Purification of phycobiliprotein

Abstract

Provided are novel methods for purifying phycobiliproteins, particularly acid pH-resistant phycobiliproteins, the resulting phycobiliproteins, and uses thereof. [Solution] A method for purifying acid pH-resistant phycobiliproteins from a crude extract of acid pH-resistant phycobiliproteins from cells of phycobiliprotein-producing microorganisms, characterized in that the method comprises the steps of: a) adjusting the pH of the crude extract of acid pH-resistant phycobiliproteins to a pH of less than 6 to precipitate organic matter other than the acid pH-resistant phycobiliproteins; b) recovering a supernatant containing the acid pH-resistant phycobiliproteins; and c) separating the acid pH-resistant phycobiliproteins from the supernatant.

Description

Technical Field

[0001] The present invention relates to a novel method for purifying phycobiliproteins, particularly acid pH-resistant phycobiliproteins, the obtained phycobiliproteins, and their use.

Background Art

[0002] The purification of phycobiliproteins from Galdieria Sulphuraria, particularly C-phycocyanin (C-PC), is much more complex than that from Arthrospira platensis (Spirulina) or other cyanobacteria. One reason for this is the composition of the Galdieria Sulphuraria cell wall, which requires mechanical action to break it down (Sorensen et al., 2013). As a result of mechanical lysis, micelles are formed, which are only partially removed by ultracentrifugation. If chlorophyll a and dissolved carotenoids are present in these micelles, the absorbance value at 280 nm (the UV absorbance characteristic of proteins) increases, which can 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 the micelles and soluble proteins other than phycobiliproteins.

[0003] Purification methods for phycobiliproteins extracted from Cyanidioschyzon merolae, Cyanidium caldarium, Galdieria sulphuraria and Spirulina by precipitation with ammonium sulfate have been described in the literature (WO2016099261; Eisele et al., 2000; Kao et al. Moon et al., 1975; 2015; Cruz de Jesus et al., 2006), but they require a large amount of ammonium sulfate and cause serious problems such as reprocessing of this ammonium sulfate and the supernatant, making their application on an industrial scale very difficult.

[0004] Other known purification methods for obtaining purity levels, such as chromatographic methods, were very costly to implement.

[0005] Accordingly, the present invention relates to a method for purifying phycobiliproteins produced by bioreactor culture of phycobiliprotein-producing microorganisms, which method is easy to implement and economically suitable for industrial-scale implementation.

[0006] Furthermore, phycobiliproteins, especially phycocyanin, are a mixture of c-phycocyanin and allophycocyanin. With known purification methods, they cannot be separated under industrial control. In purification by precipitation with ammonium sulfate, both proteins are carried along without control, making it difficult to obtain a pigment with stable properties. As a result of this precipitation method, a substantial loss in extraction yield also occurs (Cruz de Jesus et al., 2006).

[0007] Accordingly, the present invention also relates to the preparation of acid pH-resistant phycobiliproteins in a controlled composition in phycocyanin, especially purified phycobiliproteins, in particular purified phycocyanins essentially containing c-phycocyanin or essentially allophycocyanin, and more particularly to the acid pH resistance that does not require the addition of stabilizers such as ascorbic acid (WO2005 / 065697) or polyphenols (WO2015 / 090697). Summary of the Invention

[0008] Accordingly, the present invention relates to a method for purifying an acid pH-resistant phycobiliprotein from a crude extract of an acid pH-resistant phycobiliprotein, the method comprising: a) adjusting the pH of the crude extract of the acid pH-resistant phycobiliprotein to a pH of less than 6 to precipitate organic substances other than the acid pH-resistant phycobiliprotein; b) recovering the supernatant containing the acid pH-resistant phycobiliprotein; c) separating the acid pH-resistant phycobiliprotein from the supernatant and is characterized by comprising.

