Purification of Phycobiliproteins

Abstract

The present invention relates to a novel method for purifying phycobiliproteins, in particular phycobiliproteins resistant to acidic pH, the obtained phycobiliproteins and the use thereof.

Description

Field of the Invention

[0001] The present invention relates to a novel method for purifying phycobiliproteins, in particular phycobiliproteins resistant to acidic pH, the obtained phycobiliproteins and the use thereof.

[0002] Current status of the field

[0003] The purification of phycobiliproteins of Galdieria sulphuraria, in particular C-phycocyanin (C-PC), is much more complicated than that of phycobiliproteins of Arthrospira platensis (Spirulina) or other cyanobacteria. This is partly due to the composition of the cell wall of Galdieria sulphuraria, which requires mechanical action to rupture (Sorensen et al., 2013). Mechanical lysis leads to the formation of micelles, which are only partially removed by ultracentrifugation. The presence of chlorophyll a and dissolved carotenoids in these micelles contributes to the increase in absorbance values ​​at 280 nm (protein-specific UV absorbance), which may explain the low purity level of the C-PC crude extract compared to the Spirulina crude extract (Sorensen et al., 2013). Therefore, the purity level of the crude extract can be increased by removing micelles and soluble proteins other than phycobiliproteins.

[0004] The purification of phycobiliproteins extracted from Cyanidioschyzon merolae, Cyanidium caldarium, Galdieria sulphuraria and Spirulina by precipitation with ammonium sulfate has been described in the literature (WO 2016 / 099261; Eisele et al., 2000; Kao et al., Moon et al., 1975; 2015; Cruz de Jesús et al., 2006), but it is very difficult to apply on an industrial scale because it requires a lot of ammonium sulfate, which poses a major problem for the reprocessing of the ammonium sulfate and the supernatant.

[0005] Other purification methods are described which make the implementation of chromatographic methods very expensive in order to obtain a certain purity level.

[0006] Therefore, the present invention relates to a method for purifying phycobiliproteins produced by culturing phycobiliprotein-producing microorganisms in a bioreactor, which method is easy to carry out and economically suitable for implementation on an industrial scale.

[0007] Furthermore, phycobiliproteins, in particular phycocyanins, are mixtures of c-phycocyanin and allophycocyanin. Known purification methods do not allow their separation in an industrially controllable manner. Purification by precipitation with ammonium sulfate entrains both proteins in an uncontrolled manner, making it difficult to obtain pigments with stable properties. This precipitation method also results in substantial extraction yield losses (Cruz de Jesús et al., 2006).

[0008] Therefore, the present invention also relates to the preparation of purified phycobiliproteins, in particular the preparation of purified phycocyanin essentially comprising c-phycocyanin or essentially comprising allophycocyanin, in particular the preparation of phycobiliproteins resistant to acidic pH in a controlled composition in the phycocyanin, the resistance to acidic pH not requiring the addition of stabilizers such as ascorbic acid (WO 2005 / 065697) or polyphenols (WO 2015 / 090697).

[0009] Invention Disclosure

[0010] Therefore, the present invention relates to a method for purifying acid pH-resistant phycobiliproteins from a crude extract of acid pH-resistant phycobiliproteins, characterized in that it comprises the following steps

[0011] a) adjusting the pH of the crude extract of the acidic pH-resistant phycobiliprotein to a pH below 6 so that organic matter other than the acidic pH-resistant phycobiliprotein is precipitated,

[0012] b) recovering the supernatant containing the acidic pH resistant phycobiliprotein, and

[0013] c) isolating the acidic pH-resistant phycobiliprotein from the supernatant.

[0014] The present invention also relates to the acidic pH-resistant phycobiliprotein obtained by the method, in particular an acidic pH-resistant phycocyanin comprising a mixture of c-phycocyanin and allophycocyanin, more particularly an acidic pH-resistant phycocyanin having a molar ratio of c-phycocyanin to allophycocyanin of at least 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 .Increase in purity index of crude extracts as a function of pH of lysates from fresh cells.

[0017] Figure 2 : The concentrations of C-PC and APC were measured in different crude extracts obtained by centrifugation of fresh cell lysates at different pH levels. The APC concentration (mg / ml) is shown in grey and the C-PC concentration (mg / ml) is shown in black.

[0018] Figure 3: The concentrations of C-PC and APC at different pH levels were measured in micelles obtained by centrifugation of fresh cell lysates. APC concentration (mg / g DM) is shown in grey and C-PC concentration (mg / g DM) is shown in black.

[0019] Figure 4 . Increase in the purity index of crude extracts as a function of pH of lysates from lyophilized and rehydrated cells.

[0020] Figure 5 : The concentration of C-PC and APC at different pH in different crude extracts obtained by centrifuging lysates of freeze-dried and rehydrated cells was measured. APC concentration (mg / ml) is shown in gray and C-PC concentration (mg / ml) is shown in black.

[0021] Figure 6 Purification and concentration of C-PC by tangential filtration The crude extract previously purified by precipitation at acidic pH was filtered using a hollow fiber system.

