purity index measured by making the absorbance ratio at 618 nm on the absorbance at 280 nm with a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).
  • Example 3 Purification and concentration of C-PC by tangential filtration.
  • the crude extract after acid precipitation and centrifugation is filtered on a tangential filtration module of the hollow fiber type. Filtration through this mesh makes it possible to eliminate some of the proteins other than C-PC and thus to increase the purity index (FIG. 6).
  • the purity index that can be reached by this method approaches the values normally obtained by much more complex methods involving biphasic extractions, or precipitation with ammonium sulphate, or even chromatography methods (Soresen et al., 2013 Cruz de Jesùs et al., 2006).
  • this filtration step makes it possible to eliminate the water from the C-PC extract (FIG. 7), and to facilitate the drying of the product thereafter.

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Abstract

The present invention relates to a novel method for the purification of phycobiliproteins, in particular acid-pH-resistant phycobiliproteins, the resulting phycobiliproteins, and the uses thereof.

Description

PURIFICATION DES PHYCOBILIPROTEINES  PURIFICATION OF PHYCOBILIPROTEINS
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
La présente invention concerne un nouveau procédé de purification des phycobiliprotéines, en particulier résistantes aux pH acides, les phycobiliprotéines obtenues et leurs utilisations.  The present invention relates to a novel process for the purification of phycobiliproteins, in particular resistant to acidic pH, the phycobiliproteins obtained and their uses.
ETAT DE LA TECHNIQUE STATE OF THE ART
La purification de phycobiliprotéines de Galdieria sulphuraria, en particulier de C-Phycocyanine (C-PC), est beaucoup plus complexe que celle 6'Arthrospira platensis (Spiruline) ou que d'autres cyanobactéries. Cela est partiellement dû à la composition de la paroi cellulaire de Galdieria sulphuraria qui nécessite une action mécanique pour être rompue (Sorensen et al., 2013). La lyse mécanique engendre la formation de micelles qui ne sont que partiellement éliminés par ultracentrifugation. La présence de chlorophylle a et de caroténoïdes dissouts dans ces micelles contribue à augmenter les valeurs d'absorbance à 280 nm (absorbance dans les UV spécifique des protéines) ce qui peut expliquer les faibles taux de pureté des extraits brut de C-PC comparativement à ceux de Spiruline (Sorensen et al., 2013). Le taux de pureté de l'extrait brut peut donc être augmenté en éliminant les micelles et les protéines solubles autres que les phycobiliprotéines.  The purification of Galdieria sulphuraria phycobiliproteins, particularly C-Phycocyanin (C-PC), is much more complex than that of Arthrospira platensis (Spirulina) or other cyanobacteria. This is partly due to the composition of the cell wall of Galdieria sulphuraria which requires a mechanical action to be broken (Sorensen et al., 2013). Mechanical lysis results in the formation of micelles that are only partially removed by ultracentrifugation. The presence of chlorophyll a and dissolved carotenoids in these micelles helps to increase the absorbance values at 280 nm (protein-specific UV absorbance) which may explain the low purity levels of the crude C-PC extracts compared to those of Spirulina (Sorensen et al., 2013). The purity level of the crude extract can therefore be increased by eliminating micelles and soluble proteins other than phycobiliproteins.
La purification de phycobiliprotéines extraites de Cyanidioschyzon merolae, de Cyanidium caldarium, de Galdieria sulphuraria et de Spiruline par précipitation au sulfate d'ammonium a déjà été décrite dans la littérature (WO 2016/099261 ; Eisele et al., 2000 ; Kao et al. Moon et al., 1975 ; 2015 ; Cruz de Jésus et al., 2006) mais elle est très difficilement applicable à l'échelle industrielle car elle demande beaucoup de sulfate d'ammonium, ce qui pose de gros problèmes de retraitement du sulfate d'ammonium et du surnageant.  The purification of phycobiliproteins extracted from Cyanidioschyzon merolae, Cyanidium caldarium, Galdieria sulphuraria and Spirulina by ammonium sulfate precipitation has already been described in the literature (WO 2016/099261, Eisele et al., 2000, Kao et al. Moon et al., 1975, 2015; Cruz de Jesus et al., 2006) but it is very difficult to apply on an industrial scale because it requires a lot of ammonium sulphate, which poses big problems of reprocessing of sulphate of ammonium and supernatant.
Les autres méthodes de purification décrites permettant d'obtenir un taux de pureté telles que des méthodes de chromatographie sont très coûteuses à mettre en œuvre.  The other purification methods described making it possible to obtain a degree of purity such as chromatography methods are very expensive to implement.
L'invention concerne donc un procédé de purification de phycobiliprotéines produites par culture en bioréacteur de microorganismes producteurs de phycobiliprotéines, aisé à mettre en œuvre et économiquement adapté pour une mise en œuvre à l'échelle industrielle. Par ailleurs, les phycobiliprotéines, en particulier les phycocyanines, sont des mélanges de c-phycocyanine et d'allophycocyanine. Les procédés connus de purification ne permettent pas de les séparer de manière contrôlée industriellement. La purification par précipitation au sulfate d'ammonium entraîne les deux protéines de manière non contrôlée, de sorte qu'il peut être difficile d'obtenir un pigment aux propriétés stables. Cette méthode de précipitation engendre également des pertes de rendement d'extraction importante (Cruz de Jésus et al., 2006). The invention therefore relates to a method for purifying phycobiliproteins produced by bioreactor culture of phycobiliprotein-producing microorganisms, easy to implement and economically adapted for implementation on an industrial scale. On the other hand, phycobiliproteins, in particular phycocyanins, are mixtures of α-phycocyanin and allophycocyanin. The known methods of purification do not make it possible to separate them in an industrially controlled manner. Ammonium sulfate precipitation purification causes the two proteins uncontrollably, so that it may be difficult to obtain a pigment with stable properties. This method of precipitation also generates losses of important extraction yield (Cruz de Jesus et al., 2006).
L'invention concerne donc aussi la préparation de phycobiliprotéines purifiées, en particulier de phycocyanine purifiée comprenant essentiellement de la c- phycocyanine ou bien essentiellement de l'allophycocyanine, en particulier de phycobiliprotéines résistantes aux pH acides de composition contrôlée en phycocyanines, la résistance aux pH acides ne nécessitant pas l'ajout d'agents stabilisants comme l'acide ascorbique (WO 2005/065697) ou les polyphénols (WO 2015/090697).  The invention therefore also relates to the preparation of purified phycobiliproteins, in particular purified phycocyanin essentially comprising cypycocyanin or essentially allophycocyanin, in particular phycobiliproteins resistant to acidic pHs of controlled phycocyanin composition, pH resistance acids that do not require the addition of stabilizing agents such as ascorbic acid (WO 2005/065697) or polyphenols (WO 2015/090697).
EXPOSE DE L'INVENTION SUMMARY OF THE INVENTION
L'invention concerne donc un procédé de purification de phycobiliprotéines résistantes aux pH acides à partir d'un extrait brut de phycobiliprotéines résistantes aux pH acides, caractérisé en ce qu'il comprend les étapes de  The invention therefore relates to a method for purifying phycobiliproteins resistant to acidic pH from a crude extract of phycobiliproteins resistant to acidic pH, characterized in that it comprises the steps of
a) ajustement du pH de l'extrait brut de phycobiliprotéines résistantes aux pH acides à un pH inférieur à 6 de manière à faire précipiter les matières organiques autres que les phycobiliprotéines résistantes aux pH acides, a) adjusting the pH of the crude phycobiliprotein extract resistant to acidic pH to a pH below 6 so as to precipitate organic material other than phycobiliproteins resistant to acidic pH,
b) récupération du surnageant comprenant les phycobiliprotéines résistantes aux pH acides et b) recovery of the supernatant comprising phycobiliproteins resistant to acidic pH and
c) isolation des phycobiliprotéines résistantes aux pH acides à partir du surnageant. c) isolation of phycobiliproteins resistant to acidic pH from the supernatant.
L'invention concerne aussi les phycobiliprotéines résistantes aux pH acides obtenues par le procédé et en particulier, des phycocyanines résistantes aux pH acides comprenant un mélange de c-phycocyanine et d'allophycocyanine, plus particulièrement dont le rapport molaire c-phycocyanine / allophycocyanine est d'au moins 2.  The invention also relates to phycobiliproteins resistant to acidic pHs obtained by the process and in particular phycocyanines resistant to acidic pH comprising a mixture of c-phycocyanin and allophycocyanin, more particularly whose molar ratio c-phycocyanin / allophycocyanin is at least 2.
DESCRIPTION DES FIGURES DESCRIPTION OF THE FIGURES
Figure 1 . Augmentation de l'indice de pureté de l'extrait brut en fonction du pH à partir d'un lysat de cellules fraîches. Figure 2. Mesure de la concentration en C-PC et APC dans différents extraits bruts obtenus par centrifugation d'un lysat de cellules fraîches à différents pH. En gris sont représentées les concentrations en APC en mg/ml, et en noir les concentrations en C-PC en mg/ml. Figure 1 . Increasing the purity index of the raw extract as a function of pH from a fresh cell lysate. Figure 2. Measurement of the concentration of C-PC and APC in different crude extracts obtained by centrifugation of a lysate of fresh cells at different pH. APC concentrations in mg / ml are represented in gray, and concentrations of C-PC in mg / ml are black.
Figure 3. Mesure de la concentration en C-PC et APC dans les culots obtenus par centrifugation d'un lysat de cellules fraîches à différents pH. En gris sont représentées les concentrations en APC en mg/g de MS, et en noir les concentrations en C-PC en mg/g de MS.  Figure 3. Measurement of the concentration of C-PC and APC in pellets obtained by centrifugation of a lysate of fresh cells at different pH. APC concentrations in mg / g of MS are shown in gray, and C-PC concentrations in mg / g of MS in black.
Figure 4. Augmentation de l'indice de pureté de l'extrait brut en fonction du pH à partir d'un lysat de cellules lyophilisées et réhydratées.  Figure 4. Increase in the purity index of the crude extract as a function of pH from a lysate of lyophilized and rehydrated cells.