[0009] The present invention also relates to an acid pH-resistant phycobiliprotein obtained by this method, in particular an acid pH-resistant phycocyanin containing a mixture of c-phycocyanin and allophycocyanin, more specifically one having a molar ratio of c-phycocyanin to allophycocyanin of at least 2. Brief Description of the Drawings

[0010]

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DETAILED DESCRIPTION OF THE INVENTION

[0011] Accordingly, the present invention relates to a method for purifying an acid pH-resistant phycobiliprotein from a crude extract of an acid pH-resistant phycobiliprotein.

[0012] The acquisition of a crude extract of phycobiliprotein is generally carried out from the cells of microorganisms grown industrially in a high-capacity bioreactor, preferably such that a fermentation broth containing a high density of phycobiliprotein-producing microorganisms is obtained (high density is generally defined as more than 50 g of dry matter (DM) per liter of fermentation broth, preferably more than 100 g / L). These culture methods are known to those skilled in the art and can be carried out in particular in autotrophic, heterotrophic or mixotrophic manner as described in WO2017 / 050917, WO2017 / 050918 and PCT / EP2016 / 079325 filed on 30 November 2016. The phycobiliprotein produced by the cultured microorganisms must be released after cell lysis. In fact, the microbial cells contain a large amount of phycobiliprotein (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.

[0013] The aqueous extract can probably be prepared directly from the fermentation broth since an appropriate amount of water is added and it is recovered from the reactor at the end of fermentation.

[0014] The extract can be prepared from fresh cells separated from the fermentation broth by any separation method well known to those skilled in the art. The extract can also be prepared from freeze-dried or dried cells for storage.

[0015] According to a preferred embodiment of the invention, the aqueous extract is prepared from fresh cells separated from the fermentation broth after culture.

[0016] Cell lysis can be carried out by any means of cell lysis known to those skilled in the art. Cell lysis can be carried out while the cells are suspended in water, fermentation broth or reconstituted suspension.

[0017] According to a preferred embodiment of the present invention, cell lysis is performed on the cells separated from the fermentation broth before they are resuspended.

[0018] Preferably, the aqueous extract is obtained from the suspension containing the lysed cells by separating the solids by any separation means known to those skilled in the art for removing the solid residues of cell lysis, particularly filtration.

[0019] As a result, an aqueous extract called "crude extract of phycobiliprotein" or "crude extract" is obtained, which contains, in addition to the desired phycobiliprotein, other organic substances such as acid pH-resistant phycobiliprotein, micelles, and other water-soluble proteins.

[0020] The crude extract of phycobiliprotein can be prepared from fresh lysed cells (directly in the fermentation broth or after separation of the fermentation broth), or from freeze-dried or dried cells, where cell lysis is performed before and / or after freeze-drying or drying.

[0021] According to a preferred embodiment of the present invention, the crude extract is prepared from fresh cells.

[0022] The purification method according to the present invention consists in separating phycobiliprotein, particularly acid pH-resistant phycobiliprotein, from other organic substances such as micelles and other water-soluble proteins.

[0023] Microorganisms cultured for producing phycobiliprotein are well known to those skilled in the art and are particularly selected from the group of cyanobacteria such as Arthrospira platensis (Spirulina), Spirulina maxima, Synechococcus elongatus, or from the group of the class Cyanidiales such as Cyanidioschyzon merolae, Cyanidium caldarium, Galdieria sulphuraria.

[0024] Preferably, the phycobiliprotein is an acid pH-resistant phycocyanin. The acid pH-resistant phycobiliprotein or acid pH-resistant phycocyanin is defined as a phycobiliprotein that resists precipitation at acid pH. According to the present invention, the acid pH means a pH less than 7, preferably 6 or less. Advantageously, the acid pH-resistant phycobiliprotein does not precipitate in an aqueous solution at a pH level of less than 6. This protein can also be described as being resistant or stable at acid pH.