[0022] Figure 7 .Increase in C-PC concentration in the retentate during hollow fiber filtration. DETAILED DESCRIPTION OF THE INVENTION

[0024] Therefore, the present invention relates to a method for purifying an acidic pH-resistant phycobiliprotein from a crude extract of the acidic pH-resistant phycobiliprotein.

[0025] Crude extracts of phycobiliproteins are usually obtained from cells of microorganisms grown industrially in high-capacity bioreactors, preferably in a manner to obtain a fermentation broth with a high density of phycobiliprotein-producing microorganisms (high density is usually defined as more than 50 g dry matter (DM) per liter of fermentation broth, preferably more than 100 g / L). These cultivation methods are known to those skilled in the art and can be carried out autotrophically, heterotrophically or mixotrophically, in particular as described in applications WO 2017 / 050917, WO 2017 / 050918 and PCT / EP2016 / 079325 filed on November 30, 2016. The phycobiliproteins produced by the cultivated microorganisms must be released after cell lysis. In fact, microbial cells contain large amounts of phycobiliproteins (Moon et al., 2015, Sorensen et al., 2013, Eriksen 2008). Therefore, the implementation of the method according to the invention first requires the preparation of an aqueous extract from the fermentation broth.

[0026] The aqueous extract can be prepared directly from the fermentation broth, as the aqueous extract can be recovered from the reactor at the end of the fermentation, which may require the addition of an appropriate amount of water.

[0027] It can be prepared from fresh cells isolated from a fermentation broth by any isolation method known to those skilled in the art. It can also be prepared from cells that have been lyophilized or dried for storage.

[0028] According to a preferred embodiment of the present invention, the aqueous extract is prepared from fresh cells separated from the fermentation broth after cultivation.

[0029] Cell lysis can be performed by any cell lysis means known to the skilled person.Cell lysis can be performed while the cells are suspended in water, fermentation broth or rehydrated suspension.

[0030] According to a preferred embodiment of the present invention, cell lysis is performed on cells separated from the fermentation broth before the cells are resuspended.

[0031] Preferably, the aqueous extract is obtained from a suspension comprising lysed cells by separation of solids, by any separation means known to the skilled person to remove solid residues of a cell lysate, in particular filtration.

[0032] This results in an aqueous extract called "crude extract of phycobiliproteins" or "crude extract" which, besides the desired phycobiliproteins, in particular phycobiliproteins resistant to acidic pH, also contains other organic matter such as micelles and other water-soluble proteins.

[0033] Crude extracts of phycobiliproteins can be prepared from freshly lysed cells (directly in the fermentation broth or after separation of the fermentation broth) or from lyophilized or dried cells (wherein cell lysis occurs before or after lyophilization or drying).

[0034] According to a preferred embodiment of the present invention, the crude extract is prepared from fresh cells.

[0035] The purification method according to the present invention involves separating phycobiliproteins, particularly phycobiliproteins resistant to acidic pH, from other organic substances such as micelles and other water-soluble proteins.

[0036] Microorganisms cultivated to produce phycobiliproteins are well known to the person skilled in the art, in particular selected from the group of the Cyanophyceae such as Arthrospira platensis (Spirulina spp.), Spirulina maxima, Synechococcus elongatus, or from the group of the Cyanidiophyceae such as Galdieria sulphuraria, Cyanidium caldiarium, Cyanidioschyzon merolae.

[0037] Preferably, the phycobiliprotein is an acid pH resistant phycocyanin. Acid pH resistant phycobiliproteins or acid pH resistant phycocyanins are defined as phycobiliproteins that resist precipitation at acidic pH. According to the present invention, acidic pH means a pH below 7, advantageously 6 or lower. Advantageously, the acid pH resistant phycobiliproteins do not precipitate in aqueous solution at pH levels below 6. They may also be described as tolerant or stable at acidic pH.

[0038] Of course, the purified phycobiliproteins according to the invention will be more or less stable depending on the pH considered. Some will be stable in the pH range of about 6, while others will be stable at pH values ​​well below 6. Therefore, acidic pH resistant phycobiliproteins are also defined as mixtures of phycobiliproteins, the majority of which will not precipitate at a pH below 7, advantageously below 6 or lower.

[0039] Advantageously, the present invention relates to phycobiliproteins that are stable at a pH of less than 5, preferably less than or equal to 4, more preferably in the range of 4 to 2, even more preferably less than or equal to 3.5.

[0040] Such phycocyanins resistant to acidic pH are known to the person skilled in the art and are more particularly described in application WO 2016 / 099261 or in application WO 2017 / 050918. In particular, these are phycocyanins produced by strains of microalgae of the genus Cyanidioschyzon, Cyanidium or Galdieria, in particular chosen from the species Cyanidioschyzon merolae 10D, Cyanidioschyzonmerolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, in particular the strains Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merola.

[0041] These phycocyanins are a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC).