Figure 5. Mesure de la concentration en C-PC et APC dans différents extraits bruts obtenus par centrifugation d'un lysat de cellules, lyophilisées et réhydratée, à différents pH. En gris sont représentées les concentrations en APC en mg/ml, et en noir les concentrations en C-PC en mg/ml.  Figure 5. Measurement of the concentration of C-PC and APC in different crude extracts obtained by centrifugation of a cell lysate, lyophilized and rehydrated, at different pH. APC concentrations in mg / ml are represented in gray, and concentrations of C-PC in mg / ml are black.
Figure 6. Purification et concentration de la C-PC par filtration tangentielle. Figure 6. Purification and concentration of C-PC by tangential filtration.
L'extrait brut préalablement purifié par précipitation à pH acide, est filtré en utilisant un système de fibre creuse. The crude extract, previously purified by precipitation at acidic pH, is filtered using a hollow fiber system.
Figure 7. Augmentation de la concentration en C-PC dans le rétentat lors de la filtration sur fibre creuse.  Figure 7. Increased concentration of C-PC in the retentate during hollow fiber filtration.
DESCRIPTION DETAILLEE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION
L'invention concerne donc un procédé de purification de phycobiliprotéines résistantes aux pH acides à partir d'un extrait brut de phycobiliprotéines résistantes aux pH acides.  The invention therefore relates to a method for purifying phycobiliproteins resistant to acidic pH from a crude extract of phycobiliproteins resistant to acidic pH.
L'extrait brut de phycobiliprotéines est généralement obtenu à partir de cellules de microorganismes cultivés de manière industrielle dans des bioréacteurs de grande capacité, de préférence de manière à obtenir des moûts de fermentation comprenant de grandes densités de microorganismes producteurs de phycobiliprotéines (par grandes densités on entend généralement plus de 50g de matière sèche (MS) par litre de moût de fermentaiton, préférentiellement plus de 10Og/L). Ces méthodes de culture sont connues de l'homme du métier, pouvant être menées en autotrophie, hétérotrophie ou mixotrophie, notamment décrites dans les demandes WO 2017/050917, WO 2017/050918 et PCT/EP2016/079325 déposée le 30 novembre 2016. Les phycobiliprotéines produites par les microorganismes cultivés doivent être libérées après lyse cellulaire. En effet, les cellules de microorganismes contiennent de grandes quantités de phycobiliprotéines (Moon et al., 2015, Sorensen et al., 2013, Eriksen 2008). Par conséquent, la mise en œuvre du procédé selon l'invention, nécessite d'abord la préparation d'un extrait aqueux provenant du moût de fermentation. The crude extract of phycobiliproteins is generally obtained from industrially grown microorganism cells in high capacity bioreactors, preferably to obtain fermentation musts comprising high densities of phycobiliprotein-producing microorganisms (in large densities). generally means more than 50 g dry matter (DM) per liter of fermentaiton must, preferably more than 10Og / L). These culture methods are known to those skilled in the art, which can be carried out in autotrophy, heterotrophy or mixotrophy, in particular described in the applications WO 2017/050917, WO 2017/050918 and PCT / EP2016 / 079325 filed on November 30, 2016. The Phycobiliproteins produced by the microorganisms grown must be released after cell lysis. Indeed, the microorganism cells contain large amounts of phycobiliproteins (Moon et al., 2015, Sorensen et al., 2013, Eriksen 2008). Therefore, the implementation of the process according to the invention first requires the preparation of an aqueous extract from the fermentation must.
L'extrait aqueux peut être préparé directement à partir du moût de fermentation tel qu'il est récupéré du réacteur en fin de fermentation, éventuellement additionné d'une quantité appropriée d'eau.  The aqueous extract can be prepared directly from the fermentation broth as it is recovered from the reactor at the end of fermentation, optionally supplemented with a suitable amount of water.
Il peut être préparé à partir de cellules fraîches séparées du moût de fermentation par toute méthode de séparation bien connue de l'homme du métier. Il peut également être préparé à partir de cellules préalablement lyophilisées ou séchées pour leur conservation.  It can be prepared from fresh cells separated from the fermentation must by any separation method well known to those skilled in the art. It can also be prepared from cells previously lyophilized or dried for preservation.
Selon un mode préféré de réalisation de l'invention, l'extrait aqueux est préparé à partir de cellules fraîches séparées du moût de fermentation après culture.  According to a preferred embodiment of the invention, the aqueous extract is prepared from fresh cells separated from the fermentation must after culturing.
La lyse cellulaire peut se faire par tout moyen de lyse cellulaire connu de l'homme du métier. Elle peut se faire alors que les cellules sont en suspension dans l'eau, moût de fermentation ou suspension reconstituée.  Cell lysis can be by any means of cell lysis known to those skilled in the art. It can be done while the cells are suspended in water, fermentation wort or reconstituted suspension.
Selon un mode préféré de réalisation de l'invention, la lyse cellulaire est faite sur les cellules séparées du moût de fermentation, avant leur remise en suspension.  According to a preferred embodiment of the invention, the cell lysis is made on cells separated from the fermentation must, before resuspension.
De préférence, l'extrait aqueux est obtenu à partir de la suspension comprenant les cellules lysées en séparant les solides, par tout moyen de séparation connu de l'homme du métier pour éliminer les résidus solides de lyse cellulaire, notamment de filtration.  Preferably, the aqueous extract is obtained from the suspension comprising the lysed cells by separating the solids, by any means of separation known to those skilled in the art to remove solid residues of cell lysis, especially filtration.
On obtient alors un extrait aqueux appelé « extrait de brut de phycobiliprotéines » ou encore « extrait brut » qui comprend, outre les phycobiliprotéines recherchées, en particulier résistantes aux pH acides, d'autres matières organiques comme les micelles et d'autres protéines hydrosolubles.  An aqueous extract is thus obtained called "phycobiliprotein crude extract" or "crude extract" which comprises, in addition to the phycobiliproteins sought, in particular resistant to acidic pH, other organic materials such as micelles and other water-soluble proteins.
L'extrait brut de phycobiliprotéines peut être préparé à partir de cellules fraîches lysées (directement dans le moût de fermentation ou après séparation du moût de fermentation) ou à partir de cellules lyophilisées ou séchées, la lyse cellulaire intervenant avant ou après lyophilisation ou séchage.  The crude extract of phycobiliproteins can be prepared from fresh cells lysed (directly in the fermentation must or after separation of the fermentation must) or from freeze-dried or dried cells, the cell lysis taking place before or after lyophilization or drying.
Selon un mode préféré de réalisation de l'invention, l'extrait brut est préparé à partir de cellules fraîches.  According to a preferred embodiment of the invention, the crude extract is prepared from fresh cells.
Le procédé de purification selon l'invention consiste à séparer les phycobiliprotéines, en particulier résistantes aux pH acides, recherchées de ces autres matières organiques telles que micelles et les autres protéines hydrosolubles. The purification process according to the invention consists in separating the phycobiliproteins, particularly resistant to acidic pH, sought from these other organic materials such as micelles and other water-soluble proteins.
Les microorganismes cultivés pour produire des phycobiliprotéines sont bien connus de l'homme du métier, en particulier choisis parmi le groupe des Cyanophyceae comme Arthrospira platensis (Spiruline), Spirulina maxima, Synechococcus elongatus, ou bien le groupe des cyanidiophyceae tel que Galdieria sulphuraria, Cyanidium caldiarium, Cyanidioschyzon merolae.  The microorganisms cultivated to produce phycobiliproteins are well known to those skilled in the art, in particular chosen from the group of Cyanophyceae such as Arthrospira platensis (Spirulina), Spirulina maxima, Synechococcus elongatus, or the group of cyanidiophyceae such as Galdieria sulphuraria, Cyanidium caldiarium, Cyanidioschyzon merolae.
De manière préférée, les phycobiliprotéines sont des phycocyanines résistantes aux pH acides. Par phycobiliprotéines résistantes aux pH acides ou phycocyanines résistantes aux pH acides, on entend des phycobiliprotéines qui résistent à la précipitation aux pH acides. Par pH acide, on entend selon l'invention un pH inférieur à 7, avantageusement de 6 ou moins. Avantageusement, les phycobiliprotéines résistantes aux pH acides ne précipitent pas dans une solution aqueuse à des pH inférieurs à 6. On pourra également les qualifier indifféremment de résistantes ou de stables aux pH acides.  Preferably, phycobiliproteins are phycocyanins resistant to acidic pH. Phycobiliproteins resistant to acidic pH or phycocyanin resistant to acidic pH are phycobiliproteins that are resistant to precipitation at acidic pH. By acid pH is meant according to the invention a pH of less than 7, preferably 6 or less. Advantageously, phycobiliproteins resistant to acidic pH do not precipitate in an aqueous solution at pH lower than 6. They can also be described indifferently as resistant or stable at acidic pH.
Bien entendu, les phycobiliprotéines purifiées selon l'invention seront plus ou moins stables selon le pH acide considéré. Certaines seront stables dans une plage de valeur de pH voisine de 6. D'autres seront stables à des valeurs de pH bien inférieures à 6. Par conséquent, par phycobiliprotéine résistante au pH acide, on entend également un mélange de phycobiliprotéines dont la majorité ne précipite pas à un pH inférieur à 7, avantageusement inférieur à 6 ou moins.  Of course, the purified phycobiliproteins according to the invention will be more or less stable depending on the acid pH considered. Some will be stable in a range of pH values close to 6. Others will be stable at pH values well below 6. Therefore, phycobiliprotein resistant to acidic pH also means a mixture of phycobiliproteins, the majority of which not precipitate at a pH below 7, preferably below 6 or less.
De manière avantageuse, l'invention concerne des phycobiliprotéines stables à des pH inférieurs à 5, préférentiellement inférieurs ou égaux à 4, plus préférentiellement allant de 4 à 2, encore plus préférentiellement inférieurs ou égaux à 3,5.  Advantageously, the invention relates to stable phycobiliproteins at pHs of less than 5, preferably less than or equal to 4, more preferably ranging from 4 to 2, even more preferentially less than or equal to 3.5.
