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    • 专利摘要:
    The present invention relates to a novel process for the purification of phycobiliproteins, in particular resistant to acidic pH, the phycobiliproteins obtained and their uses.
    公开号:FR3064635A1
    申请号:FR1752674
    申请日:2017-03-30
    公开日:2018-10-05
    发明作者:Olivier CAGNAC；Axel Athane；Julien Demol
    申请人:Fermentalg SA；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    The present invention relates to a new process for the purification of phycobiliproteins, in particular resistant to acid pH, the phycobiliproteins obtained and their uses.
    STATE OF THE ART
    The purification of Galdieria sulphuraria phycobiliproteins, particularly C-Phycocyanin (C-PC), is much more complex than that of Arthrospira platensis (Spirulina) or other cyanobacteria. This is partially due to the composition of the cell wall of Galdieria sulphuraria which requires mechanical action to be broken (Sorensen et al., 2013). Mechanical lysis leads to the formation of micelles which are only partially eliminated 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 low 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 eliminating micelles and soluble proteins other than phycobiliproteins.
    The purification of phycobiliproteins extracted from Galdieria sulphuraria and Spirulina by precipitation with ammonium sulphate has already been described in the literature (Moon et al., 2015; Cruz de Jesûs et al., 2006) but it is very difficult to apply to the 'industrial scale because it requires a lot of ammonium sulfate, which poses major problems of reprocessing of ammonium sulfate and the supernatant.
    The other purification methods described which make it possible to obtain a level of purity such as chromatography methods are very expensive to implement.
    The invention therefore relates to a process for purifying phycobiliproteins produced by bioreactor culture of microorganisms producing phycobiliproteins, easy to use and economically suitable for use on an industrial scale.
    Furthermore, phycobiliproteins, in particular phycocyanins, are mixtures of c-phycocyanin and allophycocyanin. Known purification methods do not make it possible to separate them in an industrially controlled manner. Purification by precipitation with ammonium sulfate causes the two proteins to run in an uncontrolled manner, so it can be difficult to obtain a pigment with stable properties. This precipitation method also generates significant extraction yield losses (Cruz de Jesûs et al., 2006)
    The invention therefore also relates to the preparation of purified phycobiliproteins, in particular purified phycocyanin comprising essentially cphycocyanin or even essentially allophycocyanin, in particular phycobiliproteins resistant to acid pH of composition controlled as phycocyanins.
    STATEMENT OF THE INVENTION
    The invention therefore relates to a process for purifying phycobiliproteins resistant to acid pH from a crude extract of phycobiliproteins resistant to acid pH, characterized in that it comprises the steps of
    a) adjustment of the pH of the raw extract of phycobiliproteins resistant to acidic pHs to a pH below 6 so as to precipitate organic materials other than phycobiliproteins resistant to acidic pHs,
    b) recovery of the supernatant comprising phycobiliproteins resistant to acid pH and
    c) isolation of phycobiliproteins resistant to acidic pH from the supernatant.
    The invention also relates to phycobiliproteins resistant to acidic pHs obtained by the process and in particular, phycocyanins resistant to acidic pHs comprising a mixture of c-phycocyanin and allophycocyanin, more particularly whose molar ratio c-phycocyanin / allophycocyanin is d '' at least 2.
    DESCRIPTION OF THE FIGURES
    Figure 1. Increase in the purity index of the crude extract as a function of pH from a lysate of fresh cells.
    Figure 2. Measurement of the concentration of C-PC and APC in different crude extracts obtained by centrifugation of a lysate of fresh cells at different pH. In gray are represented the APC concentrations in mg / ml, and in black the C-PC concentrations in mg / ml.
    Figure 3. Measurement of the concentration of C-PC and APC in the pellets obtained by centrifugation of a lysate of fresh cells at different pH. In gray are the APC concentrations in mg / g DM, and in black the C-PC concentrations in mg / g DM.
    Figure 4. Increase in the purity index of the crude extract as a function of pH from a lysate of lyophilized and rehydrated cells.
    Figure 5. Measurement of the concentration of C-PC and APC in different crude extracts obtained by centrifugation of a lysate of cells, lyophilized and rehydrated, at different pH. In gray are represented the APC concentrations in mg / ml, and in black the C-PC concentrations in mg / ml.