[0025] Of course, the purified phycobiliprotein according to the present invention will be more or less stable depending on the acid pH considered. Some are stable in the pH value range of about 6, while others are stable at pH values well below 6. As a result, the acid pH-resistant phycobiliprotein is also defined as a mixture of phycobiliproteins, most of which do not precipitate at a pH less than 7, preferably 6 or less.

[0026] Advantageously, the present invention relates to a phycobiliprotein that is stable at a pH of less than 5, preferably less than 4, more preferably in the range of 4 to 2, and even more preferably 3.5 or less.

[0027] Such acid pH-resistant phycocyanins are known to those skilled in the art and are described in particular in the applications WO2016 / 099261 or WO2017 / 050918. In particular, these are phycocyanins produced by microalgae strains of the genus Cyanidioschyzon, Cyanidium or Galdieria, especially strains of Cyanidioschyzon merolae 10D species, Cyanidioschyzon merolae DBV201 species, Cyanidium caldarium species, Cyanidium daedalum species, Cyanidium maximum species, Cyanidium partitum species, Cyanidium rumpens species, Galdieria daedala species, Galdieria maxima species, Galdieria partita species, Galdieria sulphuraria species, especially Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae.

[0028] These phycocyanins are a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC).

[0029] Advantageously, the apoprotein of C-PC contains the 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 contains the protein of SEQ ID NO: 1, and the apoprotein of the β subunit of C-PC contains the protein of SEQ ID NO: 2 or a variant thereof:

Chemical formula

[0030] Advantageously also, the α subunit of said APC contains SEQ ID NO: 3 or a variant thereof, and the apoprotein of the β subunit of said APC contains SEQ ID NO: 4 or a variant thereof:

Chemical formula

[0031] The apo-proteins of C-PC and APC from the same phycocyanin source generally have different isoelectric points. By lowering the pH, it becomes possible to at least partially separate C-PC from APC.

[0032] In fact, the inventors have found that the lower the pH of the crude extract is adjusted, the purer the obtained C-PC becomes.

[0033] Advantageously, when the phycobiliprotein is an acid pH-tolerant phycocyanin, lowering the pH below the isoelectric point of APC results in a phycocyanin containing a C-PC / APC mixture having a molar ratio of at least 5, preferably at least 10, more preferably at least 15.

[0034] Preferably, the pH of the crude extract in step a) is adjusted to a pH of less than 5. Thus, it is possible to obtain an acid pH-tolerant phycocyanin containing less than 5 mol%, preferably less than 1 mol%, more preferably less than 0.1 mol% of APC, where the percentage represents the ratio to the total of APC and C-PC.

[0035] In step a), pH adjustment is carried out by adding strong or weak mineral or organic acids in solid or solution form, where the amount of acid added is determined by the pH of the crude extract to be treated and the pH value that the person skilled in the art is trying to obtain. Among the mineral acids well known to those skilled in the art, hydrochloric acid and phosphoric acid can be specifically mentioned. Among the organic acids well known to those skilled in the art, acetic acid, citric acid, tartaric acid, lactic acid, preferably citric acid can be specifically mentioned. Also, acidic polyphenols such as rosmarinic acid, tannic acid, digallic acid, casuarina tannic acid, gallotannic acid, and acidic tannins such as quercetin, ellagitannin, castalagin, castalin, casuarictin, grandinin, punicalagin, punicalin, roburin A, terimagrandin II, terflavin B, vescatalagin, pezunculagin, casuarinin, castrin, vescarin, preferably tannic acid can also be mentioned. Preferably, the acid used is an acid approved for use in food, particularly phosphoric acid, citric acid or tannic acid.

[0036] Regarding step b) of recovering the supernatant containing the acid pH-resistant phycobiliprotein, any separation method known to those skilled in the art can be used, particularly tangential filtration with an organic membrane such as a ceramic membrane or a polyethersulfone hollow fiber membrane. The cut-off of these filters can be selected to separate molecules with a molecular weight larger or smaller than the target phycobiliprotein.