De telles phycocyanines résistantes aux pH acides sont connues de l'homme du métier, notamment décrites dans la demande WO 2016/099261 ou la demande WO 2017/050918. Il s'agit en particulier de phycocyanines produites par des souches de microalgues des genres Cyanidioschyzon, Cyanidium ou Galdieria, notamment choisis parmi les espèces Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201 , Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, en particulier des souches de Galdieria sulphuraria, Cyanidium caldarium et Cyanidioschyzon merolae. Ces phycocyanines sont un mélange de c-phycocyanine (C-PC) et d'allophycocyanine (APC). Such phycocyanines resistant to acidic pH are known to those skilled in the art, in particular described in the application WO 2016/099261 or the application WO 2017/050918. In particular, these are phycocyanins produced by microalgae strains of the genera Cyanidioschyzon, Cyanidium or Galdieria, chosen in particular from the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, especially strains of Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae. These phycocyanins are a mixture of α-phycocyanin (C-PC) and allophycocyanin (APC).
Avantageusement, l'apoprotéine de la C-PC comprend la protéine de SEQ ID NO 1 ou de SEQ ID NO 2 ou un variant de celles-ci. En particulier, l'apoprotéine de la sous-unité a de la C-PC comprend la protéine de SEQ ID NO 1 et l'apoprotéine de la sous-unité β de la C-PC comprend la protéine de SEQ ID NO 2 ou des variants de celles-ci.  Advantageously, the apoprotein of C-PC comprises the protein of SEQ ID NO 1 or SEQ ID NO 2 or a variant thereof. In particular, the α-subunit apoprotein of C-PC comprises the protein of SEQ ID NO 1 and the apoprotein of the β subunit of C-PC comprises the protein of SEQ ID NO 2 or variants thereof.
SEQ ID 1 : MKTPITEAIA AADNQGRFLS NTELQAVNGR YQRAAASLEA ARSLTSNAQR LINGAAQAVY SKFPYTSQMP GPQYASSAVG KAKCARDIGY YLRMVTYCLV VGGTGPMDEY LIAGLEEINR TFDLSPSWYV EALNYVKSNH GLSGQAANEA NTYIDYAINA LS SEQ ID 1: MKTPITEAIA AADNQGRFLS NTELQAVNGR YQRAAASLEA ARSLTSNAQR LINGAAQAVY SKFPYTSQMP GPQYASSAVG KAKCARDIGY YLRMVTYCLV VGGTGPMDEY LIAGLEEINR TFDLSPSWYV EALNYVKSNH GLSGQAANEA NTYIDYAINA LS
SEQ ID 2 : MLDAFAKVVA QADARGEFLS NTQLDALSKM VSEGNKRLDV VNRITSNASA IVTNAARALF SEQPQLIQPG GNAYTNRRMA ACLRDMEI IL RYVSYAI IAG DSSVLDDRCL NGLRETYQAL GVPGASVAVG VEKMKDSAIA IANDPSGITT GDCSALMAEV GTYFDRAATA VQ SEQ ID 2: MLDAFAKVVA QADARGEFLS NTQLDALSKM VSEGNKRLDV VNRITSNASA IVTNAARALF SEQPQLIQPG GNAYTNRRMA ACLRDMEI IL RYVSYAI IAG DSSVLDDRCL NGLRETYQAL GVPGASVAVG VEKMKDSAIA IANDPSGITT GDCSALMAEV GTYFDRAATA VQ
Également avantageusement, la sous-unité a de ladite APC comprend la SEQ ID NO 3 ou des variants de celles-ci et l'apoprotéine de la sous-unité β de ladite APC comprend la SEQ ID NO 4 ou des variants de celles-ci.  Also advantageously, the α subunit of said APC comprises SEQ ID NO 3 or variants thereof and the apoprotein of the β subunit of said APC comprises SEQ ID NO 4 or variants thereof .
SEQ ID 3 : MSLISQIINT ADEELRYPNG GELSTLIYFF NTANTRINII NKLKEREKDI IQNASKKLFQ LHPEYVSSGG NASGPKQRAL CLRDYGWYLR SEQ ID NO: MSLISQIINT ADEELRYPNG GELSTLIYFF NTANTRINII NKLKEREKDI IQNASKKLFQ LHPEYVSSGG NASGPKQRAL CLRDYGWYLR
LVTYGILAGD ITPIEKIGII GVKDMYNSLG VPIIGMYDAI KCLKEAS INILVTYGILAGD ITPIEKIGII GVKDMYNSLG VPIIGMYDAI KCLKEAS INI
FELSEEKDLI I PYFDYLSNA ILSFELSEEKDLI I PYFDYLSNA ILS
SEQ ID 4 : MSIVTKSIVN ADAEARYLSP GELDRIKSFV LSGQRRLRIASEQ ID 4: MSIVTKSIVN ADAEARYLSP GELDRIKSFV LSGQRRLRIA
QILTDNRERI VKQAGQQLFQ QRPDIVSPGG NAYGEEMTAT CLRDLDYYLR LVTYGVVAGD ISPIEEIGLE DFMQDAITAV INTADVQGKY LDNSSIEKLKQILTDNRERI VKQAGQQLFQ QRPDIVSPGG NAYGEEMATAT CLRDLDYYLR LVTYGVVAGD ISPIEEIGLE DFMQDAITAV INTADVQGKY LDNSSIEKLK
GYFQTGELRV RAAATIAANA AGI IKDAVAK SLLYSDITRP GGNMYTTRRYGYFQTGELRV RAAATIAANA AGI IKDAVAK SLLYSDITRP GGNMYTTRRY
AACIRDLDYY LRYATYSMLA GDPS ILDERV LNGLKETYNS LGVPIGATIQAACIRDLDYY LRYATYSMLA GDPS ILDERV LNGLKETYNS LGVPIGATIQ
SIQAMKEVTS SLV SIQAMKEVTS SLV
Les apoprotéines des C-PC et APC d'une même source de phycocyanines ont généralement des points isoélectriques différents. En baissant le pH, il sera possible de séparer au moins en partie les C-PC des APC.  The apoproteins of C-PC and APC from the same source of phycocyanins generally have different isoelectric points. By lowering the pH, it will be possible to separate at least part of the C-PCs from the APCs.
En effet, les inventeurs ont constaté que plus le pH de l'extrait brut était ajusté bas, plus les C-PC obtenues étaient pures. De manière avantageuse, lorsque la phycobiliprotéine est une phycocyanine résistante aux pH acides, l'abaissement du pH en deçà du point isoélectrique de l'APC permet d'obtenir une phycocyanine comprenant un mélange C-PC/APC dont le rapport molaire est d'au moins 5, préférentiellement d'au moins 10, plus préférentiellement d'au moins 15. Indeed, the inventors have found that the lower the pH of the crude extract was adjusted, the more C-PC obtained were pure. Advantageously, when the phycobiliprotein is phycocyanin resistant to acidic pH, lowering the pH below the isoelectric point of the APC makes it possible to obtain a phycocyanin comprising a C-PC / APC mixture whose molar ratio is at least 5, preferably at least 10, more preferably at least 15.
De préférence, le pH de l'extrait brut dans l'étape a) est ajusté à un pH inférieur à 5. On peut obtenir alors de la phycocyanine résistante aux pH acides comprenant moins de 5% Molaires d'APC, préférentiellement moins de 1 %, plus préférentiellement moins de 0,1 % d'APC, les pourcentages étant exprimés par rapport à la somme totale d'APC et de C-PC.  Preferably, the pH of the crude extract in step a) is adjusted to a pH of less than 5. Phycocyanin resistant to acidic pH comprising less than 5 mol% of APC, preferably less than 1, can be obtained. %, more preferably less than 0.1% APC, the percentages being expressed relative to the total sum of APC and C-PC.
Dans l'étape a), l'ajustement du pH se fait par l'ajout d'un acide, minéral ou organique, fort ou faible, sous forme de solide ou de solution, la quantité d'acide ajoutée étant déterminée par le pH de l'extrait brut à traiter et la valeur de pH que l'homme du métier cherchera à obtenir. Parmi les acides minéraux bien connus de l'homme du métier, on citera plus particulièrement l'acide chlorhydrique et l'acide phosphorique. Parmi les acides organiques bien connus de l'homme du métier, on citera en particulier l'acide acétique, l'acide citrique, l'acide tartrique, l'acide lactique, de préférence l'acide citrique. On peut également citer les polyphénols acides comme l'acide rosmarinique, l'acide tannique, l'acide digallique, l'acide quercitannic, l'acide gallotannique, les tannins acides comme la quercitine, les ellagitannines, castalagine, castaline, casuariticine, grandinine, punicaligine punicaline, roburine A, tellimagrandine II, terflavine B, vescaligine, pendunculagine, casua ine, castline, vescaline, de préférence l'acide tannique. De préférence, les acides employés sont des acides autorisés pour un usage alimentaire, en particulier l'acide phophorique, l'acide citrique ou l'acide tannique.  In step a), the pH adjustment is done by adding an acid, mineral or organic, strong or weak, in the form of solid or solution, the amount of acid added being determined by the pH the raw extract to be treated and the pH value that the skilled person will seek to obtain. Among the mineral acids well known to those skilled in the art, mention will be made more particularly of hydrochloric acid and phosphoric acid. Among the organic acids well known to those skilled in the art, mention will in particular be made of acetic acid, citric acid, tartaric acid, lactic acid, preferably citric acid. Acidic polyphenols such as rosmarinic acid, tannic acid, digallic acid, quercitannic acid, gallotannic acid, acidic tannins such as quercitin, ellagitannines, castalagine, castaline, casuariticin, grandinine may also be mentioned. Punicalin, punicalin, roburin A, tellimagrandin II, terflavin B, vescalin, pendunculagin, casua ine, castline, vescaline, preferably tannic acid. Preferably, the acids used are acids authorized for food use, in particular phophoric acid, citric acid or tannic acid.