[0042] Advantageously, the apoprotein of C-PC comprises a protein of SEQ ID NO 1 or SEQ ID NO 2 or a variant thereof. In particular, the apoprotein of the α subunit of C-PC comprises a protein of SEQ ID NO 1, and the apoprotein of the β subunit of C-PC comprises a protein of SEQ ID NO 2 or a variant thereof.

[0043] SEQ ID1:

[0044] SEQ ID2:

[0045] Also advantageously, the α subunit of the APC comprises SEQ ID NO 3 or a variant thereof, and the apoprotein of the β subunit of the APC comprises SEQ ID NO 4 or a variant thereof.

[0046] SEQ ID3:

[0047] SEQ ID 4:

[0048] The apoproteins of C-PC and APC from the same phycocyanin source usually have different isoelectric points. By lowering the pH, it will be possible to at least partially separate C-PC from APC.

[0049] Indeed, the inventors found that the more the pH of the crude extract was adjusted downward, the purer the C-PC obtained.

[0050] Advantageously, when the phycobiliprotein is an acidic pH resistant phycocyanin, lowering the pH below the isoelectric point of APC results in a phycocyanin comprising a C-PC / APC mixture in a molar ratio of at least 5, preferably at least 10, more preferably at least 15.

[0051] Preferably, the pH of the crude extract in step a) is adjusted to a pH below 5. It is thereby possible to obtain an acidic pH-resistant phycocyanin containing less than 5 mol % APC, preferably less than 1 %, more preferably less than 0.1 % APC, the percentage being expressed relative to the sum of APC and C-PC.

[0052] In step a), the pH adjustment is carried out by adding a strong or weak mineral or organic acid in solid form or in solution, the amount of acid added being determined by the pH of the crude 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 may be made in particular of hydrochloric acid and phosphoric acid. Among the organic acids well known to those skilled in the art, mention may be made in particular of acetic acid, citric acid, tartaric acid, lactic acid, preferably citric acid. Mention may also be made of acidic polyphenols such as rosmarinic acid, tannic acid, digallic acid, quercitannicacid, gallotannic acid, acidic tannins such as quercetin, ellagitannin, castaline, casuaritin, grandinin, punicaligin, punicarin, roburin A, tellimagrandin II, terflavin B, vescaligin, pendunculagin, casuarin, castlin, vescalin, preferably tannic acid. Preferably, the acid used is an acid approved for use in foods, in particular phosphoric acid, citric acid or tannic acid.

[0053] For step b) of recovering the supernatant containing the phycobiliproteins resistant to acidic pH, any separation method known to the skilled person can be used, in particular by tangential filtration on ceramic membranes or organic membranes such as polyethersulfone hollow fibers. The cut-off values ​​of these filters can be selected to separate molecules with a higher or lower molecular weight than the targeted phycobiliprotein.

[0054] According to a particular embodiment of the invention, the separation in step b) is performed by tangential filtration. This step concentrates and removes some of the proteins other than phycobiliproteins, thereby increasing the purity level of the final product.

[0055] Step c) of drying / dehydrating the acidic pH resistant phycobiliproteins from the supernatant is carried out by any method of removing the solvent, in this case water, for example by evaporation at atmospheric pressure or under vacuum. Mention may in particular be made of nebulization, freeze drying, zeodratation, infrared drying or refractive window drying.

[0056] In the case of evaporation by heating, the technician should be careful not to use too high a temperature, which may cause denaturation of the phycobiliproteins.

[0057] After recovering the supernatant in step b), it is possible to recycle the phycobiliproteins contained in the precipitate. To this end, the remaining phycobiliproteins are dissolved in an acidic aqueous solution at a pH of about 6 or less, at which the impurities remain insoluble, while the phycobiliproteins are soluble.

[0058] These residual phycobiliproteins are then separated from the impurities and isolated by repeating steps b) and c) of the method. This is an iterative process that can be repeated as many times as desired. When the conditions used in the method according to the invention allow for preferential purification of C-PC, the residual phycobiliproteins are a C-PC / APC mixture enriched in APC.

[0059] By recycling the precipitate, phycobiliproteins are obtained which comprise a C-PC / APC mixture with a molar ratio of less than 5, in particular less than 4, advantageously ranging from 3 to 0.1.

[0060] By repeating steps a) to c) of the above method, it is possible by an iterative process to deplete the precipitate of C-PC, which can be added to the previously obtained fractions to enrich their content, and to enrich the residual mixture of phycobiliproteins in APC, wherein the APC / C-PC ratio is at least 5, preferably at least 10, more preferably at least 15.

[0061] In this way it is possible to obtain APC mixtures containing less than 5 mol % CPC, preferably less than 1 %, more preferably less than 0.1 % CPC, the percentage being expressed relative to the sum of APC and C-PC.

[0062] The APCs separated from the precipitate can then be further purified by preparative chromatography techniques well known to the skilled person for the production of allophycocyanin which can be used, for example, in the field of medical imaging due to these fluorescent properties.

[0063] The present invention also relates to acidic pH-resistant phycobiliproteins, in particular phycocyanins, obtainable by the purification method.