[0037] According to a specific embodiment of the present invention, the separation in step b) is carried out by tangential filtration. In this step, a part of the proteins other than the phycobiliprotein is concentrated and removed, and thus the purity level of the final product is increased.

[0038] Step c) of drying / dehydrating the acid pH-resistant phycobiliprotein from the supernatant is carried out by any method of removing the solvent, in this case water, for example by evaporation under atmospheric pressure or vacuum. Atomization, freeze-drying, zeodratation using zeolite, infrared drying, or refractive window drying can be specifically mentioned.

[0039] In the case of evaporation by heating, those skilled in the art should pay attention not to use an overly high temperature that may cause denaturation of the phycobiliprotein.

[0040] After recovering the supernatant in step b), it is possible to reuse the phycobiliprotein contained in the precipitate. To do this, the residual phycobiliprotein is solubilized in an aqueous solution with an acidic pH of about 6 or less, at which pH the phycobiliprotein is soluble while the impurities remain insoluble.

[0041] 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 the required number of times. If the conditions used in the method according to the invention allow for the preferential purification of C-PC, the residual phycobiliprotein is a C-PC / APC mixture rich in APC.

[0042] By reusing the precipitate, a phycobiliprotein containing a C-PC / APC mixture with a molar ratio of less than 5, particularly less than 4, and preferably in the range of 3 to 0.1 is obtained.

[0043] By repeating steps a) to c) of the above method, it is possible to discharge the precipitate of C-PC by an iterative process, add this precipitate to the previously obtained fraction to enrich its content, and also enrich the residual mixture of phycobiliprotein in APC with an APC / C-PC ratio of at least 5, preferably at least 10, and more preferably at least 15.

[0044] Thereby, it is possible to obtain an APC mixture containing less than 5 mol%, preferably less than 1 mol%, and more preferably less than 0.1 mol% of CPC, where the percentage represents the ratio to the total of APC and C-PC.

[0045] These APCs isolated from the precipitate can then be further purified by preparative chromatography techniques well known to those skilled in the art for the production of allophycocyanin, which can be used, for example, in the field of medical imaging due to these fluorescence properties.

[0046] The present invention also relates to acid pH-resistant phycobiliproteins, particularly phycocyanin, which can be obtained by a purification process.

[0047] The present invention also relates to a purified acid pH-resistant phycocyanin comprising a C-PC / APC mixture having a molar ratio of at least 2.

[0048] In particular, the present invention relates to an acid 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, where the percentages represent the ratio to the total of APC and C-PC.

[0049] These C-PCs are known to those skilled in the art and are particularly defined above. In particular, the α subunit of C-PC contains the protein of SEQ ID NO: 1, and the apoprotein of the β subunit of C-PC contains the protein of SEQ ID NO: 2 or a variant thereof.

[0050] Advantageously, the variant according to the present invention has at least 83% sequence identity to the α subunit of C-PC and at least 82% sequence identity to the β subunit of C-PC.

[0051] Preferably, the variant according to the present invention has at least 90% identity to the α (SEQ ID NO: 1) and β (SEQ ID NO: 2) subunits.

[0052] The present invention also relates to a purified phycocyanin rich in APC obtained by the method according to the present invention.

[0053] In particular, the present invention relates to a purified phycocyanin comprising a mixture rich in APC with a C-PC / APC molar ratio of less than 5, particularly 4, advantageously in the range of 3 to 0.1.

[0054] According to a particular embodiment of the invention, the mixture rich in APC has an APC / C-PC ratio of at least 5, preferably at least 10, more preferably at least 15.

[0055] According to a more specific embodiment of the invention, the phycocyanin consists essentially of APC and contains at least 95 mol% of APC and less than 5 mol% of C-PC, preferably at least 99 mol% of APC and less than 1 mol% of C-PC, where the percentages represent the ratio with respect to the total of APC and C-PC.