Pour l'étape b) de récupération du surnageant comprenant les phycobiliprotéines résistantes aux pH acides, toute méthode de séparation connue de l'homme du métier pourra être employée, notamment par filtration tangentielle sur membranes céramiques ou membranes organiques tel que les fibres creuses en polyethersulfone. Les seuils de ces filtres peuvent être choisis pour séparer des molécules de poids moléculaire supérieur ou inférieur aux phycobiliprotéines ciblées.  For step b) of recovering the supernatant comprising phycobiliproteins resistant to acidic pH, any separation method known to those skilled in the art may be employed, in particular by tangential filtration on ceramic membranes or organic membranes such as hollow fibers polyethersulfone . The thresholds of these filters can be chosen to separate molecules of molecular weight higher or lower than targeted phycobiliproteins.
Selon un mode particulier de réalisation de l'invention, la séparation à l'étape b) est faite par filtration tangentielle. Cette étape permet de concentrer et d'éliminer une partie des protéines autre que les phycobiliprotéines, augmentant ainsi le taux de pureté du produit final. According to a particular embodiment of the invention, the separation in step b) is done by tangential filtration. This step helps to focus and eliminate a part of the proteins other than phycobiliproteins, thus increasing the degree of purity of the final product.
L'étape c) de séchage/déshydratation des phycobiliprotéines résistantes aux pH acides à partir du surnageant se fait par toute méthode d'élimination du solvant, l'eau en l'occurrence, par exemple par évaporation à pression atmosphérique ou sous vide. On citera en particulier l'atomisation, la lyophilisation, la zéodratation, le séchage par infra rouge, ou le séchage par fenêtre de réfraction.  Step c) drying / dehydration phycobiliproteins resistant to acidic pH from the supernatant is by any method of removing the solvent, the water in this case, for example by evaporation at atmospheric pressure or under vacuum. In particular, atomization, lyophilization, zeodration, infra-red drying, or refractive window drying are mentioned.
Dans l'éventualité d'une évaporation par chauffage, l'homme du métier fera attention à ne pas employer des températures trop élevées susceptibles de provoquer une dénaturation des phycobiliprotéines.  In the event of evaporation by heating, those skilled in the art will be careful not to use too high temperatures likely to cause denaturation of phycobiliproteins.
Il est possible, après récupération du surnageant à l'étape b), de recycler les phycobiliprotéines contenues dans le précipité. Pour ce faire, les phycobilliprotéines résiduelles sont solubilisées dans une solution aqueuse de pH acide, d'environ 6, ou moins, pH auquel les impuretés restent insolubles alors que les phycobiliprotéines sont solubles.  It is possible, after recovery of the supernatant in step b), to recycle the phycobiliproteins contained in the precipitate. To do this, the residual phycobilliproteins are solubilized in an aqueous solution of acidic pH, about 6 or less, pH at which the impurities remain insoluble while the phycobiliproteins are soluble.
Ces phycobiliprotéines résiduelles sont alors séparées des impuretés et isolées en reprenant les étapes b) et c) du procédé. C'est un processus itératif qui peut être répété autant de fois que nécessaire. Lorsque les conditions employées pour le procédé selon l'invention permettent une purification préférentielle des C-PC, les phycobiliprotéines résiduelles sont un mélange C-PC/APC enrichi en APC.  These residual phycobiliproteins are then separated from the impurities and isolated by repeating steps b) and c) of the process. It is an iterative process that can be repeated as many times as necessary. When the conditions employed for the process according to the invention allow a preferential purification of the C-PCs, the residual phycobiliproteins are an APC enriched C-PC / APC mixture.
Par le recyclage du précipité on obtient alors des phycobiliprotéines comprenant un mélange C-PC/APC dont le rapport Molaire est inférieur à 5, en particulier inférieur à 4, avantageusement de l'ordre de 3 à 0,1 .  By recycling the precipitate, phycobiliproteins comprising a C-PC / APC mixture whose molar ratio is less than 5, in particular less than 4, advantageously of the order of 3 to 0.1, are obtained.
En reprenant les étapes a) à c) du procédé, précédemment décrit, on peut par un processus itératif, d'une part épuiser le précipité en C-PC, qui peut être jointe aux fractions précédemment obtenues pour en enrichir la teneur et d'autre part enrichir le mélange résiduel de phycobiliprotéines en APC, avec un rapport APC/C-PC d'au moins 5, préférentiellement d'au moins 10, plus préférentiellement d'au moins 15.  By repeating steps a) to c) of the process described above, it is possible, by an iterative process, firstly to deplete the C-PC precipitate, which can be combined with the fractions previously obtained in order to enrich the content thereof. on the other hand, enriching the residual mixture of phycobiliproteins with APC, with an APC / C-PC ratio of at least 5, preferably of at least 10, more preferably of at least 15.
On peut obtenir alors un mélange d'APC comprenant moins de 5% Molaires de CPC, préférentiellement moins de 1 %, plus préférentiellement moins de 0,1 % de CPC, les pourcentages étant exprimés par rapport à la somme totale d'APC et de C- PC.  A mixture of APC comprising less than 5 mol% of CPC, preferably less than 1%, more preferably less than 0.1% of CPC, can then be obtained, the percentages being expressed with respect to the total sum of APC and C-PC.
Ces APC isolées à partir du précipité peuvent ensuite être à nouveau purifiées par des techniques de chromatographie préparative bien connues de l'homme du métier pour la production d'allophycocyanines susceptible d'être employées comme dans le domaine de l'imagerie médicale par exemple de part ces propriétés fluorescentes These APCs isolated from the precipitate can then be purified again by preparative chromatography techniques well known to humans. profession for the production of allophycocyanins that can be used as in the field of medical imaging for example from these fluorescent properties
L'invention concerne également les phycobiliprotéines résistantes aux pH acides, en particulier phycocyanines, susceptibles d'être obtenues par le procédé de purification.  The invention also relates to phycobiliproteins resistant to acidic pH, in particular phycocyanin, which can be obtained by the purification process.
L'invention concerne aussi des phycocyanines purifiées résistantes aux pH acides comprenant un mélange C-PC/APC dont le rapport molaire est d'au moins 2.  The invention also relates to purified phycocyanins resistant to acidic pH comprising a C-PC / APC mixture having a molar ratio of at least 2.
En particulier, l'invention concerne une phycocyanine résistante aux pH acides qui comprend au moins 95% Molaires de C-PC et moins de 5% Molaires d'APC, préférentiellement au moins 99% Molaires de C-PC et moins de 1 % Molaires d'APC, les pourcentages étant exprimés par rapport à la somme totale d'APC et de C-PC.  In particular, the invention relates to phycocyanin resistant to acidic pH which comprises at least 95 mol% of C-PC and less than 5 mol% of APC, preferably at least 99 mol% of C-PC and less than 1 mol% of APC, the percentages being expressed in relation to the total sum of APC and C-PC.
Ces C-PC sont connues de l'homme du métier et notamment définies précédemment, en particulier celles dont la sous-unité a de la C-PC comprend la protéine de SEQ ID NO 1 et l'apoprotéine de la sous-unité β de la C-PC comprend la protéine de SEQ ID 2 ou des variants de celles-ci.  These C-PCs are known to those skilled in the art and in particular defined above, in particular those whose α-subunit of C-PC comprises the protein of SEQ ID NO 1 and the apoprotein of the β subunit of C-PC comprises the protein of SEQ ID 2 or variants thereof.
De manière avantageuse, les variants selon l'invention présentent une identité de séquence d'au moins 83 % pour les sous-unités a de la C-PC, et d'au moins 82% pour les sous-unités β de la C-PC.  Advantageously, the variants according to the invention have a sequence identity of at least 83% for the α-subunits of C-PC, and at least 82% for the β subunits of the C-PC. PC.
De manière préférentielle, les variants selon l'invention ont une identité d'au moins 90% pour les sous-unités a (SEQ ID NO 1 ) et β (SEQ ID NO 2).  Preferably, the variants according to the invention have an identity of at least 90% for the α (SEQ ID NO 1) and β (SEQ ID NO 2) subunits.
L'invention concerne également une phycocyanine purifiée enrichie en APC susceptible d'être obtenue par le procédé selon l'invention.  The invention also relates to a purified phycocyanin enriched in APC that can be obtained by the process according to the invention.
En particulier, l'invention concerne de la phycocyanine purifiée qui comprend un mélange enrichi en APC dont le rapport Molaire C-PC/APC est inférieur à 5, en particulier 4, avantageusement de l'ordre 3 à 0,1 .  In particular, the invention relates to purified phycocyanin which comprises a mixture enriched in APC whose Molar ratio C-PC / APC is less than 5, in particular 4, advantageously of the order 3 to 0.1.
Selon un mode particulier de réalisation de l'invention, le mélange enrichi en APC a un rapport APC/C-PC d'au moins 5, préférentiellement d'au moins 10, plus préférentiellement d'au moins 15.  According to a particular embodiment of the invention, the mixture enriched with APC has an APC / C-PC ratio of at least 5, preferably at least 10, more preferably at least 15.
Selon un mode plus particulier de réalisation de l'invention, la phycocyanine est constituée essentiellement d'APC, avec au moins 95% Molaires d'APC et moins de 5% Molaires de C-PC, préférentiellement au moins 99% Molaires d'APC et moins de 1 % Molaires de C-PC, les pourcentages étant exprimés par rapport à la somme totale d'APC et de C-PC. According to a more particular embodiment of the invention, the phycocyanin consists essentially of APC with at least 95% APC Molar and less than 5% C-PC Molar, preferably at least 99% APC Molar. and less 1 mol% of C-PC, the percentages being expressed with respect to the total sum of APC and C-PC.
Ces APC sont connues de l'homme du métier et notamment définies précédemment, en particulier celles dont la sous-unité a de ladite APC comprend la SEQ ID NO 3 ou des variants de celles-ci et l'apoprotéine de la sous-unité β de ladite APC comprend la SEQ ID NO 4 ou des variants de celles-ci.  These APCs are known to those skilled in the art and in particular defined above, in particular those whose α subunit of said APC comprises SEQ ID NO 3 or variants thereof and the apoprotein of the β subunit. of said APC comprises SEQ ID NO 4 or variants thereof.