[0064] The present invention also relates to purified acidic pH resistant phycocyanin comprising a C-PC / APC mixture having a molar ratio of at least 2.

[0065] In particular, the present invention relates to acidic pH resistant phycocyanin comprising at least 95 mol % C-PC and less than 5 mol % APC, preferably at least 99 mol % C-PC and less than 1 mol % APC, the percentages being expressed relative to the sum of APC and C-PC.

[0066] These C-PCs are known to those skilled in the art, and are in particular the C-PCs described above, in particular those C-PCs, i.e., the α subunit of C-PC comprises a protein of SEQ ID NO 1, and the apoprotein of the β subunit of C-PC comprises a protein of SEQ ID 2 or a variant thereof.

[0067] Advantageously, the variant according to the invention has at least 83% sequence identity with the alpha subunit of C-PC and at least 82% sequence identity with the beta subunit of C-PC.

[0068] Preferably, the variant according to the invention has at least 90% identity with the alpha (SEQ ID NO 1) and beta (SEQ ID NO 2) subunits.

[0069] The present invention also relates to purified phycocyanin enriched in APC obtainable by the method of the present invention.

[0070] In particular, the present invention relates to purified phycocyanin comprising an APC-enriched mixture having a C-PC / APC molar ratio of less than 5, in particular 4, advantageously in the range of 3 to 0.1.

[0071] According to a particular embodiment of the present invention, the APC-rich mixture has an APC / C-PC ratio of at least 5, preferably at least 10, more preferably at least 15.

[0072] According to a more specific embodiment of the present invention, the phycocyanin consists essentially of APC, wherein at least 95 mol % APC and less than 5 mol % C-PC, preferably at least 99 mol % APC and less than 1 mol % C-PC, the percentages being expressed relative to the sum of APC and C-PC.

[0073] These APCs are known to the skilled person and are in particular the APCs described above, in particular those APCs whose α subunit comprises SEQ ID NO 3 or a variant thereof and whose apoprotein of the β subunit comprises SEQ ID NO 4 or a variant thereof.

[0074] Advantageously, the variant according to the invention has at least 83% sequence identity with the alpha subunit of APC and at least 82% sequence identity with the beta subunit of APC.

[0075] The skilled person knows how to measure the identity of protein sequences using conventional methods at his or her disposal, in particular the BLASTP program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0076] Similarly, the skilled person knows how to identify variants of the sequence and how to verify that they retain the same structural properties by a simple stability test at acidic pH, for example by a test such as the one shown in Example 3 of application WO 2017 / 050918.

[0077] It is known to the skilled person that a polypeptide can be modified by substitution, insertion and / or deletion of at least one amino acid without substantially modifying its function.

[0078] For example, substitution of an amino acid at a given position by another chemically equivalent amino acid is a known example of a sequence change that does not substantially affect the properties of the protein.

[0079] These "conservative" substitutions can be defined as exchanges within the following amino acid groups:

[0080] -Ala, Ser, Thr, Pro, Gly

[0081] -Asp, Asn, Glu, Gln

[0082] -His, Arg, Lys

[0083] -Met, Leu, Ile, Val, Cys and

[0084] -Phe, Tyr, Trp

[0085] Thus, variants of the apoproteins of phycocyanin and / or allophycocyanin according to the invention may comprise from 1 to 30 amino acids that differ in number compared to the corresponding so-called reference sequence, in particular with regard to the α and / or β subunit of phycocyanin, provided that the variants obtained retain the properties of the reference protein and the above-mentioned homology / identity percentages.

[0086] More precisely, according to the present invention,

[0087] - variants (derived from substitutions, insertions and / or deletions) of the apoprotein of the alpha subunit of phycocyanin that can be used in the acidic composition according to the invention, they may contain from 1 to 27 amino acids that differ from the corresponding so-called reference sequence, provided that the variant obtained retains the properties of the reference protein and the above-mentioned percentages of identity;

[0088] - variants (derived from substitutions, insertions and / or deletions) of the apoprotein of the β-subunit of phycocyanin that can be used in the acidic composition according to the invention, they may contain from 1 to 30 amino acids that differ from the corresponding so-called reference sequence, provided that the variant obtained retains the properties of the reference protein and the percentages of identity stated above;

[0089] - variants (derived from substitutions, insertions and / or deletions) of the apoprotein of the alpha subunit of allophycocyanin that can be used in the acidic composition according to the invention, they may contain from 1 to 24 amino acids that differ from the corresponding so-called reference sequence, provided that the variant obtained retains the properties of the reference protein and the percentages of identity stated above;

[0090] - As for the variants (derived from substitutions, insertions and / or deletions) of the apoprotein of the β subunit of allophycocyanin that can be used in the acidic composition according to the invention, they may contain from 1 to 20 amino acids that differ from the corresponding so-called reference sequence, provided that the variant obtained retains the properties of the reference protein and the percentages of identity stated above.