[0056] These APCs are known to those skilled in the art, particularly as defined above, in particular where the α subunit of said APC contains SEQ ID NO: 3 or a variant thereof, and the apoprotein of the β subunit of said APC contains SEQ ID NO: 4 or a variant thereof.

[0057] Advantageously, the variants according to the invention have at least 83% sequence identity with respect to the α subunit of APC and at least 82% sequence identity with respect to the β subunit of APC.

[0058] Those skilled in the art know the usual methods that can be freely used, particularly the method of measuring protein sequence identity using the BLASTP program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0059] Similarly, those skilled in the art know the method of identifying variants of said sequences and verifying that they retain the same structural properties by means of simple stability tests at acidic pH, for example by performing tests such as those shown in Example 3 of the application WO2017 / 050918.

[0060] It is known to those skilled in the art that a polypeptide can be modified by substitution, insertion and / or deletion of at least one amino acid without substantially altering its function.

[0061] For example, substitution of an amino acid with another chemically equivalent amino acid at a given position is a known example of a sequence variation that does not substantially affect the properties of the protein.

[0062] These "conservative" substitutions can be defined as exchanges among the following amino acid groups: - Ala, Ser, Thr, Pro, Gly - Asp, Asn, Glu, Gln - His, Arg, Lys - Met, Leu, Ile, Val, Cys and - Phe, Tyr, Trp

[0063] Thus, variants of the apoprotein of phycocyanin and / or allophycocyanin according to the invention may contain 1 to 30 amino acids that differ in number compared to the corresponding so-called reference sequence, particularly with respect to the α and / or β subunits of phycocyanin, provided that the resulting variant retains the properties of the reference protein and the percentage of homology / identity described above.

[0064] More precisely, according to the invention, - In the case of a variant of the apoprotein of the α subunit of phycocyanin that can be used in the acidic composition according to the invention, resulting from substitution, insertion and / or deletion, the variant may contain 1 to 27 amino acids that differ from the corresponding so-called reference sequence, provided that the resulting variant retains the properties of the reference protein and the percentage of identity described above; - In the case of a variant of the apoprotein of the β subunit of phycocyanin that can be used in the acidic composition according to the invention, resulting from substitution, insertion and / or deletion, the variant may contain 1 to 30 amino acids different from the corresponding so-called reference sequence, provided that the resulting variant retains the properties of the reference protein and the percentage of identity described above; - In the case of a variant of the apoprotein of the α subunit of allophycocyanin that can be used in the acidic composition according to the invention, resulting from substitution, insertion and / or deletion, the variant may contain 1 to 24 amino acids different from the corresponding so-called reference sequence, provided that the resulting variant retains the properties of the reference protein and the percentage of identity described above; - In the case of a variant of the apoprotein of the β subunit of allophycocyanin that can be used in the acidic composition according to the invention, resulting from substitution, insertion and / or deletion, the variant may contain 1 to 20 amino acids different from the corresponding so-called reference sequence, provided that the resulting variant retains the properties of the reference protein and the percentage of identity described above.

[0065] In particular, according to the present invention, regardless of the reference sequence considered (α and / or β subunits of phycocyanin and / or α and / or β subunits of allophycocyanin), the variant of said subunit preferably contains 1 to 15 different amino acids, preferably 1 to 10 different amino acids, particularly 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 resulting variant retains the properties of the reference protein and the percentage of identity described above.

[0066] Preferably, the present invention relates to C-PC in which the subunit α protein consists of the protein of SEQ ID NO: 1 and the β subunit protein consists of the protein of SEQ ID NO: 2.

[0067] According to another preferred embodiment, the present invention relates to an APC in which the subunit α protein consists of the protein of SEQ ID NO: 3 and the subunit β protein consists of the protein of SEQ ID NO: 4.

[0068] Phycobiliprotein is a natural pigment mainly used for coloring food.