De manière avantageuse, les variants selon l'invention présentent une identité de séquence d'au moins 83 % pour les sous-unités a de l'APC, et d'au moins 82% pour les sous-unités S de l'APC.  Advantageously, the variants according to the invention have a sequence identity of at least 83% for the α subunits of APC, and at least 82% for the S subunits of APC.
L'homme du métier sait comment mesurer une identité de séquences protéiques selon les méthodes usuelles à sa disposition, notamment le programme BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi).  Those skilled in the art know how to measure an identity of protein sequences according to the usual methods at their disposal, in particular the BLASTP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
De même, l'homme du métier sait comment identifier des variants desdites séquences et vérifier qu'ils conservent les mêmes propriétés structurelles par simple essai de stabilité en pH acide, par exemple en pratiquant un essai comme l'essai présenté à l'exemple 3 de la demande WO 2017/050918.  Similarly, those skilled in the art know how to identify variants of said sequences and verify that they retain the same structural properties by simple acid pH stability test, for example by performing a test such as the test presented in Example 3 of the application WO 2017/050918.
Il est connu de l'homme du métier qu'un polypeptide peut être modifié par substitution, insertion et/ou délétion d'au moins un acide aminé sans modifier de manière substantielle sa fonction.  It is known to those skilled in the art that a polypeptide may be modified by substitution, insertion and / or deletion of at least one amino acid without substantially modifying its function.
Par exemple, la substitution d'un acide aminé à une position donnée par un autre acide aminé chimiquement équivalent est un exemple connu de variation de séquence qui n'affecte pas de manière substantielle les propriétés de la protéine.  For example, the substitution of an amino acid at a given position by another chemically equivalent amino acid is a known example of sequence variation that does not substantially affect the properties of the protein.
Ces substitutions "conservatrices" peuvent être définies comme des échanges à l'intérieur des groupes d'acides aminés suivants  These "conservative" substitutions can be defined as exchanges within the following amino acid groups
- Ala, Ser, Thr, Pro, Gly - Ala, Ser, Thr, Pro, Gly
- Asp, Asn, Glu, Gin  - Asp, Asn, Glu, Gin
- His, Arg, Lys  - His, Arg, Lys
- Met, Leu, Ile, Val, Cys et  - Met, Leu, Ile, Val, Cys and
- Phe, Tyr, Trp  - Phe, Tyr, Trp
Ainsi, les variants des apoprotéines des phycocyanines et/ou des allophycocyanines selon l'invention peuvent comprendre de 1 à 30 acides aminés de différence en nombre par rapport à la séquence dite de référence correspondante, particulièrement pour ce qui concerne les sous unités a et/ou β de la phycocyanine, dans la mesure où le variant obtenu conserve les propriétés de la protéine de référence et les pourcentages d'homologie/identité énoncés ci-dessus. Thus, the apoprotein variants of the phycocyanins and / or allophycocyanins according to the invention may comprise from 1 to 30 amino acids of difference in number with respect to the corresponding reference sequence, particularly with regard to the subunits a and / or or β of phycocyanin, to the extent that the variant obtained retains the properties of the reference protein and the percentages of homology / identity stated above.
Plus précisément selon l'invention,  More precisely according to the invention,
pour les variants des apoprotéines de la sous-unité a des phycocyanines utilisables dans les compositions acides selon l'invention, issus de substitutions, d'insertions et/ou de délétions, ils peuvent comprendre de 1 à 27 acides aminés de différence par rapport à la séquence dite de référence correspondante, dans la mesure où le variant obtenu conserve les propriétés de la protéine de référence et les pourcentages d'identité énoncés ci-dessus;  for the apoprotein variants of the phycocyanin subunit usable in the acid compositions according to the invention, resulting from substitutions, insertions and / or deletions, they may comprise from 1 to 27 amino acids of difference with respect to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;
- pour les variants des apoprotéines de la sous-unité β des phycocyanines utilisables dans les compositions acides selon l'invention, issus de substitutions, d'insertions et/ou de délétions, ils peuvent comprendre de 1 à 30 acides aminés de différence par rapport à la séquence dite de référence correspondante, dans la mesure où le variant obtenu conserve les propriétés de la protéine de référence et les pourcentages d'identité énoncés ci-dessus; for variants of the apoproteins of the β subunit of the phycocyanins that can be used in the acid compositions according to the invention, derived from substitutions, insertions and / or deletions, they may comprise from 1 to 30 amino acids of difference relative to to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;
pour les variants des apoprotéines de la sous-unité a des allophycocyanines utilisables dans les compositions acides selon l'invention, issus de substitutions, d'insertions ou de délétions, ils peuvent comprendre de 1 à 24 acides aminés de différence par rapport à la séquence dite de référence correspondante, dans la mesure où le variant obtenu conserve les propriétés de la protéine de référence et les pourcentages d'identité énoncés ci-dessus;  for the apoprotein variants of the α-subunit of allophycocyanines that may be used in the acid compositions according to the invention, derived from substitutions, insertions or deletions, they may comprise from 1 to 24 amino acids of difference relative to the sequence so-called reference reference, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;
pour les variants des apoprotéines de la sous-unité β des allophycocyanines utilisables dans les compositions acides selon l'invention, issus de substitutions, d'insertions et/ou de délétions, ils peuvent comprendre de 1 à 20 acides aminés de différence par rapport à la séquence dite de référence correspondante, dans la mesure où le variant obtenu conserve les propriétés de la protéine de référence et les pourcentages d'identité énoncés ci-dessus.  for the apoprotein variants of the β subunit of the allophycocyanines that can be used in the acid compositions according to the invention, resulting from substitutions, insertions and / or deletions, they may comprise from 1 to 20 amino acids of difference with respect to the so-called reference reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above.
Très particulièrement selon l'invention, et quelle que soit la séquence de référence considérée (sous-unité a et/ou β de la phycocyanine et/ou sous-unité a et/ou β de l'allophycocyanine) les variants desdites sous-unités peuvent comprendre avantageusement de 1 à 15 acides aminés de différence, de préférence de 1 à 10 acides aminés de différence, en particulier 1 ou 2 ou 3 ou 4 ou 5 ou 6 ou 7 ou 8 ou 9 ou 10 acides aminés de différence par rapport à la séquence dite de référence correspondante, dans la mesure où le variant obtenu conserve les propriétés de la protéine de référence et les pourcentages d'identité énoncés ci-dessus. Very particularly according to the invention, and whatever the reference sequence considered (subunit a and / or β of phycocyanin and / or subunit a and / or β of allophycocyanin) the variants of said subunits may advantageously comprise from 1 to 15 amino acids of difference, preferably from 1 to 10 amino acids of difference, in particular 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids of difference relative to to the so-called reference sequence corresponding, to the extent that the obtained variant retains the properties of the reference protein and the percentages of identity stated above.
De préférence, l'invention concerne la C-PC dont la protéine de la sous unité alpha consiste en la protéine de SEQ ID 1 et la protéine de la sous unité bêta consiste en la protéine de SEQ ID 2.  Preferably, the invention relates to C-PC whose alpha subunit protein consists of the protein of SEQ ID 1 and the subunit beta protein consists of the protein of SEQ ID 2.
Selon un autre mode préféré, l'invention concerne l'APC dont la protéine de la sous unité alpha consiste en la protéine de SEQ ID 3 et la protéine de la sous unité bêta consiste en la protéine de SEQ ID 4.  According to another preferred embodiment, the invention relates to APC whose alpha subunit protein consists of the protein of SEQ ID 3 and the subunit beta protein consists of the protein of SEQ ID 4.
Les phycobiliprotéines sont des colorants naturels essentiellement employés pour la coloration des aliments.  Phycobiliproteins are natural dyes essentially used for coloring foods.
L'invention concerne aussi l'utilisation des phycobiliprotéines résistantes aux pH acides obtenues par le procédé selon l'invention et en particulier les phycocyanines résistantes aux pH acides définies ci-dessus comme colorant dans un produit alimentaire.  The invention also relates to the use of phycobiliproteins resistant to acidic pH obtained by the method according to the invention and in particular phycocyanines resistant to acidic pH defined above as a colorant in a food product.
L'invention concerne aussi une composition en particulier alimentaire comprenant de la phycobiliprotéine résistante aux pH acides obtenue par le procédé selon l'invention et en particulier de la phycocyanine résistante aux pH acides définies ci-dessus.  The invention also relates to a particular food composition comprising phycobiliprotein resistant to acidic pH obtained by the process according to the invention and in particular phycocyanin resistant to acidic pH defined above.
De telles utilisations et de telles compositions sont connues de l'homme du métier.  Such uses and compositions are known to those skilled in the art.
De manière préférentielle, le produit alimentaire ou la composition alimentaire est une composition acide, telle que définie dans la demande WO 2017/050918.  Preferably, the food product or the food composition is an acid composition, as defined in the application WO 2017/050918.
Par composition acide on entend selon l'invention toute composition comprenant un acide minéral ou organique et de la phycocyanine. Cette composition peut être liquide, fluide ou visqueuse, pâteuse ou solide qui présente un pH acide et dans laquelle de la phycocyanine résistante au pH acide est incorporée.  By acid composition is meant according to the invention any composition comprising a mineral or organic acid and phycocyanin. This composition may be liquid, fluid or viscous, pasty or solid which has an acidic pH and in which phycocyanin resistant to acidic pH is incorporated.
Pour les compositions liquides aqueuses, le pH est mesuré de manière usuelle. Pour les compositions liquides non aqueuses ou pour les compositions pâteuses ou solides, le pH est mesuré après dissolution de la composition dans une quantité d'eau suffisante pour dissoudre les composés solubles qu'elle contient, dont les acides minéraux ou organiques et la phycocyanine.  For aqueous liquid compositions, the pH is measured in the usual manner. For non-aqueous liquid compositions or for pasty or solid compositions, the pH is measured after dissolution of the composition in an amount of water sufficient to dissolve the soluble compounds it contains, including inorganic or organic acids and phycocyanin.