[0091] According to the invention, very particularly, and irrespective of the reference sequence under consideration (alpha and / or beta subunit of phycocyanin and / or alpha and / or beta subunit of allophycocyanin), the variants of said subunits may advantageously comprise from 1 to 15 different amino acids, preferably from 1 to 10 different amino acids, in particular 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 different amino acids compared to the corresponding so-called reference sequence, provided that the variant obtained retains the properties of the reference protein and the identity percentages stated above.

[0092] Preferably, the present invention relates to C-PC, wherein the subunit α protein consists of the protein of SEQ ID 1, and the β subunit protein consists of the protein of SEQ ID 2.

[0093] According to another preferred embodiment, the present invention relates to APC, whose subunit α protein consists of the protein of SEQ ID 3, and whose subunit β protein consists of the protein of SEQ ID 4.

[0094] Phycobiliproteins are natural dyes mainly used for food coloring.

[0095] The present invention also relates to the use of the acid pH resistant phycobiliprotein obtained by the process according to the invention, in particular the acid pH resistant phycocyanin as defined above, as a dye in food products.

[0096] The present invention also relates to a composition, in particular a food composition, comprising an acid pH resistant phycobiliprotein obtained by the process according to the present invention, in particular an acid pH resistant phycocyanin as defined above.

[0097] Such uses and such compositions are known to those skilled in the art.

[0098] Preferably, the food product or food composition is an acidic composition as defined in application WO 2017 / 050918.

[0099] According to the present invention, an acidic composition is defined as any composition comprising an inorganic acid or an organic acid and phycocyanin. This composition may be a liquid, a fluid or a mucus or a solid having an acidic pH, and into which phycocyanin resistant to acidic pH is incorporated.

[0100] For aqueous liquid compositions, the pH is measured in a conventional manner. For non-aqueous liquid compositions or for pasty or solid compositions, the pH is measured after the composition has been dissolved in water sufficient to dissolve the soluble compounds it contains, including the inorganic or organic acid and the phycocyanin.

[0101] Advantageously, the composition according to the invention is an aqueous liquid composition, optionally in the form of a gel composition or a paste composition or a solid composition intended to be dissolved in an aqueous solution, or a solid or paste composition comprising water. According to another advantageous embodiment of the invention, the acidic composition is a paste or solid composition intended to be used and / or stored in a humid environment.

[0102] The inorganic or organic acids that can be used in the composition according to the invention are well known to those skilled in the art. Among the inorganic acids, mention may be made in particular of carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulfonic acid and nitric acid. Among the organic acids, mention may be made in particular of citric acid, lactic acid, malic acid, tartaric acid, succinic acid, advantageously citric acid.

[0103] According to the present invention, an acidic food composition is taken to mean any composition falling within the above definition intended for human or animal ingestion. In the sense of the present invention, nutraceutical acidic compositions must be considered to fall within the definition of an acidic food composition.

[0104] Acidic food compositions according to the present invention are well known to those skilled in the art. They may include a vehicle, which may include a structural composition associated with an active compound, which is identified because of its nutritional contribution or its properties that are beneficial to human or animal health. Acidic food compositions according to the present invention may also include food additives such as thickeners (texturizing agent), flavoring agents, preservatives, all of which are well known to those skilled in the art. The vehicle may include water and / or protein and / or fat and / or fiber and / or sugar. The composition of the vehicle may only have structural properties, but it is usually known for its nutritional contribution.

[0105] The acidic food composition according to the present invention may be ready-to-use or in the form of a food additive to be added to a solid, paste or liquid preparation to prepare a food that can be ingested.

[0106] For food compositions, the acid should preferably be selected from the list of approved dietary acidulants, in particular carbonic acid, phosphoric acid, citric acid, malic acid, tartaric acid and lactic acid, more preferably citric acid.

[0107] As regards the acidic compositions other than the food compositions according to the invention, they may in particular be pharmaceutical, veterinary or cosmetic and may further comprise any additives and / or active agents known and used in compositions of this type.

[0108] In the solid composition, liquid composition or pasty composition according to the invention, phycocyanin can be incorporated, for example, in the form of a powder. The acidic composition, in particular the acidic food composition, can then be in any known conventional form such as creams, gels, foams, pastes, etc. In particular for solid food compositions, mention may be made of cakes or biscuits, dry cooked foods, powders to be diluted, solid or "jelly" gel-like compositions, foams, etc.

[0109] According to the invention, the liquid 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, in particular for ingestion or to be added to solid food for its preparation or for ingestion, such as a liquid coating concentrate or a "topping" composition that can be placed on a cake to color it. Among these concentrated compositions, mention may be made of syrups, optionally containing ethanol.

[0110] The liquid acidic composition according to the invention may have a variable viscosity and may or may not include additives such as viscosity agents, gelling agents and other structuring additives known to those skilled in the art and commonly used in the preparation of liquid food compositions.