[0069] The present invention also relates to the use of the acid pH-resistant phycobiliprotein obtained by the method according to the present invention, in particular the acid pH-resistant phycocyanin defined above as a pigment in food.

[0070] The present invention also relates to a composition containing the acid pH-resistant phycobiliprotein obtained by the method according to the present invention, in particular the acid pH-resistant phycocyanin defined above, especially a food composition.

[0071] Such uses and such compositions are known to those skilled in the art.

[0072] Preferably, as defined in the application of WO2017 / 050918, the food or food composition is an acidic composition.

[0073] According to the present invention, the acidic composition is defined as any composition containing a mineral acid or an organic acid and phycocyanin. This composition may be a liquid, fluid or viscous, paste-like or solid having an acidic pH, and the acid pH-resistant phycocyanin is incorporated therein.

[0074] In the case of an aqueous liquid composition, the pH is measured as usual. In the case of a non-aqueous liquid composition or a paste-like or solid composition, the pH is measured after dissolving the composition in an amount of water sufficient to dissolve the soluble compound containing the inorganic acid or organic acid and phycocyanin.

[0075] Advantageously, the composition according to the invention is optionally an aqueous liquid composition in the form of a gel, or a paste or solid composition intended to dissolve in an aqueous solution or a solid or paste composition containing 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.

[0076] Mineral or organic acids that can be used in the composition according to the invention are well known to those skilled in the art. Among mineral acids, carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulfonic acid and nitric acid can be particularly mentioned. Among organic acids, citric acid, lactic acid, malic acid, tartaric acid, succinic acid, especially citric acid can be particularly mentioned.

[0077] According to the invention, an acidic food composition means any composition included in the above definition and intended for ingestion by humans or animals. Acidic compositions of dietary supplements must be considered to be included in the definition of acidic food compositions in the meaning of the present invention.

[0078] The acidic food composition according to the invention is well known to those skilled in the art. This composition may contain excipients that may contain structural components related to active compounds identified for their nutrient contribution or properties beneficial to human or animal health. The acidic food composition according to the invention may also contain food additives such as texturizing agents, flavoring agents, preservatives, etc., all of which are well known to those skilled in the art. Excipients may contain water and / or protein and / or fat and / or fiber and / or sugar. The components of the excipients have only structural properties, but they are generally known for contributing nutrients.

[0079] The acidic food composition according to the invention may be in the form of a food additive that can be used immediately or added to a solid, paste or liquid preparation to prepare a food that can be ingested.

[0080] In the case of food compositions, the acid is preferably selected from the list of approved food acidulants, in particular carbonic acid, phosphoric acid, citric acid, malic acid, tartaric acid and lactic acid, more specifically citric acid.

[0081] Regarding acidic compositions other than the food compositions according to the invention, they may be, inter alia, for pharmaceutical, veterinary or cosmetic use and may further contain any additives and / or active agents known and used in this type of composition.

[0082] In the solid, liquid or paste-like acidic compositions according to the invention, phycocyanin can be incorporated, for example, in powder form. The acidic composition, in particular the acidic food composition, can be in any known conventional form such as cream, gel, foam, paste and the like. In particular, in the case of solid food compositions, mention may be made especially of cakes or biscuits, dried cooked foods, diluted powders, solid or "jelly" gelatin-like compositions, foams and the like.

[0083] According to the invention, the liquid acidic composition may be an aqueous composition in which phycocyanin is dissolved. This composition may be in the form of a ready-to-use composition, or a liquid concentrate diluted especially for ingestion or a liquid concentrate added to solid foods for their preparation or ingestion, for example a liquid coating concentrate or a "topping" composition placed on a cake to impart coloration. Among these concentrated compositions, mention may be made, inter alia, of syrups optionally containing alcohol.

[0084] The liquid acidic composition according to the invention may have a variable viscosity and may or may not contain additives such as thickeners, gelling agents and other structuring additives known to those skilled in the art and customary in the preparation of liquid food compositions.