De manière avantageuse la composition selon l'invention est une composition liquide aqueuse, éventuellement sous forme d'un gel, ou une composition pâteuse ou solide destinée à être dissoute dans une solution aqueuse ou dans une composition solide ou pâteuse comprenant de l'eau. Selon un autre mode avantageux de réalisation de l'invention, la composition acide composition pâteuse ou solide destinée à être employée et/ou stockée dans un environnement humide. Advantageously, the composition according to the invention is an aqueous liquid composition, optionally in the form of a gel, or a pasty or solid composition intended to be dissolved in an aqueous solution or in a composition solid or pasty including water. According to another advantageous embodiment of the invention, the pasty or solid acid composition composition intended to be used and / or stored in a humid environment.
Les acides minéraux ou organiques susceptibles d'être employés dans les compositions selon l'invention sont bien connus de l'homme du métier. Parmi les acides minéraux, on citera en particulier les acides carbonique, phosphorique, chlorhydrique, sulfurique, perchlorique, sulfonique et nitrique. Parmi les acides organiques, on citera en particulier les acides citrique, lactique, malique, tartrique, succinique, avantageusement l'acide citrique.  The mineral or organic acids that can be used in the compositions according to the invention are well known to those skilled in the art. Among the mineral acids, mention will in particular be made of carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulphonic acid and nitric acid. Among the organic acids, mention will be made in particular of citric, lactic, malic, tartaric and succinic acids, advantageously citric acid.
Par composition alimentaire acide selon l'invention on entend toute composition destinée à être ingérée par l'homme ou les animaux qui entre dans la définition précédente. Les compositions acides neutraceutiques doivent être considérées comme entrant dans la définition des compositions alimentaires acides au sens de l'invention.  By acidic food composition according to the invention is meant any composition intended to be ingested by humans or animals which falls within the above definition. Nutraceutical acid compositions must be considered as falling within the definition of acidic food compositions within the meaning of the invention.
Les compositions alimentaires acides selon l'invention sont bien connues de l'homme du métier. Elles peuvent comprendre un véhicule pouvant comprendre des constituants structurels associé à des composés actifs identifiés au regard de leurs apports nutritifs ou encore pour leurs propriétés bénéfiques à la santé de l'homme ou de l'animal. La composition alimentaire acide selon l'invention peut également comprendre des additifs alimentaires comme des agents de texture, des agents de saveur, des agents conservateurs, tous constituants bien connus de l'homme du métier. Le véhicule peut comprendre de l'eau et/ou des protéines et/ou des matières grasses et/ou des fibres et/ou des sucres. Les constituants du véhicule peuvent avoir uniquement des propriétés structurelles mais ils sont généralement connus pour leurs apports nutritifs.  The acidic food compositions according to the invention are well known to those skilled in the art. They may comprise a vehicle that may comprise structural constituents associated with active compounds identified with regard to their nutritive contributions or for their properties beneficial to the health of humans or animals. The acidic food composition according to the invention may also comprise food additives such as texturizing agents, flavoring agents, preserving agents, all components well known to those skilled in the art. The vehicle may comprise water and / or proteins and / or fats and / or fibers and / or sugars. The constituents of the vehicle may have only structural properties but they are generally known for their nutrient inputs.
La composition alimentaire acide selon l'invention peut être prête à l'emploi ou bien sous forme d'un additif alimentaire que l'on ajoute à une préparation solide, pâteuse ou liquide pour préparer l'aliment qui pourra être ingéré.  The acidic food composition according to the invention can be ready for use or in the form of a food additive that is added to a solid, pasty or liquid preparation to prepare the food that can be ingested.
Pour les compositions alimentaires, l'acide sera choisi de préférence dans la liste des acidifiants autorisés dans l'alimentation, en particulier les acides carbonique, phosphorique, citrique, malique, tartrique et lactique, plus particulièrement l'acide citrique.  For food compositions, the acid will preferably be selected from the list of acidifiers authorized in the diet, in particular carbonic acid, phosphoric acid, citric acid, malic acid, tartaric acid and lactic acid, more particularly citric acid.
Concernant les compositions acides autres qu'alimentaires selon l'invention elles peuvent être entre autres pharmaceutiques, vétérinaires ou cosmétiques et comprendre en outre tout additifs et/ou actifs connus et utilisés dans ce genre de composition. As regards the non-food acidic compositions according to the invention, they may be, for example, pharmaceutical, veterinary or cosmetic and further include any additives and / or active known and used in this kind of composition.
Dans une composition acide solide, liquide ou pâteuse selon l'invention, la phycocyanine peut être incorporée par exemple sous forme de poudre. Ladite composition acide, particulièrement ladite composition alimentaire acide pourra alors se présenter sous toute forme usuelle connue telle que des crèmes, gels, mousses, pâtes, etc .. Particulièrement pour une composition alimentaire solide on peut citer notamment les gâteaux ou biscuits, les aliments secs à cuire, les poudres à diluer, les compositions gélatineuses solides ou "jelly", des mousses etc.  In a solid, liquid or pasty acid composition according to the invention, the phycocyanin may be incorporated, for example, in the form of a powder. Said acid composition, particularly said acidic food composition may then be in any known known form such as creams, gels, foams, pastes, etc. Especially for a solid food composition include cakes or biscuits, dry foods for cooking, powders for dilution, solid gelatinous compositions or "jelly", foams, etc.
Selon l'invention, ladite composition acide liquide pourra être une composition aqueuse dans laquelle la phycocyanine est dissoute. Elle peut se présenter sous la forme d'une composition prête à l'emploi ou comme un concentré liquide à diluer, notamment pour son ingestion ou à ajouter à un aliment solide soit pour sa préparation, soit pour son ingestion, par exemple une composition liquide concentrée d'enrobage ou "topping" qui sera déposée sur un gâteau pour lui apporter sa couleur. Parmi ces compositions concentrées, on peut citer les sirops, alcoolisés ou non.  According to the invention, said liquid acid composition may be an aqueous composition in which the phycocyanin is dissolved. It may be in the form of a ready-to-use composition or as a liquid concentrate to be diluted, especially for its ingestion or to be added to a solid food either for its preparation or for its ingestion, for example a liquid composition. concentrated coating or "topping" which will be deposited on a cake to bring its color. Among these concentrated compositions include syrups, alcoholic or not.
La composition acide liquide selon l'invention peut être de viscosité variable et comprendre ou non des additifs tels que des agents de viscosité, des gélifiants, et autres additifs structurants connus de l'homme du métier et usuels pour la préparation de compositions alimentaires liquides.  The liquid acid composition according to the invention may be of variable viscosity and may or may not comprise additives such as viscosity agents, gelling agents, and other structuring additives known to those skilled in the art and customary for the preparation of liquid food compositions.
Selon un mode particulier de réalisation de l'invention, la composition alimentaire liquide peut être une boisson acide, gazeuse ou non. On citera en particulier les sodas, les jus, les boissons pour sportifs, boissons d'effort, boissons de récupération, etc. Les compositions de ces boissons sont bien connues de l'homme du métier et peuvent comprendre, notamment, des sucres, des sels minéraux, des additifs alimentaires, du gaz dissout, etc. La boisson selon l'invention est une boisson acide usuelle dans laquelle le colorant habituellement employé a été remplacé en totalité ou en partie par une phycocyanine résistante à un pH acide selon l'invention.  According to a particular embodiment of the invention, the liquid food composition may be an acidic beverage, gaseous or not. Examples include sodas, juices, sports drinks, exercise drinks, salvage drinks, etc. The compositions of these drinks are well known to those skilled in the art and may include, in particular, sugars, mineral salts, food additives, dissolved gas, etc. The beverage according to the invention is a usual acidic beverage in which the dye usually employed has been replaced in whole or in part by phycocyanin resistant to acidic pH according to the invention.
Selon l'invention la teneur en phycocyanine dans les compositions selon l'invention pourra être conforme aux usages de l'homme du métier.  According to the invention, the phycocyanin content in the compositions according to the invention may be in accordance with the practice of those skilled in the art.
Par exemple lorsque la phycocyanine sera utilisée pour colorer la composition acide alors la teneur en phycocyanine dans ladite composition pourra être conforme aux usages de l'homme du métier en matière de coloration.  For example, when the phycocyanin will be used to color the acidic composition, then the phycocyanin content in said composition may be in accordance with the practices of a person skilled in the art of coloring.
Dans une composition acide liquide au sens de l'invention la teneur en phycocyanine peut être comprise entre 2,5 mg/L et 2500 mg/L, préférentiellement entre 25 mg/L et 300 mg/L. In a liquid acid composition within the meaning of the invention, the content of phycocyanin may be between 2.5 mg / L and 2500 mg / L, preferably between 25 mg / L and 300 mg / L.
Dans une composition liquide de type boisson prête à l'emploi, la teneur en phycocyanine peut être généralement comprise entre 25 mg/l et 300 mg/L, préférentiellement entre 50 mg/L et 100 mg/L.  In a ready-to-use beverage liquid composition, the phycocyanin content may generally be between 25 mg / l and 300 mg / l, preferably between 50 mg / l and 100 mg / l.
Dans une composition liquide concentrée à diluer pour son usage, comme un sirop, la teneur en phycocyanine peut être généralement comprise entre 250 mg/l et 2500 mg/l, préférentiellement entre 500 mg/L et 1000 mg/L.  In a concentrated liquid composition to be diluted for its use, such as a syrup, the phycocyanin content may generally be between 250 mg / l and 2500 mg / l, preferably between 500 mg / l and 1000 mg / l.
Dans une composition solide, la teneur en phycocyanine peut être généralement comprise entre 0,01 mg/g et 10 mg/g, préférentiellement entre 0,1 mg/g et 5,0 mg/g, très préférentiellement entre 0,25 mg/g et 2,5 mg/g.  In a solid composition, the phycocyanin content may generally be between 0.01 mg / g and 10 mg / g, preferentially between 0.1 mg / g and 5.0 mg / g, very preferably between 0.25 mg / g g and 2.5 mg / g.