[0111] According to a particular embodiment of the invention, the liquid food composition may be an acidic beverage, optionally a carbonated beverage. These acidic beverages include sodas, fruit juices, sports drinks, sports drinks, fitness drinks, recovery drinks, etc. The compositions of these beverages are well known to those skilled in the art and may include, in particular, sugars, inorganic salts, food additives, dissolved gases, etc. The beverage according to the invention is a common acidic beverage in which the dyes commonly used have been replaced in whole or in part by the phycocyanin according to the invention.

[0112] According to the present invention, the content of phycocyanin in the composition according to the present invention may be based on the practice of skilled artisans.

[0113] For example, when phycocyanin is used to color an acidic composition, the amount of phycocyanin in the composition may be based on the coloring practice of a skilled artisan.

[0114] In the liquid acidic composition as defined in the present invention, the phycocyanin content may be between 2.5 mg / L and 2500 mg / L, preferably between 25 mg / L and 300 mg / L.

[0115] In a ready-to-use liquid beverage composition, the phycocyanin content may typically be between 25 mg / l and 300 mg / L, preferably between 50 mg / L and 100 mg / L.

[0116] In concentrated liquid compositions to be diluted for use, such as syrups, the phycocyanin content may generally be between 250 mg / l and 2500 mg / l, preferably between 500 mg / L and 1000 mg / L.

[0117] In solid compositions, the phycocyanin content may generally be between 0.01 mg / g and 10 mg / g, preferably between 0.1 mg / g and 5.0 mg / g, very preferably between 0.25 mg / g and 2.5 mg / g. Example

[0118] Example 1. Purification by acid precipitation of fresh cells

[0119] The fermentation broth of Galdieria sulphuraria cells, centrifuged and washed with an equal volume of water, was mechanically ground to release the phycobiliproteins in the aqueous phase at pH 6. The ground material was acidified by adding citric acid in 0.5 pH unit steps. At each level, a sample of the mixture was taken and then centrifuged at 11,000 g for 10 min. The supernatant containing the phycobiliproteins was collected and the purity index was measured by measuring the ratio of the absorbance at 618 nm to the absorbance at 280 nm using a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).

[0120] Obviously, the lower the pH, the higher the purity index ( Figure 1 The increase in the purity index reflects the reduction of contaminating proteins in the supernatant, while C-PC remains mainly in the supernatant. Due to its resistance to acidic pH, the C-PC content in the supernatant is essentially not lost ( Figure 2 ). Surprisingly, at pH values ​​below 5, allophycocyanin (APC) completely disappeared from the supernatant and eventually precipitated in the pellet along with other precipitated proteins and cell debris ( Figure 3 ). This precipitate also contains C-PC and APC, of ​​which APC has a higher content ( Figure 3 ).

[0121] Acidification also results in better separation of liquid and solid phases, as well as precipitates of cell debris and proteins which are more compact and easier to separate from the aqueous phase.

[0122] Example 2. Purification by acid precipitation of lyophilized and rehydrated cells

[0123] The fermentation broth of Galdieria sulphuraria cells, centrifuged and rinsed with an equal volume of water, was mechanically ground to release the phycobiliproteins in the aqueous phase at pH 6. The ground material was then lyophilized. The lyophilized dry material was suspended in a volume of water equal to the initial volume of the fermentation broth and then acidified in 0.5 pH unit steps by adding citric acid. At each level, a sample of the mixture was taken and then centrifuged at 11,000 g for 10 min. The supernatant containing the phycobiliproteins was collected and the purity index was measured by measuring the ratio of the absorbance at 618 nm to the absorbance at 280 nm with a spectrophotometer (Amersham Biosciences Ultra Spec 2100Pro).

[0124] As previously described in Example 1, we can see that an increase in the purity index correlates with a decrease in pH ( Figure 4 In this case, too, acidification also resulted in a better separation of the liquid and solid phases, as well as a precipitate of cell debris and proteins that was more compact and easier to separate from the aqueous phase. Similar to what was observed in Example 1, it was found that at pH values ​​below 5, APC was in the precipitate and not in the aqueous phase ( Figure 5 ). For pH values ​​between 6 and 5, the amount of APC in the supernatant decreased as the pH decreased.

[0125] Example 3. Purification and concentration of C-PC by tangential filtration

[0126] The crude extract after acid precipitation and centrifugation was filtered on a hollow fiber tangential filtration module. Some of the proteins other than C-PC were filtered through this filter, thereby increasing the purity index ( Figure 6 ). The purity index that can be achieved by this method is close to the values ​​usually obtained by more complex methods involving two-phase extraction or ammonium sulfate precipitation or even chromatographic methods (Soresen et al., 2013; Cruz de Jesús et al., 2006). In parallel with the purification process, this filtration step removes water from the C-PC extract ( Figure 7 ), and promote subsequent product drying.

[0127] References

[0128] -Cruz de Jesús et al.,“Methods for Extraction,Isolation andPurification of C-phycocyanin:50years of Research in Review”(2016)Int J FoodNutr Sci 3(3):1-10.

[0129] -Eisele et al.,“Studies on C-phycocyanin from Cyanidium caldarium,aeukaryote at the extremes of habitat”Biochemica et Biophysica Acta1456(2000)1456,2-3,99-107.