[0085] According to certain embodiments of the present invention, the liquid food composition may be an optionally carbonated acidic beverage. These may include sodas, juices, sports drinks, fitness drinks, recovery drinks, and the like. The composition of these beverages is well-known to those skilled in the art and may include, in particular, sugars, mineral salts, food additives, dissolved gases, and the like. The beverages according to the present invention are normal acidic beverages in which all or part of the commonly used coloring agents have been replaced by the acid pH-resistant phycocyanin according to the present invention.

[0086] According to the present invention, the phycocyanin content in the composition according to the present invention can be in accordance with the practice of those skilled in the art.

[0087] For example, when phycocyanin is used to color an acidic composition, the phycocyanin content in said composition can be in accordance with the practice of those skilled in the art regarding coloring.

[0088] In the liquid acid composition as defined in the present invention, the phycocyanin content can be between 2.5 mg / L and 2500 mg / L, preferably between 25 mg / L and 300 mg / L.

[0089] In a ready-to-use liquid beverage composition, the phycocyanin content can generally be between 25 mg / L and 300 mg / L, preferably between 50 mg / L and 100 mg / L.

[0090] In a concentrated liquid composition that is diluted for use such as a syrup, the phycocyanin content can generally be between 250 mg / l and 2500 mg / l, preferably between 500 mg / L and 1000 mg / L.

[0091] In a solid composition, the phycocyanin content can generally be between 0.01 mg / g and 10 mg / g, preferably between 0.1 mg / g and 5.0 mg / g, and very preferably between 0.25 mg / g and 2.5 mg / g.

Examples

[0092] Example 1. Purification by Acid Precipitation in Fresh Cells Centrifuge and mechanically grind the fermentation mash of Galdieria sulphuraria cells, rinsed with an equal volume of water, in an aqueous phase at pH 6 to release the phycobiliprotein. Acidify the ground material in 0.5 pH unit increments by the addition of citric acid. At each level, collect a sample of the mixture and then centrifuge at 11,000 g for 10 minutes. Collect the supernatant containing the phycobiliprotein and measure the purity index by measuring the ratio of absorbance at 618 nm to absorbance at 280 nm using a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).

[0093] It is clear that the lower the pH, the higher the purity index (Figure 1). This increase in the purity index reflects a decrease in contaminating proteins in the supernatant, while C-PC remains mainly in the supernatant. Due to its resistance to acidic pH, there is no substantial loss of C-PC content in the supernatant (Figure 2). Surprisingly, allophycocyanin (APC) completely disappears from the supernatant at pH values below 5 and forms a pellet containing other precipitated proteins and cell debris (Figure 3). This pellet also contains C-PC and has a high APC content (Figure 3).

[0094] As a result of acidification, the separation of the liquid and solid phases is improved, the pellet of cell debris and protein becomes more compact, and it is easier to separate from the aqueous phase.

[0095] Example 2. Purification by Acid Precipitation in Lyophilized and Rehydrated Cells Centrifuge and mechanically grind the fermentation mash of Galdieria sulphuraria cells rinsed with an equal volume of water to release phycobiliprotein in an aqueous phase with a pH of 6. The ground material is then freeze-dried. The freeze-dried dry matter is suspended in an amount of water corresponding to the initial volume of the mash and acidified in 0.5 pH unit increments by the addition of citric acid. At each level, a sample of the mixture is taken and then centrifuged at 11,000 g for 10 minutes. The supernatant containing phycobiliprotein is collected, and the purity index is measured by measuring the ratio of absorbance at 618 nm to absorbance at 280 nm using a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).

[0096] As described above in Example 1, an increase in the purity index correlated with a decrease in pH can be confirmed (Figure 4). Also in this case, as a result of acidification, the separation of the liquid phase and the solid phase is improved, the pellet of cell debris and protein becomes denser, and it becomes easier to separate from the aqueous phase. Similar to what was observed in Example 1, for pH values less than 5, APC is found in the pellet rather than in the aqueous phase (Figure 5). When the pH value is between 6 and 5, as the pH decreases, the amount of APC in the supernatant decreases.

