Purification of phycobiliproteins

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. More specifically, a method for purifying acid-pH-resistant phycocyanins from Cyanidioschyzon, Cyanidium or Galdieria, wherein the phycocyanins are stable at a pH of less than 5.

Description

[0001] PHYCOBILIPROTEIN PURIFICATION

[0002] FIELD OF INVENTION

[0003] The present invention relates to a new process for purifying phycobiliproteins, in particular those resistant to acidic pH, the phycobiliproteins obtained and their uses.

[0004] STATE OF THE ART

[0005] The purification of phycobiliproteins from Galdieria sulphuraria, 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 Galdieria sulphuraria cell wall, which requires mechanical action to be broken down (Sorensen et al., 2013). Mechanical lysis leads to the formation of micelles that are only partially removed by ultracentrifugation. The presence of chlorophyll a and carotenoids dissolved in these micelles contributes to increasing the absorbance values ​​at 280 nm (protein-specific UV absorbance), which may explain the lower purity levels of 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 removing micelles and soluble proteins other than phycobiliproteins.

[0006] The purification of phycobiliproteins extracted from Cyanidioschyzon merolae, Cyanidium caldarium, Galdieria sulphuraria and Spirulina by precipitation with ammonium sulfate 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 sulfate, which poses big problems for reprocessing the ammonium sulfate and the supernatant.

[0007] Other purification methods described that achieve a certain level of purity, such as chromatography methods, are very expensive to implement.

[0008] The invention relates to a purification process for phycobiliproteins produced by bioreactor culture of phycobiliprotein-producing microorganisms, which is easy to implement and economically suitable for industrial-scale application. Furthermore, phycobiliproteins, particularly phycocyanins, are mixtures of c-phycocyanin and allophycocyanin. Existing purification processes do not allow for their controlled separation on an industrial scale. Purification by ammonium sulfate precipitation carries both proteins in an uncontrolled manner, making it difficult to obtain a pigment with stable properties. This precipitation method also results in significant extraction yield losses (Cruz de Jesus et al., 2006).

[0009] The invention therefore also relates to the preparation of purified phycobiliproteins, in particular purified phycocyanin comprising essentially c-phycocyanin or essentially allophycocyanin, in particular acid pH resistant phycobiliproteins of controlled phycocyanin composition, acid pH resistance not requiring the addition of stabilizing agents such as ascorbic acid (WO 2005 / 065697) or polyphenols (WO 2015 / 090697).

[0010] DESCRIPTION OF THE INVENTION

[0011] The invention therefore relates to a process for purifying acid pH-resistant phycobiliproteins from a crude extract of acid pH-resistant phycobiliproteins, characterized in that it comprises the steps of

[0012] a) adjusting the pH of the crude extract of acid pH resistant phycobiliproteins to a pH below 6 so as to precipitate organic matter other than acid pH resistant phycobiliproteins,

[0013] b) recovery of the supernatant comprising phycobiliproteins resistant to acidic pH and

[0014] c) isolation of acid pH resistant phycobiliproteins from the supernatant.

[0015] The invention also relates to acid pH resistant phycobiliproteins obtained by the process and in particular, acid pH resistant phycocyanins comprising a mixture of c-phycocyanin and allophycocyanin, more particularly of which the c-phycocyanin / allophycocyanin molar ratio is at least 2.

[0016] DESCRIPTION OF THE FIGURES

[0017] Figure 1. Increase in the purity index of the crude extract as a function of pH from a fresh cell lysate. Figure 2. Measurement of C-PC and APC concentrations in different crude extracts obtained by centrifugation of a fresh cell lysate at different pH values. APC concentrations in mg / ml are shown in gray, and C-PC concentrations in mg / ml are shown in black.

[0018] Figure 3. Measurement of C-PC and APC concentrations in pellets obtained by centrifugation of a fresh cell lysate at different pH values. APC concentrations in mg / g DM are shown in grey, and C-PC concentrations in mg / g DM are shown in black.