[0130] -Eriksen NT.“Production of phycocyanin-a pigment with applications inbiology,biotechnology,foods and medicine”Appl Microbiol Biotechnol.2008Aug;80(1):1-14.

[0131] -Kao et al.,“Physical-chemical properties of C-phycocyanin isolatedfrom an acido-thermophilic eukaryote,Cyanidium caldarium”Biochem.J.(1975)147,63-70

[0132] -Myounghoon et al.“Isolation and Characterization of ThermostablePhycocyanin from Galdieria Sulphuraria,”Korean journal of chemicalengineering,31(2014):1–6.

[0133] - et al. "Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria" J Sci Food Agric.2013Sep;93(12):2933-8

[0134] -WO 2005 / 065697,WO 2015 / 090697,WO 2016 / 099261,WO 2017 / 050917,WO 2017 / 050918 et PCT / EP2016 / 079325 filed on 30November 2016 Sequence Listing <110> Fehrmantag <120> Purification of Phycobiliproteins <130> P100090PC01 <150> FR1752674 <151> 2017-03-30 <160> 4 <170> BiSSAP 1.3.6 <210> 1 <211> 162 <212> PRT <213> Galderia sulphuraria <400> 1 Met Lys Thr Pro Ile Thr Glu Ala Ile Ala Ala Ala Asp Asn Gln Gly 1 5 10 15 Arg Phe Leu Ser Asn Thr Glu Leu Gln Ala Val Asn Gly Arg Tyr Gln 20 25 30 Arg Ala Ala Ala Ser Leu Glu Ala Ala Arg Ser Leu Thr Ser Asn Ala 35 40 45 Gln Arg Leu Ile Asn Gly Ala Ala Gln Ala Val Tyr Ser Lys Phe Pro 50 55 60 Tyr Thr Ser Gln Met Pro Gly Pro Gln Tyr Ala Ser Ser Ala Val Gly 65 70 75 80 Lys Ala Lys Cys Ala Arg Asp Ile Gly Tyr Tyr Leu Arg Met Val Thr 85 90 95 Tyr Cys Leu Val Val Gly Gly Thr Gly Pro Met Asp Glu Tyr Leu Ile 100 105 110 Ala Gly Leu Glu Glu Ile Asn Arg Thr Phe Asp Leu Ser Pro Ser Trp 115 120 125 Tyr Val Glu Ala Leu Asn Tyr Val Lys Ser Asn His Gly Leu Ser Gly 130 135 140 Gln Ala Ala Asn Glu Ala Asn Thr Tyr Ile Asp Tyr Ala Ile Asn Ala 145 150 155 160 Leu Ser <210> 2 <211> 172 <212> PRT <213> Galderia sulphuraria <400> 2 Met Leu Asp Ala Phe Ala Lys Val Val Ala Gln Ala Asp Ala Arg Gly 1 5 10 15 Glu Phe Leu Ser Asn Thr Gln Leu Asp Ala Leu Ser Lys Met Val Ser 20 25 30 Glu Gly Asn Lys Arg Leu Asp Val Val Asn Arg Ile Thr Ser Asn Ala 35 40 45 Ser Ala Ile Val Thr Asn Ala Ala Arg Ala Leu Phe Ser Glu Gln Pro 50 55 60 Gln Leu Ile Gln Pro Gly Gly Asn Ala Tyr Thr Asn Arg Arg Met Ala 65 70 75 80 Ala Cys Leu Arg Asp Met Glu Ile Ile Leu Arg Tyr Val Ser Tyr Ala 85 90 95 Ile Ile Ala Gly Asp Ser Ser Val Leu Asp Asp Arg Cys Leu Asn Gly 100 105 110 Leu Arg Glu Thr Tyr Gln Ala Leu Gly Val Pro Gly Ala Ser Val Ala 115 120 125 Val Gly Val Glu Lys Met Lys Asp Ser Ala Ile Ala Ile Ala Asn Asp 130 135 140 Pro Ser Gly Ile Thr Thr Gly Asp Cys Ser Ala Leu Met Ala Glu Val 145 150 155 160 Gly Thr Tyr Phe Asp Arg Ala Ala Thr Ala Val Gln 165 170 <210> 3 <211> 163 <212> PRT <213> Galderia sulphuraria <400> 3 Met Ser Leu Ile Ser Gln Ile Ile Asn Thr Ala Asp Glu Glu Leu Arg 1 5 10 15 Tyr Pro Asn Gly Gly Glu Leu Ser Thr Leu Ile Tyr Phe Phe Asn Thr 20 25 30 Ala Asn Thr Arg Ile Asn Ile Ile Asn Lys Leu Lys Glu Arg Glu Lys 35 40 45 Asp Ile Ile Gln Asn Ala Ser Lys Lys Leu Phe Gln Leu His Pro Glu 50 55 60 Tyr Val Ser Ser Gly Gly Asn Ala Ser Gly Pro Lys Gln Arg Ala Leu 65 70 75 80 Cys Leu Arg Asp Tyr Gly Trp Tyr Leu Arg Leu Val Thr Tyr Gly Ile 85 90 95 Leu Ala Gly Asp Ile Thr Pro Ile Glu Lys Ile Gly Ile Ile Gly Val 100 105 110 Lys Asp Met Tyr Asn Ser Leu Gly Val Pro Ile Ile Gly Met Tyr Asp 115 120 125 Ala Ile Lys Cys Leu Lys Glu Ala Ser Ile Asn Ile Phe Glu Leu Ser 130 135 140 Glu Glu Lys Asp Leu Ile Ile Pro Tyr Phe Asp Tyr Leu Ser Asn Ala 145 150 155 160 Ile Leu Ser <210> 4 <211> 253 <212> PRT <213> Galderia sulphuraria <400> 4 Met Ser Ile Val Thr Lys Ser Ile Val Asn Ala Asp Ala Glu Ala Arg 1 5 10 15 Tyr Leu Ser Pro Gly Glu Leu Asp Arg Ile Lys Ser Phe Val Leu Ser 20 25 30 Gly Gln Arg Arg Leu Arg Ile Ala Gln Ile Leu Thr Asp Asn Arg Glu 35 40 45 Arg Ile Val Lys Gln Ala Gly Gln Gln Leu Phe Gln Gln Arg Pro Asp 50 55 60 Ile Val Ser Pro Gly Gly Asn Ala Tyr Gly Glu Glu Met Thr Ala Thr 65 70 75 80 Cys Leu Arg Asp Leu Asp Tyr Tyr Leu Arg Leu Val Thr Tyr Gly Val 85 90 95 Val Ala Gly Asp Ile Ser Pro Ile Glu Glu Ile Gly Leu Glu Asp Phe 100 105 110 Met Gln Asp Ala Ile Thr Ala Val Ile Asn Thr Ala Asp Val Gln Gly 115 120 125 Lys Tyr Leu Asp Asn Ser Ser Ile Glu Lys Leu Lys Gly Tyr Phe Gln 130 135 140 Thr Gly Glu Leu Arg Val Arg Ala Ala Ala Thr Ile Ala Ala Asn Ala 145 150 155 160 Ala Gly Ile Ile Lys Asp Ala Val Ala Lys Ser Leu Leu Tyr Ser Asp 165 170 175 Ile Thr Arg Pro Gly Gly Asn Met Tyr Thr Thr Arg Arg Tyr Ala Ala 180 185 190 Cys Ile Arg Asp Leu Asp Tyr Tyr Leu Arg Tyr Ala Thr Tyr Ser Met 195 200 205 Leu Ala Gly Asp Pro Ser Ile Leu Asp Glu Arg Val Leu Asn Gly Leu 210 215 220 Lys Glu Thr Tyr Asn Ser Leu Gly Val Pro Ile Gly Ala Thr Ile Gln 225 230 235 240 Ser Ile Gln Ala Met Lys Glu Val Thr Ser Ser Leu Val 245 250