    Figure 6. Purification and concentration of C-PC by tangential filtration. The crude extract previously purified by precipitation at acidic pH is filtered using a hollow fiber system.
    Figure 7. Increased concentration of C-PC in the retentate during filtration on hollow fiber.
    DETAILED DESCRIPTION OF THE INVENTION
    The invention therefore relates to a process for the purification of phycobiliproteins resistant to acid pH from a crude extract of phycobiliproteins resistant to acid pH.
    The raw extract of phycobiliproteins is generally obtained from cells of microorganisms cultured industrially in large capacity bioreactors, preferably so as to obtain fermentation musts comprising large densities of microorganisms producing phycobiliproteins (by large densities one generally means more than 50g of dry matter (DM) per liter of fermenting wort, preferably more than 10g / L). These culture methods are known to those skilled in the art, which can be carried out in autotrophy, heterotrophy or mixotrophy, in particular described in applications WO 2017/050917, WO 2017/050918 and PCT / EP2016 / 079325 filed on November 30, 2016. Phycobiliproteins produced by cultured microorganisms must be released after cell lysis. Indeed, the cells of microorganisms contain large quantities of phycobiliproteins (Moon et al., 2015, Sorensen et al., 2013, Eriksen 2008). Consequently, the implementation of the method according to the invention first requires the preparation of an aqueous extract from the fermentation must.
    The aqueous extract can be prepared directly from the fermentation must as it is recovered from the reactor at the end of fermentation, optionally with the addition of an appropriate amount of water.
    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 which have been lyophilized or dried beforehand for their preservation.
    According to a preferred embodiment of the invention, the aqueous extract is prepared from fresh cells separated from the fermentation must after culture.
    Cell lysis can be done by any cell lysis means known to the skilled person. It can be done while the cells are in suspension in water, fermentation wort or reconstituted suspension.
    According to a preferred embodiment of the invention, cell lysis is carried out on the cells separated from the fermentation wort, before their resuspension.
    Preferably, the aqueous extract is obtained from the suspension comprising the lysed cells by separating the solids, by any separation means known to a person skilled in the art to remove solid residues from cell lysis, in particular from filtration.
    This gives an aqueous extract called "phycobiliprotein crude extract" or even "crude extract" which comprises, in addition to the desired phycobiliproteins, in particular resistant to acid pH, other organic materials such as micelles and other water-soluble proteins.
    The raw extract of phycobiliproteins can be prepared from fresh lysed cells (directly in the fermentation must or after separation from the fermentation must) or from lyophilized or dried cells, cell lysis taking place before or after lyophilization or drying.
    According to a preferred embodiment of the invention, the crude extract is prepared from fresh cells.
    The purification process according to the invention consists in separating the phycobiliproteins, in particular resistant to acidic pH, which are sought after from these other organic materials such as micelles and the other water-soluble proteins.
    The microorganisms cultivated to produce phycobiliproteins are well known to those skilled in the art, in particular chosen from the group of Cyanophyceae such as Arthrospira platensis (Spirulina), Spirulina maxima, Synechococcus elongatus, or else the group of cyanidiophyceae such as Galdieria sulphuraria, Cyanidium caldiarium, Cyanidioschyzon merolae.
    Preferably, the phycobiliproteins are phycocyanins resistant to acid pH. By phycobiliproteins resistant to acidic pHs or phycocyanins resistant to acidic pHs is meant phycobiliproteins which resist precipitation at acidic pHs. By acid pH is meant according to the invention a pH less than 7, advantageously 6 or less. Advantageously, phycobiliproteins resistant to acidic pHs do not precipitate in an aqueous solution at pHs lower than 6. They can also be described indifferently as resistant or stable at acidic pHs.
    Of course, the purified phycobiliproteins according to the invention will be more or less stable depending on the acid pH considered. Some will be stable in a pH range close to 6. Others will be stable at pH values much lower than 6. Consequently, by acid-resistant phycobiliprotein, we also mean a mixture of phycobiliproteins, the majority of which do not not precipitate at a pH below 7, advantageously below 6 or less.
    Advantageously, the invention relates to phycobiliproteins stable at pH less than 5, preferably less than or equal to 4, more preferably ranging from 4 to 2, even more preferably less than or equal to 3.5.