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

[0020] Figure 5. Measurement of C-PC and APC concentrations in different crude extracts obtained by centrifugation of a lyophilized and rehydrated cell lysate at different pH levels. APC concentrations in mg / ml are shown in grey, and C-PC concentrations in mg / ml are shown in black.

[0021] Figure 6. Purification and concentration of C-PC by tangential flow filtration.

[0022] The crude extract, previously purified by precipitation at acidic pH, is filtered using a hollow fiber system.

[0023] Figure 7. Increase in C-PC concentration in the retentate during hollow fiber filtration.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] The invention therefore relates to a process for purifying acid pH resistant phycobiliproteins from a crude extract of acid pH resistant phycobiliproteins.

[0026] The crude phycobiliprotein extract is generally obtained from microbial cells cultured industrially in large-capacity bioreactors, preferably in such a way as to obtain fermentation mash containing high densities of phycobiliprotein-producing microorganisms (high densities generally mean more than 50 g of dry matter (DM) per liter of fermentation mash, preferably more than 100 g / L). These culture methods are known to those skilled in the art and can be carried out under autotrophic, heterotrophic, or mixotrophic conditions, as described in particular in applications WO 2017 / 050917, WO 2017 / 050918, and PCT / EP2016 / 079325 filed on November 30, 2016. The phycobiliproteins produced by the cultured microorganisms must be released after cell lysis. Indeed, microorganism cells contain large quantities 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.

[0027] The aqueous extract can be prepared directly from the fermentation must as it is recovered from the reactor at the end of fermentation, possibly with an appropriate amount of water added.

[0028] It can be prepared from fresh cells separated from the fermentation wort by any separation method well known to those skilled in the art. It can also be prepared from cells that have been previously freeze-dried or dried for preservation.

[0029] According to a preferred embodiment of the invention, the aqueous extract is prepared from fresh cells separated from the fermentation must after culture.

[0030] Cell lysis can be performed by any method of cell lysis known to those skilled in the art. It can be carried out while the cells are suspended in water, fermentation wort, or a reconstituted suspension.

[0031] According to a preferred embodiment of the invention, cell lysis is performed on the cells separated from the fermentation must, before their resuspension.

[0032] 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, in particular filtration.

[0033] This yields an aqueous extract called "crude phycobiliprotein extract" or simply "crude extract" which includes, in addition to the desired phycobiliproteins, particularly those resistant to acidic pH, other organic matter such as micelles and other water-soluble proteins.

[0034] The crude extract of phycobiliproteins can be prepared from fresh lysed cells (directly in the fermentation must or after separation from the fermentation must) or from freeze-dried or dried cells, with cell lysis occurring before or after freeze-drying or drying.

[0035] According to a preferred embodiment of the invention, the crude extract is prepared from fresh cells.

[0036] The purification process according to the invention consists of separating the phycobiliproteins, in particular those resistant to acidic pH, of interest from other organic matter such as micelles and other water-soluble proteins.

[0037] The microorganisms cultivated to produce phycobiliproteins are well known to those skilled in the art, particularly those chosen from the Cyanophyceae group such as Arthrospira platensis (Spirulina), Spirulina maxima, Synechococcus elongatus, or the cyanidiophyceae group such as Galdieria sulphuraria, Cyanidium caldiarium, Cyanidioschyzon merolae.

[0038] Preferably, the phycobiliproteins are acid-resistant phycocyanins. Acid-resistant phycobiliproteins, or acid-resistant phycocyanins, are defined as phycobiliproteins that resist precipitation at acidic pH. According to the invention, acidic pH is defined as a pH below 7, advantageously 6 or less. Advantageously, acid-resistant phycobiliproteins do not precipitate in aqueous solution at pH levels below 6. They may also be referred to interchangeably as acid-resistant or acid-stable.

[0039] Naturally, the phycobiliproteins purified according to the invention will be more or less stable depending on the acidic pH considered. Some will be stable within a pH range close to 6. Others will be stable at pH values ​​well below 6. Therefore, by acid-resistant phycobiliprotein, we also mean a mixture of phycobiliproteins, the majority of which do not precipitate at a pH below 7, advantageously below 6 or less.