EXEMPLES EXAMPLES
Exemple 1. Purification par précipitation acide sur cellule fraîche.  Example 1. Purification by acid precipitation on fresh cell.
Un moût de fermentation de cellule de Galdieria sulphuraria centrifugé puis rincé avec un volume équivalent d'eau à subit un broyage mécanique afin de libérer les phycobiliprotéines dans une phase aqueuse à pH 6. Le broyât est acidifié par pallier de 0,5 unité pH par ajout d'acide citrique. A chaque pallier, un prélèvement du mélange est effectué puis centrifugé 10 min à 1 1 000 g. Le surnageant contenant les phycobiliprotéines est prélevé et l'indice de pureté mesuré en faisant le rapport d'absorbance à 618 nm sur l'absorbance à 280 nm avec un spectrophotomètre (Amersham Biosciences Ultra Spec 2100 Pro).  A galdieria sulphuraria cell fermentation broth centrifuged and then rinsed with an equivalent volume of water undergoes a mechanical grinding in order to release the phycobiliproteins in an aqueous phase at pH 6. The ground material is acidified by adjusting the pH unit by 0.5. addition of citric acid. At each step, a sample of the mixture is made and then centrifuged for 10 min at 1000 g. The supernatant containing the phycobiliproteins is removed and the purity index measured by making the absorbance ratio at 618 nm on the absorbance at 280 nm with a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).
Il apparaît clairement que plus le pH baisse plus le l'indice de pureté augmente (Figure 1 ). Cette augmentation de l'indice de pureté traduit une baisse des protéines contaminantes dans le surnageant, alors que la C-PC, elle, reste majoritairement dans le surnageant. De par sa résistance au pH acide il n'y a pas de perte importante de la teneur en C-PC dans le surnageant (Figure 2). De façon surprenante, l'allophycocyanine (APC) elle disparaît totalement du surnageant pour des valeurs de pH inférieures à 5, pour se retrouver dans le culot avec les autres protéines précipitées et les débris cellulaires (Figure 3). Dans ce culot on retrouve également de la C-PC et de l'APC avec une teneur plus élevée en APC (Figure 3).  It is clear that the lower the pH, the higher the purity index (Figure 1). This increase in the purity index indicates a decrease of the contaminating proteins in the supernatant, while the C-PC remains predominantly in the supernatant. Due to its resistance to acid pH there is no significant loss of C-PC content in the supernatant (Figure 2). Surprisingly, allophycocyanin (APC) disappears completely from the supernatant at pH values below 5, ending up in the pellet with other precipitated proteins and cell debris (Figure 3). This pellet also contains C-PC and APC with a higher content of APC (Figure 3).
L'acidification permet également d'obtenir une meilleure séparation des phases liquide et solide et d'obtenir un culot de débris cellulaire et de protéines plus compacte et plus facile à séparer de la phase aqueuse. Exemple 2. Purification par précipitation acide sur cellule lyophilisée et réhydratées. The acidification also makes it possible to obtain a better separation of the liquid and solid phases and to obtain a pellet of cellular debris and proteins which is more compact and easier to separate from the aqueous phase. Example 2. Purification by acid precipitation on lyophilized cell and rehydrated.
Un moût de fermentation de cellule de Galdieria sulphuraria centrifugé puis rincé avec un volume équivalent d'eau à subit un broyage mécanique afin de libérer les phycobiliprotéines dans une phase aqueuse à pH 6. Le broyât ensuite lyophilisé. La matière sèche lyophilisée est suspendue dans un volume d'eau équivalent au volume initial du moût puis acidifié par pallier de 0,5 unité pH par ajout d'acide citrique. A chaque pallier, un prélèvement du mélange est effectué puis centrifugé 10 min à 1 1 000 g. Le surnageant contenant les phycobiliprotéines est prélevé et l'indice de pureté mesuré en faisant le rapport d'absorbance à 618 nm sur l'absorbance à 280 nm avec un spectrophotomètre (Amersham Biosciences Ultra Spec 2100 Pro).  A fermentation broth of Galdieria sulphuraria cell centrifuged and then rinsed with an equivalent volume of water undergoes a mechanical grinding to release the phycobiliproteins in an aqueous phase at pH 6. The ground then freeze-dried. The freeze-dried dry matter is suspended in a volume of water equivalent to the initial volume of the must and then acidified by adjusting 0.5 pH unit by addition of citric acid. At each step, a sample of the mixture is made and then centrifuged for 10 min at 1000 g. The supernatant containing the phycobiliproteins is removed and the purity index measured by making the absorbance ratio at 618 nm on the absorbance at 280 nm with a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).
Comme ce qui a été précédemment décrit dans l'exemple 1 , nous pouvons constater une augmentation de l'indice de pureté corrélée avec une baisse du pH (Figure 4). Dans ce cas également, l'acidification permet également d'obtenir une meilleure séparation des phases liquides et solides et d'obtenir un culot de débris cellulaire et de protéines plus compacte et plus facile à séparer de la phase aqueuse. De façon similaire à ce qui a été observé dans l'exemple 1 , l'APC est retrouvée dans le culot et non dans la phase aqueuse (Figure 5) pour des pH inférieurs à 5. Pour des pH compris entre 6 et 5, la quantité d'APC diminue dans le surnageant en fonction de la baisse du pH.  As was previously described in Example 1, we can see an increase in purity index correlated with a decrease in pH (Figure 4). In this case also, the acidification also makes it possible to obtain a better separation of the liquid and solid phases and to obtain a pellet of cellular debris and proteins which is more compact and easier to separate from the aqueous phase. In a manner similar to that observed in Example 1, the APC is found in the pellet and not in the aqueous phase (FIG. 5) for pH values below 5. For pH values of between 6 and 5, the The amount of APC decreases in the supernatant as the pH decreases.
Exemple 3. Purification et concentration la C-PC par filtration tangentielle.Example 3. Purification and concentration of C-PC by tangential filtration.
L'extrait brut après précipitation acide et centrifugation est filtré sur un module de filtration tangentielle de type fibre creuse. La filtration à travers ce maillage permet d'éliminer une partie des protéines autres que la C-PC et d'augmenter ainsi l'indice de pureté (Figure 6). L'indice de pureté pouvant être atteint par cette méthode approche des valeurs normalement obtenues par des méthodes beaucoup plus complexe faisant intervenir des extractions biphasiques, ou des précipitations au sulfate d'ammonium, voir même des méthodes de chromatographie (Soresen et al., 2013 ; Cruz de Jesùs et al., 2006). En parallèle de la purification cette étape de filtration permet d'éliminer l'eau de l'extrait de C-PC (Figure 7), et de faciliter le séchage du produit par la suite. REFERENCES The crude extract after acid precipitation and centrifugation is filtered on a tangential filtration module of the hollow fiber type. Filtration through this mesh makes it possible to eliminate some of the proteins other than C-PC and thus to increase the purity index (FIG. 6). The purity index that can be reached by this method approaches the values normally obtained by much more complex methods involving biphasic extractions, or precipitation with ammonium sulphate, or even chromatography methods (Soresen et al., 2013 Cruz de Jesùs et al., 2006). In parallel with the purification, this filtration step makes it possible to eliminate the water from the C-PC extract (FIG. 7), and to facilitate the drying of the product thereafter. REFERENCES
- Cruz de Jesùs et al., "Methods for Extraction, Isolation and Purification of C- phycocyanin: 50 years of Research in Review" (2016) Int J Food Nutr Sci 3(3): 1 - 10. Cruz de Jesues et al., "Methods for Extraction, Isolation and Purification of Cypycocyanin: 50 Years of Research in Review" (2016) Int J Food Nutr Sci 3 (3): 1-10.
- Eisele et al., "Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extrêmes of habitat" Biochemica et Biophysica Acta 1456 (2000) 1456, 2-3, 99-107. Eisele et al., "Studies on C-phycocyanin from Cyanidium caldarium, eukaryote at the extremes of habitat" Biochemica and Biophysica Acta 1456 (2000) 1456, 2-3, 99-107.
- Eriksen NT. "Production of phycocyanin - a pigment with applications in biology, biotechnology, foods and medicine" Appl Microbiol Biotechnol. 2008 Aug;80(1 ):1 - 14.  - Eriksen NT. "Production of phycocyanin - a pigment with applications in biology, biotechnology, food and medicine" Appl Microbiol Biotechnol. 2008 Aug; 80 (1): 1 - 14.
- Kao et al., "Physical-chemical properties of C-phycocyanin isolated from an acido- thermophilic eukaryote, Cyanidium caldarium" Biochem. J. (1975) 147, 63-70 Kao et al., "Physical-chemical properties of C-phycocyanin isolated from eukaryotic acid-thermophilic, Cyanidium caldarium" Biochem. J. (1975) 147, 63-70
- Myounghoon et al. "Isolation and Characterization of Thermostable Phycocyanin from Galdieria Sulphuraria," Korean journal of chemical engineering, 31 (2014): 1 - 6. - Myounghoon et al. "Isolation and Characterization of Thermostable Phycocyanin from Galdieria Sulfuraria," Korean Journal of Chemical Engineering, 31 (2014): 1-6.