[0097] Example 3. Purification and Concentration of C-PC by Tangential Filtration The crude extract after acid precipitation and centrifugation is filtered through a hollow fiber membrane tangential filtration module. Filtration through this mesh removes some of the proteins other than C-PC and thus increases the purity index (Figure 6). The purity index that can be achieved by this method approximates the values usually obtained by much more complex methods including two-phase extraction, or precipitation with ammonium sulfate, or even chromatography methods (Soresen et al., 2013; Cruz de Jesus et al., 2006). In parallel with the purification process, this filtration step removes water from the C-PC extract (Figure 7) and facilitates the subsequent drying of the product.

[0098] References

Table 1

Claims

**Claim 1**: A method for purifying acid pH-resistant phycocyanin from a crude extract of acid pH-resistant phycocyanin from cells of a phycocyanin-producing microorganism of the genus Cyanidioschyzon, Cyanidium, or Galdieria, comprising: The acid pH-resistant phycocyanin is stable at a pH in the range of 4 to 2; The acid pH-resistant phycocyanin contains c-phycocyanin (C-PC) and allophycocyanin (APC), where the C-PC / APC molar ratio is at least 5; The method comprises: a) adjusting the pH of the crude extract of the acid pH-resistant phycocyanin to a pH of less than 5 to precipitate organic substances other than the acid pH-resistant phycocyanin, and obtaining a supernatant and a pellet; b) recovering the supernatant containing the acid pH-resistant phycocyanin obtained in step a); c) separating the acid pH-resistant phycocyanin from the supernatant. A method characterized by comprising the above steps. **Claim 2**: The method according to claim 1, wherein in step a), the pH of the crude extract of the acid pH-resistant phycocyanin is adjusted to a pH of less than 4. **Claim 3**: The method according to claim 1 or 2, wherein in step a), the pH of the crude extract of the acid pH-resistant phycocyanin is adjusted to a value less than the isoelectric point of allophycocyanin (APC). **Claim 4**: The method according to any one of claims 1 to 3, wherein the acid pH-resistant phycocyanin separated in step c) contains a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC), where the C-PC / APC molar ratio is at least 10. **Claim 5**: The method according to any one of claims 1 to 4, wherein the acid pH-resistant phycocyanin separated in step c) contains a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC), where the C-PC / APC molar ratio is at least 15. **Claim 6**: The method according to any one of claims 1 to 5, wherein the acid pH-resistant phycocyanin separated in step c) contains more than 95% acid pH-resistant C-PC and less than 5% APC, where the percentage represents the ratio with respect to the total of APC and C-PC. **Claim 7**: The method according to any one of claims 1 to 6, wherein the phycocyanin-producing microorganism is a strain of Galdieria sulphuraria. **Claim 8**: The method according to any one of claims 1 to 7, wherein the residual acid pH-resistant phycocyanin contained in the pellet is solubilized in an aqueous solution having an acid pH, separated from impurities, and isolated by repeating steps b) and c), and the residual acid pH-resistant phycocyanin is stable at a pH in the range of 4 to 2.

9. The method according to claim 8, wherein the pH of the aqueous solution is 6 or less.

10. The method according to claim 8 or 9, wherein the phycocyanin contained in the pellet contains c-phycocyanin (C-PC) and allophycocyanin (APC), and here, the C-PC / APC molar ratio is less than 5.

11. The method according to claim 10, wherein the phycocyanin contained in the pellet contains c-phycocyanin (C-PC) and allophycocyanin (APC), and here, the C-PC / APC molar ratio is less than 4.

12. The method according to claim 11, wherein the phycocyanin contained in the pellet contains c-phycocyanin (C-PC) and allophycocyanin (APC), and here, the C-PC / APC molar ratio is from 3 to 0.1.

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