[0040] Advantageously, the invention relates to phycobiliproteins stable at pH values ​​below 5, preferably below or equal to 4, more preferably from 4 to 2, even more preferably below or equal to 3.5.

[0041] Such acid-resistant phycocyanins are known to those skilled in the art, notably described in application WO 2016 / 099261 or application WO 2017 / 050918. These are in particular phycocyanins produced by strains of microalgae of the genera Cyanidioschyzon, Cyanidium or Galdieria, in particular selected 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, in particular strains of Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae. These phycocyanins are a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC).

[0042] Additionally, the C-PC protein includes the protein from SEQ ID NO 1 or SEQ ID NO 2 or a cell variant. In particular, the apoprotéine of the C-PC subunit includes the protein of SEQ ID NO 1 and the apoprotéine of the β subunit of the C-PC includes the protein of SEQ ID NO 2 or the cell variants.

[0043] SEQ ID 1 : MKTPITEAIA AADNQGRFLS NTELQAVNGR YQRAAASLEA ARSLTSNAQR LINGAAQAVY SKFPYTSQMP GPQYASSAVG KAKCARDIGY YLRMVTYCLV VGGTPMMDEY LIAGLEEINR TFDLSPSWYV EALNYVKSNH GLSGQAANEA NTYIDYAINA LS

[0044] SEQ ID 2 : MLDAFAKVVA QADARGEFLS NTQLDALSKM VSEGNKRLDV VNRITSNASA ITNAARALF SEQPQLIQPG GNAYTNRRMA ACLRDMEI IL RYVSYAI IAG DSSVLDDRCL NGLRETYQAL GVPGASVAVG VEKMKDSAIA IANDPSGITT GDCSALMAEV GTYFDRAATA VQ

[0045] Également avantagement, the sous-unité de ladite APC comprend la SEQ ID NO 3 ou de celles-ci and the apoprotéine de la sous-unité β de ladite APC comprend la SEQ ID NO 4 ou de celles-ci variants.

[0046] SEQ ID 3 : MSLISQIINT ADEELRYPNG GELSTLIYFF NTANTRINII NKLKEREKDI IQNASKKLFQ LHPEYVSSGG NASGPKQRAL CLRDYGWYLR

[0047] LVTYGILAGD ITPIEKIGII GVKDMYNSLG VPIIGMYDAI KCLKEAS INI

[0048] FELSEEKDLI I PYFDYLSNA ILS

[0049] SEQ ID 4: MSIVTKSIVN ADAEARYLSP GELDRIKSFV LSGQRRLRIA

[0050] QILTDNRERI VKQAGQQLFQ QRPDIVSPGG NAYGEEMTAT CLRDLDYYLR LVTYGVVAGD ISPIEEIGLE DFMQDAITAV INTADVQGKY LDNSSIEKLK

[0051] GYFQTGELRV RAAATIAANA AGI IKDAVAK SLLYSDITRP GGNMYTTRRY

[0052] AACIRDLDYY LRYATYSMLA GDPS ILDERV LNGLKETYNS LGVPIGATIQ

[0053] SIQAMKEVTS SLV

[0054] The apolipoproteins of C-PC and APC from the same phycocyanin source generally have different isoelectric points. Lowering the pH will allow for at least partial separation of the C-PC from the APC.

[0055] Indeed, the inventors observed that the lower the pH of the crude extract was adjusted, the purer the C-PC obtained. Advantageously, when the phycobiliprotein is a phycocyanin resistant to acidic pH, lowering the pH below the isoelectric point of APC makes it possible to obtain a phycocyanin comprising a C-PC / APC mixture with a molar ratio of at least 5, preferably at least 10, and more preferably at least 15.

[0056] Preferably, the pH of the crude extract in step a) is adjusted to a pH below 5. Acid pH resistant phycocyanin can then be obtained comprising less than 5 mol% of APC, preferably less than 1%, more preferably less than 0.1% of APC, the percentages being expressed relative to the total sum of APC and C-PC.