Claims

1. A method for purifying an acidic pH resistant phycocyanin from a crude extract of an acidic pH resistant phycocyanin from cells of a phycocyanin producing microorganism, the microorganism being selected from the species Galdieria sulphuraria, and the acidic pH resistant phycocyanin being a phycocyanin that is stable at a pH below 5, and characterized in that It includes the following steps: a) adjusting the pH of the crude extract of the acidic pH resistant phycocyanin to a pH below 5 so as to precipitate organic matter other than the acidic pH resistant phycocyanin, b) recovering the supernatant obtained in step a) containing the acidic pH-resistant phycocyanin, and c) isolating the acidic pH resistant phycocyanin from the supernatant obtained in step b).

2. The method according to claim 1, characterized in that The phycocyanin extracted in step c) comprises a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC), and the C-PC / APC molar ratio is at least 5.

3. The method according to claim 1, characterized in that The pH of the crude extract in step a) is adjusted to be below the isoelectric point of allophycocyanin (APC).

4. The method according to claim 1, characterized in that The phycocyanin comprises at least 95% acidic pH resistant C-PC and less than 5 mol% APC, the percentage being expressed relative to the sum of APC and C-PC.

5. The method according to claim 1, characterized in that The recovery of the supernatant in step b) is carried out by filtration.

6. The method according to claim 1, characterized in that Phycocyanin contained in the precipitate obtained after pH adjustment of the crude extract of the acidic pH-resistant phycocyanin is i) dissolved in an aqueous solution of acidic pH, ii) separated from insoluble impurities, and iii) isolated by recovering a supernatant containing the acidic pH-resistant phycocyanin and separating the acidic pH-resistant phycocyanin from the supernatant.

7. The method according to claim 6, characterized in that The pH of the aqueous solution is 6 or lower.

8. The method according to claim 6, characterized in that The acidic pH resistant phycocyanin of step iii) is recovered as a mixture comprising phycocyanin and allophycocyanin, wherein the phycocyanin / allophycocyanin molar ratio is at least 5.

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