- S0rensen et al. "Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria" J Sci Food Agric. 2013 Sep;93(12):2933-8 - Sorensen et al. "Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria" J Sci Food Agric. 2013 Sep; 93 (12): 2933-8
- WO 2005/065697, WO 2015/090697, WO 2016/099261 , WO 2017/050917, WO 2017/050918 et PCT/EP2016/079325 déposée le 30 novembre 2016 - WO 2005/065697, WO 2015/090697, WO 2016/099261, WO 2017/050917, WO 2017/050918 and PCT / EP2016 / 079325 filed on November 30, 2016

Claims

REVENDICATIONS
1 . Procédé de purification de phycobiliprotéines résistantes aux pH acides à partir d'un extrait brut de phycobiliprotéines résistantes aux pH acides provenant de cellules de microorganismes producteurs de phycobiliprotéines, caractérisé en ce qu'il comprend les étapes de 1. Process for the purification of phycobiliproteins resistant to acidic pH from a crude extract of phycobiliproteins resistant to acidic pH from phycobiliprotein-producing microorganism cells, characterized in that it comprises the steps of
a) ajustement du pH de l'extrait brut de phycobiliprotéines résistantes aux pH acides à un pH inférieur à 6 de manière à faire précipiter les matières organiques autres que les phycobiliprotéines résistantes aux pH acides,  a) adjusting the pH of the crude phycobiliprotein extract resistant to acidic pH to a pH below 6 so as to precipitate organic material other than phycobiliproteins resistant to acidic pH,
b) récupération du surnageant comprenant les phycobiliprotéines résistantes aux pH acides et  b) recovery of the supernatant comprising phycobiliproteins resistant to acidic pH and
c) isolation des phycobiliprotéines résistantes aux pH acides à partir du surnageant.  c) isolation of phycobiliproteins resistant to acidic pH from the supernatant.
2. Procédé selon la revendication 1 , caractérisé en ce que la phycobiliprotéine est de la phycocyanine résistante aux pH acides.  2. Method according to claim 1, characterized in that phycobiliprotein is phycocyanin resistant to acidic pH.
3. Procédé selon l'une des revendications 1 ou 2, caractérisé en ce que la phycocyanine comprend un mélange c-phycocyanine (C-PC) et d'allophycocyanine (APC), le rapport molaire C-PC/APC étant d'au moins 5.  3. Method according to one of claims 1 or 2, characterized in that the phycocyanin comprises a mixture c-phycocyanin (C-PC) and allophycocyanin (APC), the molar ratio C-PC / APC being from less 5.
4. Procédé selon l'une des revendications 1 à 3, caractérisé en ce que le pH du milieu de culture dans l'étape a) est ajusté à un pH inférieur à 5.  4. Method according to one of claims 1 to 3, characterized in that the pH of the culture medium in step a) is adjusted to a pH below 5.
5. Procédé selon la revendication 4, caractérisé en ce que la phycobiliprotéine comprend au moins 95% de C-PC résistante aux pH acides et moins de 5% Molaires d'APC, les pourcentages étant exprimés par rapport à la somme totale d'APC et de C-PC.  5. Method according to claim 4, characterized in that the phycobiliprotein comprises at least 95% of C-PC resistant to acidic pH and less than 5 mol% of APC, the percentages being expressed relative to the total sum of APC. and C-PC.
6. Procédé selon l'une des revendications 1 à 5, caractérisé en ce que la récupération du surnageant est faite par filtration.  6. Method according to one of claims 1 to 5, characterized in that the recovery of the supernatant is made by filtration.
7. Procédé selon l'une des revendications 1 à 6, caractérisé en ce que le microorganisme producteur de phycobiliprotéine est choisi parmi les souches de microalgues des genres Cyanidioschyzon, Cyanidium ou Galdieria.  7. Method according to one of claims 1 to 6, characterized in that the microorganism producing phycobiliprotein is selected from the microalgae strains of the genera Cyanidioschyzon, Cyanidium or Galdieria.
8. Procédé selon la revendication 7, caractérisé en ce que la microalgue est choisie parmi les souches Galdieria sulphuraria, Cyanidium caldarium et 8. Process according to claim 7, characterized in that the microalgae is chosen from the strains Galdieria sulphuraria, Cyanidium caldarium and
Cyanidioschyzon merolae. Cyanidioschyzon merolae.
9. Procédé selon l'une des revendications 1 à 8, caractérisé en ce que les phycobiliprotéines contenues dans le culot sont solubilisées dans une solution aqueuse de pH acide et séparées des impuretés et isolées en reprenant les étapes b) et c). 9. Method according to one of claims 1 to 8, characterized in that the phycobiliproteins contained in the pellet are solubilized in a solution aqueous acid pH and separated from the impurities and isolated by repeating steps b) and c).
10. Phycobiliprotéine résistante aux pH acides obtenue par le procédé selon l'une des revendications 1 à 9.  Phycobiliprotein resistant to acidic pH obtained by the process according to one of claims 1 to 9.
1 1 . Phycocyanine résistante aux pH acides comprenant un mélange c- phycocyanine (C-PC) et d'allophycocyanine (APC), caractérisée en ce que le rapport molaire C-PC/APC est d'au moins 5.  1 1. Phycocyanin resistant to acidic pH comprising a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC), characterized in that the molar ratio C-PC / APC is at least 5.
12. Phycocyanine selon la revendication 1 1 , caractérisée en ce qu'elle comprend moins de 5% Molaires d'APC.  12. Phycocyanin according to claim 1 1, characterized in that it comprises less than 5 mol% of APC.
13. Phycocyanine selon l'une des revendications 1 1 ou 12, caractérisée en ce que l'apoprotéine de la sous-unité de la C-PC comprend la SEQ ID NO 1 et l'apoprotéine de la sous-unité β de la C-PC comprend la SEQ ID 2 ou des variants de celles-ci.  13. Phycocyanin according to one of claims 1 1 or 12, characterized in that the apoprotein of the subunit of C-PC comprises SEQ ID NO 1 and the apoprotein of the β subunit of C -PC comprises SEQ ID 2 or variants thereof.
14. Utilisation d'une phycobiliprotéine selon la revendication 10 ou d'une phycocyanine selon l'une des revendications 1 1 à 13, comme colorant dans un produit alimentaire.  14. Use of a phycobiliprotein according to claim 10 or a phycocyanin according to one of claims 1 1 to 13, as a colorant in a food product.
15. Composition alimentaire caractérisée en ce qu'elle comprend une phycobiliprotéine selon la revendication 10 ou une phycocyanine selon l'une des revendications 1 1 à 13.  15. Food composition characterized in that it comprises a phycobiliprotein according to claim 10 or a phycocyanin according to one of claims 1 1 to 13.
16. Phycocyanine comprenant un mélange d'allophycocyanine (APC) et de la c-phycocyanine (C-PC) dont le rapport Molaire C-PC/APC est inférieur à 5.  16. Phycocyanin comprising a mixture of allophycocyanin (APC) and α-phycocyanin (C-PC) whose Molar ratio C-PC / APC is less than 5.
17. Phycocyanine selon la revendication 16, caractérisée en ce que l'apoprotéine de la sous-unité a de l'APC comprend la SEQ ID NO 3 et l'apoprotéine de la sous-unité β de l'APC comprend la SEQ ID 4 ou des variants de celles-ci.  17. Phycocyanin according to claim 16, characterized in that the apoprotein of the α subunit of APC comprises SEQ ID NO 3 and the apoprotein of the β subunit of APC comprises SEQ ID 4. or variants thereof.
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FR3091703B1 (en) 2019-01-11 2021-02-12 Fermentalg PHYCOCYANIN EXTRACTION PROCESS
FR3091640B1 (en) 2019-01-11 2021-06-11 Fermentalg PHYCOCYANIN PURIFICATION PROCESS
FR3092586A1 (en) 2019-02-08 2020-08-14 Fermentalg OPTIMIZED PROCESS FOR INDUSTRIAL EXPLOITATION OF SINGLE-CELLS RED ALGAE
BE1027115B1 (en) * 2019-03-12 2020-10-12 B Blue Nutraceuticals S A LIQUID COMPOSITION INCLUDING PHYCOCYANIN
EP3939437A1 (en) * 2020-07-17 2022-01-19 GNT Group B.V. A composition comprising spirulina extract
AU2022334888A1 (en) * 2021-08-24 2024-03-07 The Williamson Group, Llc Improved stabilization of phycocyanins in acidic compositions
WO2023085922A1 (en) * 2021-11-15 2023-05-19 Ful Foods B.V. A method of producing a composition, an water soluble extract composition and a non-water soluble extract composition, each composition being a phycobiliprotein containing micro-organism-based composition
WO2023112407A1 (en) * 2021-12-13 2023-06-22 Dic株式会社 Cyanidioschyzon merolae which can be heterotrophically proliferated in dark place, and use thereof
FR3130842A1 (en) 2021-12-22 2023-06-23 CarbonWorks METHOD FOR CAPTURING PHYTOTOXINS IN A BIOLOGICAL REACTOR

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS626691A (en) * 1985-07-02 1987-01-13 Dainippon Ink & Chem Inc Phycocyanin pigment a from blue-green alga, production and use thereof
JP3256318B2 (en) * 1993-03-19 2002-02-12 学校法人桐蔭学園 Separation method of cyanobacteria-containing pigment
JP4048420B2 (en) * 2002-06-26 2008-02-20 大日本インキ化学工業株式会社 Purification method of phycocyanin dye solution
FR2863615A1 (en) * 2003-12-12 2005-06-17 Minard Florence Stabilizing phycobiliproteins against light, useful in cosmetic, dermatological, neutraceutical and pharmaceutical compositions, by adding ascorbic acid to algal extracts
CN101343310B (en) * 2008-07-16 2012-01-25 广东海洋大学 Method for preparing high purity phycobiliprotein with primary column chromatography
CN102329381B (en) * 2011-09-13 2013-08-21 暨南大学 Method for simultaneously separating high-purity phycocyanin and allophycocyanin and application thereof
MX2016008084A (en) * 2013-12-18 2016-10-12 Basf Se Stabilized phycocyanin for blue color.
WO2016099261A1 (en) * 2014-12-16 2016-06-23 Rijksuniversiteit Groningen Natural blue photopigments, methods for producing them and to uses thereof as colorant.
FR3041653B1 (en) * 2015-09-25 2017-12-29 Fermentalg PROCESS FOR CULTIVATION OF ALGAE, PARTICULARLY UNICELLULAR RED ALGAE (ARUS)
JP6681065B2 (en) 2016-01-14 2020-04-15 学校法人明治大学 Food, heat treatment method of food, phycocyanin-containing material manufacturing method, and food manufacturing method

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