[0057] In step a), the pH is adjusted by adding a strong or weak mineral or organic acid, in solid or solution form. The amount of acid added is determined by the pH of the crude extract to be treated and the pH value that a person skilled in the art would seek to obtain. Among the mineral acids well known to those skilled in the art, hydrochloric acid and phosphoric acid are particularly relevant. Among the organic acids well known to those skilled in the art, acetic acid, citric acid, tartaric acid, and lactic acid are particularly relevant, with citric acid being the preferred choice.Other examples include acidic polyphenols such as rosmarinic acid, tannic acid, digallic acid, quercitannic acid, gallotannic acid, acid tannins such as quercetin, ellagitannins, castalagin, castaline, casuariticine, grandinine, punicaligine, punicaline, roburine A, tellimagrandine II, terflavin B, vescaligine, pendunculagin, casua ine, castline, vescaline, and preferably tannic acid. Preferably, the acids used are those authorized for food use, in particular phosphoric acid, citric acid, or tannic acid.

[0058] For step b) of recovering the supernatant containing the acid-resistant phycobiliproteins, any separation method known to those skilled in the art may be used, in particular tangential flow filtration through ceramic membranes or organic membranes such as hollow polyethersulfone fibers. The thresholds of these filters may be chosen to separate molecules with a molecular weight higher or lower than the target phycobiliproteins.

[0059] According to a particular embodiment of the invention, the separation in step b) is carried out by tangential flow filtration. This step makes it possible to concentrate and remove some of the proteins other than phycobiliproteins, thus increasing the purity level of the final product.

[0060] Step c) of drying / dehydrating the acid pH-resistant phycobiliproteins from the supernatant is carried out by any method of solvent removal, in this case water, for example by evaporation at atmospheric pressure or under vacuum. Examples include spray drying, freeze-drying, zeodration, infrared drying, or refraction window drying.

[0061] In the event of evaporation by heating, a person skilled in the art will take care not to use excessively high temperatures which may cause denaturation of the phycobiliproteins.

[0062] It is possible, after recovery of the supernatant in step b), to recycle the phycobiliproteins contained in the precipitate. To do this, the residual phycobiliproteins are solubilized in an acidic aqueous solution, with a pH of approximately 6 or less, a pH at which impurities remain insoluble while phycobiliproteins are soluble.

[0063] These residual phycobiliproteins are then separated from impurities and isolated by repeating steps b) and c) of the process. This is an iterative process that can be repeated as many times as necessary. When the conditions used for the process according to the invention allow for preferential purification of C-PCs, the residual phycobiliproteins are a C-PC / APC mixture enriched in APCs.

[0064] By recycling the precipitate, we then obtain phycobiliproteins comprising a C-PC / APC mixture with a molar ratio of less than 5, in particular less than 4, advantageously in the order of 3 to 0.1.

[0065] By repeating steps a) to c) of the process, previously described, it is possible, through an iterative process, on the one hand to deplete the precipitate in C-PC, which can be joined to the previously obtained fractions to enrich their content and on the other hand to enrich the residual mixture of phycobiliproteins in APC, with an APC / C-PC ratio of at least 5, preferably at least 10, more preferably at least 15.

[0066] We can then obtain a mixture of APC comprising less than 5 molar percent of CPC, preferably less than 1 percent, more preferably less than 0.1 percent of CPC, the percentages being expressed in relation to the total sum of APC and C-PC.

[0067] 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 allophycocyanins which can be used, for example in the field of medical imaging, due to their fluorescent properties.

[0068] The invention also relates to phycobiliproteins resistant to acidic pH, in particular phycocyanins, which can be obtained by the purification process.

[0069] 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.

[0070] In particular, the invention relates to a phycocyanin resistant to acidic pH comprising at least 95% molar of C-PC and less than 5% molar of APC, preferably at least 99% molar of C-PC and less than 1% molar of APC, the percentages being expressed with respect to the total sum of APC and C-PC.

[0071] These C-PCs are known to those skilled in the art and have been defined previously, in particular those whose C-PC a subunit includes the SEQ ID NO 1 protein and whose C-PC β subunit apolipoprotein includes the SEQ ID 2 protein or variants thereof.

[0072] Advantageously, the variants according to the invention exhibit a sequence identity of at least 83% for the α subunits of C-PC, and at least 82% for the β subunits of C-PC.

[0073] Preferably, the variants according to the invention have an identity of at least 90% for the subunits a (SEQ ID NO 1 ) and β (SEQ ID NO 2).

[0074] The invention also relates to a purified phycocyanin enriched in APC that can be obtained by the process according to the invention.

[0075] In particular, the 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.

[0076] According to a particular embodiment of the invention, the APC-enriched mixture has an APC / C-PC ratio of at least 5, preferably at least 10, more preferably at least 15.

[0077] According to a more particular embodiment of the invention, phycocyanin is essentially made up of APC, with at least 95 molar percent of APC and less than 5 molar percent of C-PC, preferably at least 99 molar percent of APC and less than 1 molar percent of C-PC, the percentages being expressed in relation to the total sum of APC and C-PC.

[0078] These APCs are known to those skilled in the art and defined previously, in particular those whose a subunit of said APC includes SEQ ID NO 3 or variants thereof and the apolipoprotein of the β subunit of said APC includes SEQ ID NO 4 or variants thereof.

[0079] Advantageously, the variants according to the invention exhibit a sequence identity of at least 83% for the a subunits of the APC, and of at least 82% for the S subunits of the APC.

[0080] A person skilled in the art knows how to measure protein sequence identity using the usual methods at their disposal, including the BLASTP program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0081] Similarly, a person skilled in the art knows how to identify variants of said sequences and verify that they retain the same structural properties by simple stability testing in acidic pH, for example by carrying out a test like the test shown in example 3 of application WO 2017 / 050918.

[0082] 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.

[0083] For example, substituting one amino acid at a given position with another chemically equivalent amino acid is a known example of sequence variation that does not substantially affect the properties of the protein.

[0084] These "conservative" substitutions can be defined as exchanges within the following amino acid groups

[0085] - Ala, Ser, Thr, Pro, Gly

[0086] - Asp, Asn, Glu, Gin

[0087] - His, Arg, Lys

[0088] - Met, Leu, Ile, Val, Cys and

[0089] - Phe, Tyr, Trp

[0090] Thus, the variants of the apolipoproteins of phycocyanins and / or allophycocyanins according to the invention can comprise from 1 to 30 amino acids difference in number compared to the corresponding so-called reference sequence, particularly with regard to the α and / or β subunits of phycocyanin, provided that the variant obtained retains the properties of the reference protein and the percentages of homology / identity stated above.

[0091] More specifically, according to the invention,

[0092] for the variants of the apolipoproteins of the α subunit of phycocyanins 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 corresponding so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;

[0093] - for the variants of the apolipoproteins of the β subunit of phycocyanins usable in the acid compositions according to the invention, resulting from substitutions, insertions and / or deletions, they may comprise from 1 to 30 amino acids of difference with respect to the corresponding so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;

[0094] for the variants of the apolipoproteins of the a subunit of allophycocyanins usable in the acid compositions according to the invention, resulting from substitutions, insertions or deletions, they may comprise from 1 to 24 amino acids of difference with respect to the corresponding so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above;

[0095] for the variants of the apolipoproteins of the β subunit of allophycocyanins usable 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 corresponding so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above.

[0096] Most particularly according to the invention, and regardless of the reference sequence considered (phycocyanin a and / or β subunit and / or allophycocyanin a and / or β subunit), the variants of said subunits may advantageously comprise a difference of 1 to 15 amino acids, preferably a difference of 1 to 10 amino acids, in particular 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids from the corresponding reference sequence, provided that the variant obtained retains the properties of the reference protein and the percentages of identity stated above.

[0097] Preferably, the invention relates to C-PC whose alpha subunit protein consists of the SEQ ID 1 protein and whose beta subunit protein consists of the SEQ ID 2 protein.

[0098] According to another preferred embodiment, the invention relates to the APC whose alpha subunit protein consists of the SEQ ID 3 protein and whose beta subunit protein consists of the SEQ ID 4 protein.

[0099] Phycobiliproteins are natural colorants primarily used for coloring food.

[0100] The invention also relates to the use of acid pH resistant phycobiliproteins obtained by the process according to the invention and in particular acid pH resistant phycocyanins defined above as coloring in a food product.

[0101] The invention also relates to a composition, in particular a food composition, comprising acid pH resistant phycobiliprotein obtained by the process according to the invention and in particular acid pH resistant phycocyanin as defined above.

[0102] Such uses and compositions are known to a person skilled in the art.

[0103] Preferably, the food product or food composition is an acidic composition, as defined in application WO 2017 / 050918.

[0104] According to the invention, an acidic composition is defined as any composition comprising a mineral or organic acid and phycocyanin. This composition may be liquid, fluid or viscous, pasty or solid, exhibiting an acidic pH and incorporating acid-resistant phycocyanin.

[0105] For aqueous liquid compositions, pH is measured in the usual way. For non-aqueous liquid compositions, or for paste-like or solid compositions, pH is measured after dissolving the composition in a sufficient quantity of water to dissolve the soluble compounds it contains, including mineral or organic acids and phycocyanin.

[0106] Advantageously, the composition according to the invention is an aqueous liquid composition, optionally in the form of a gel, or a paste or solid composition intended to be dissolved in an aqueous solution or in a solid or paste composition comprising water. According to another advantageous embodiment of the invention, the composition is an acidic paste or solid composition intended to be used and / or stored in a humid environment.

[0107] 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, carbonic, phosphoric, hydrochloric, sulfuric, perchloric, sulfonic, and nitric acids are particularly well known. Among the organic acids, citric, lactic, malic, tartaric, and succinic acids are particularly well known, advantageously citric acid.

[0108] The term "acidic food composition" according to the invention refers to any composition intended for human or animal consumption that falls within the preceding definition. Acidic, nutraceutical compositions are to be considered as falling within the definition of acidic food compositions as defined in the invention.

[0109] The acidic food compositions according to the invention are well known to those skilled in the art. They may comprise a vehicle that includes structural components associated with active compounds identified for their nutritional value or for their health benefits to humans or animals. The acidic food composition according to the invention may also include food additives such as texturizing agents, flavoring agents, and preservatives, all components well known to those skilled in the art. The vehicle may include water and / or proteins and / or fats and / or fibers and / or sugars. The components of the vehicle may have only structural properties, but they are generally known for their nutritional value.

[0110] The acidic food composition according to the invention can be ready-to-use or in the form of a food additive which is added to a solid, pasty or liquid preparation to prepare the food which can be ingested.

[0111] For food compositions, the acid will preferably be chosen from the list of acidifiers authorized in food, in particular carbonic, phosphoric, citric, malic, tartaric and lactic acids, more particularly citric acid.

[0112] Regarding acid compositions other than food according to the invention, they may be, among other things, pharmaceutical, veterinary or cosmetic and further include any additives and / or active ingredients known and used in this type of composition.

[0113] In a solid, liquid, or paste-like acidic composition according to the invention, phycocyanin can be incorporated, for example, in powder form. This acidic composition, particularly this acidic food composition, can then be in any known common form such as creams, gels, mousses, pastes, etc. Particularly for a solid food composition, examples include cakes or biscuits, dry foods to be cooked, powders to be diluted, solid gelatinous compositions or "jelly," mousses, etc.

[0114] According to the invention, said liquid acid composition may be an aqueous composition in which phycocyanin is dissolved. It may be in the form of a ready-to-use composition or as a liquid concentrate to be diluted, particularly for ingestion or addition to a solid food, either for its preparation or for ingestion; for example, a concentrated liquid coating or "topping" composition that will be applied to a cake to give it color. Examples of such concentrated compositions include syrups, alcoholic or non-alcoholic.

[0115] The liquid acid composition according to the invention may have variable viscosity and may or may not include additives such as viscosity-modifying agents, gelling agents, and other structuring additives known to those skilled in the art and commonly used for the preparation of liquid food compositions.

[0116] According to a particular embodiment of the invention, the liquid food composition may be an acidic beverage, carbonated or not. Examples include sodas, juices, sports drinks, energy drinks, recovery drinks, etc. The compositions of these beverages 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 common acidic beverage in which the coloring normally used has been replaced, in whole or in part, by a phycocyanin resistant to an acidic pH according to the invention.

[0117] According to the invention, the phycocyanin content in the compositions according to the invention may conform to the practices of a person skilled in the art.

[0118] For example, when phycocyanin is used to color the acid composition, then the phycocyanin content in said composition may conform to the practices of a person skilled in the art regarding coloring.

[0119] In a liquid acid composition as defined in the invention, the phycocyanin content can be between 2.5 mg / L and 2500 mg / L, preferably between 25 mg / L and 300 mg / L.

[0120] In a ready-to-use beverage type liquid composition, the phycocyanin content can generally be between 25 mg / l and 300 mg / L, preferably between 50 mg / L and 100 mg / L.

[0121] In a concentrated liquid composition to be diluted for its 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.

[0122] 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, very preferably between 0.25 mg / g and 2.5 mg / g.

[0123] EXAMPLES

[0124] Example 1. Purification by acid precipitation on fresh cell.

[0125] A fermentation mash of Galdieria sulphuraria cells, centrifuged and then rinsed with an equal volume of water, underwent mechanical grinding to release the phycobiliproteins into an aqueous phase at pH 6. The mash was acidified in 0.5 pH unit increments by adding citric acid. At each increment, a sample of the mixture was taken and centrifuged for 10 min at 1,000 g. The supernatant containing the phycobiliproteins was collected, and the purity index was measured by calculating the ratio of absorbance at 618 nm to absorbance at 280 nm using a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).

[0126] It is clear that as the pH decreases, the purity index increases (Figure 1). This increase in the purity index reflects a decrease in contaminating proteins in the supernatant, while C-PC remains predominantly present in the supernatant. Due to its resistance to acidic 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 along with other precipitated proteins and cellular debris (Figure 3). This pellet also contains C-PC and APC, with a higher APC content (Figure 3).

[0127] Acidification also allows for better separation of the liquid and solid phases, resulting in a more compact pellet of cell debris and proteins that is easier to separate from the aqueous phase. Example 2. Purification by acid precipitation on lyophilized and rehydrated cells.

[0128] A fermentation mash of Galdieria sulphuraria cells, centrifuged and then rinsed with an equal volume of water, underwent mechanical grinding to release the phycobiliproteins into an aqueous phase at pH 6. The ground material was then lyophilized. The lyophilized dry matter was suspended in a volume of water equivalent to the initial volume of the mash and then acidified in 0.5 pH unit increments by adding citric acid. At each increment, a sample of the mixture was taken and centrifuged for 10 minutes at 1,000 g. The supernatant containing the phycobiliproteins was collected, and the purity index was measured by calculating the ratio of absorbance at 618 nm to absorbance at 280 nm using a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).

[0129] As described in Example 1, we observe an increase in the purity index correlated with a decrease in pH (Figure 4). In this case as well, acidification leads to better separation of the liquid and solid phases, resulting in a more compact pellet of cell debris and proteins that is easier to separate from the aqueous phase. Similar to what was observed in Example 1, APC is found in the pellet and not in the aqueous phase (Figure 5) at pH values ​​below 5. For pH values ​​between 5 and 6, the amount of APC in the supernatant decreases as the pH decreases.

[0130] Example 3. Purification and concentration of C-PC by tangential flow filtration.

[0131] The crude extract, after acid precipitation and centrifugation, is filtered through a hollow fiber tangential flow filtration module. Filtration through this mesh removes some of the proteins other than C-PC, thus increasing the purity index (Figure 6). The purity index achievable with this method approaches the values ​​normally obtained by much more complex methods involving biphasic extractions, ammonium sulfate precipitation, or even chromatography (Soresen et al., 2013; Cruz de Jesús et al., 2006). In parallel with purification, this filtration step removes water from the C-PC extract (Figure 7) and facilitates subsequent drying of the product. REFERENCES

[0132] - 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.

[0133] - Eisele et al., "Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extremes of habitat" Biochemica et Biophysica Acta 1456 (2000) 1456, 2-3, 99-107.

[0134] - Eriksen NT. "Production of phycocyanin - a pigment with applications in biology, biotechnology, foods and medicine" Appl Microbiol Biotechnol. 2008 Aug;80(1):1-1

[0135] - Kao et al., "Physico-chemical properties of C-phycocyanin isolated from an acido- thermophilic eukaryote, Cyanidium caldarium" Biochem. J. (1975) 147, 63–70

[0136] - Myounghoon et al. “Isolation and Characterization of Thermostable Phycocyanin from Galdieria Sulphuraria,” Korean Journal of Chemical Engineering, 31 (2014): 1–6.

[0137] - S0rensen et al. "Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria" J Sci Food Agric. 2013 Sep;93(12):2933-8

[0138] - WO 2005 / 065697, WO 2015 / 090697, WO 2016 / 099261, WO 2017 / 050917, WO 2017 / 050918 and PCT / EP2016 / 079325 filed on 30 November 2016

Claims

DEMANDS 1. A process for purifying acid pH-resistant phycobiliproteins from a crude extract of acid pH-resistant phycobiliproteins from phycobiliprotein-producing microorganism cells, characterized in that it comprises the steps of a) adjusting the pH of the crude extract of acid pH resistant phycobiliproteins to a pH below 6 so as to precipitate organic matter other than acid pH resistant phycobiliproteins, b) recovery of the supernatant comprising phycobiliproteins resistant to acidic pH and c) isolation of acid pH resistant phycobiliproteins from the supernatant.

2. A process according to claim 1, characterized in that the phycobiliprotein is phycocyanin resistant to acidic pH.

3. A process according to any one of claims 1 or 2, characterized in that the phycocyanin comprises a mixture of c-phycocyanin (C-PC) and allophycocyanin (APC), the molar ratio C-PC / APC being at least 5.

4. A method according to any one of claims 1 to 3, characterized in that the pH of the culture medium in step a) is adjusted to a pH less than 5.

5. A process according to claim 4, characterized in that the phycobiliprotein comprises at least 95% acid pH resistant C-PC and less than 5 molar APC, the percentages being expressed relative to the total sum of APC and C-PC.

6. A method according to any one of claims 1 to 5, characterized in that the recovery of the supernatant is done by filtration.

7. A method according to any one of claims 1 to 6, characterized in that the phycobiliprotein-producing microorganism is selected from microalgal strains of the genera Cyanidioschyzon, Cyanidium or Galdieria.

8. A method according to claim 7, characterized in that the microalga is selected from the strains Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae.

9. A method according to any one of claims 1 to 8, characterized in that the phycobiliproteins contained in the pellet are solubilized in a solution aqueous solution of acidic pH, separated from impurities and isolated by repeating steps b) and c).

10. Acid pH resistant phycobiliprotein obtained by the process according to any one of claims 1 to 9. 1 1. Acid pH resistant phycocyanin 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. Phycocyanin according to claim 11, characterized in that it comprises less than 5 Molar % of APC.

13. Phycocyanin according to any one of claims 11 or 12, characterized in that the apolipoprotein of the C-PC subunit comprises SEQ ID NO 1 and the apolipoprotein of the β subunit of C-PC comprises SEQ ID 2 or variants thereof.

14. Use of a phycobiliprotein according to claim 10 or of a phycocyanin according to any one of claims 11 to 13, as a colorant in a food product.

15. Food composition characterized in that it comprises a phycobiliprotein according to claim 10 or a phycocyanin according to any one of claims 11 to 13.

16. Phycocyanin comprising a mixture of allophycocyanin (APC) and c-phycocyanin (C-PC) having a C-PC / APC molar ratio of less than 5.

17. Phycocyanin according to claim 16, characterized in that the apolipoprotein of the α subunit of APC comprises SEQ ID NO 3 and the apolipoprotein of the β subunit of APC comprises SEQ ID 4 or variants thereof.

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