    Such phycocyanins resistant to acid pH are known to those skilled in the art, in particular described in application WO 2016/099261 or application WO 2017/050918. These are in particular phycocyanins produced by microalgae strains of the genera Cyanidioschyzon, Cyanidium or Galdieria, in particular chosen from the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partit 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).
    Advantageously, the apoprotein of C-PC comprises the protein of SEQ ID NO 1 or SEQ ID NO 2 or a variant of these. In particular, the apoprotein of the α-subunit of C-PC comprises the protein of SEQ ID NO 1 and the apoprotein of the β subunit of C-PC comprises the protein of SEQ ID NO 2 or variants thereof.
    SEQ ID 1: MKTPITEAIA AADNQGRFLS NTELQAVNGR YQRAAASLEA ARSLTSNAQR LINGAAQAVY SKFPYTSQMP GPQYASSAVG KAKCARDIGY YLRMVTYCLV VGGTGPMDEY LIAGLEEINR TFDLSPSWYV EALNYAH
    SEQ ID 2: MLDAFAKWA QADARGEFLS NTQLDALSKM VSEGNKRLDV VNRITSNASA IVTNAARALF SEQPQLIQPG GNAYTNRRMA ACLRDMEIIL RYVSYAIIAG DSSVLDDRCL NGLRETYQAL GVPGASVAVG VEKMDDGVKGDKV
    Also advantageously, the a subunit of said APC comprises SEQ ID NO 3 or variants thereof and the apoprotein of the β subunit of said APC comprises SEQ ID NO 4 or variants thereof .
    SEQ ID 3: MSLISQIINT ADEELRYPNG GELSTLIYFF NTANTRINII NKLKEREKDI IQNASKKLFQ LHPEYVSSGG NASGPKQRAL CLRDYGWYLR LVTYGILAGD ITPIEKIGII GVKDMYNSLG VPIIGMYDAI KCLKEIN
    SEQ ID 4: MSIVTKSIVN ADAEARYLSP GELDRIKSFV LSGQRRLRIA QILTDNRERI VKQAGQQLFQ QRPDIVSPGG NAYGEEMTAT CLRDLDYYLR LVTYGWAGD ISPIEEIGLE DFMQDAITAV INTADVQGKY LDNSSIEK
    GYFQTGELRV RAAATIAANA AGIIKDAVAK SLLYSDITRP GGNMYTTRRY
    AACIRDLDYY LRYATYSMLA GDPSILDERV LNGLKETYNS LGVPIGATIQ
    SIQAMKEVTS SLV
    C-PC and APC apoproteins from the same phycocyanin source generally have different isoelectric points. By lowering the pH, it will be possible to at least partially separate the C-PCs from the APCs.
    In fact, the inventors have found that the lower the pH of the raw extract is adjusted, the purer the C-PCs obtained.
    Advantageously, when the phycobiliprotein is a phycocyanin resistant to acidic pHs, lowering the pH below the isoelectric point of the APC makes it possible to obtain a phycocyanin comprising a C-PC / APC mixture whose molar ratio is at least 5, preferably at least 10, more preferably at least 15.
    Preferably, the pH of the crude extract in step a) is adjusted to a pH of less than 5. It is then possible to obtain phycocyanin resistant to acidic pH 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.
    In step a), the pH is adjusted by adding a strong or weak acid, mineral or organic, in the form of a solid or solution, the amount of acid added being determined by the pH of the crude extract to be treated and the pH value that a person skilled in the art will seek to obtain. Among the mineral acids well known to those skilled in the art, there will be mentioned more particularly hydrochloric acid and phosphoric acid. Among the organic acids well known to a person skilled in the art, there will be mentioned in particular acetic acid, citric acid, tartaric acid, lactic acid, preferably citric acid. Mention may also be made of acid polyphenols such as rosmarinic acid, tannic acid, digallic acid, quercitannic acid, gallotannic acid, acid tannins such as quercitine, ellagitannins, castalagine, castaline, casuariticine, grandinine , punicaligine punicaline, roburine A, tellimagrandine II, terflavine B, vescaligine, pendunculagine, casuariine, castline, vescaline, preferably tannic acid. Preferably, the acids used are acids authorized for food use, in particular phophoric acid, citric acid or tannic acid.
    For step b) of recovery of the supernatant comprising phycobiliproteins resistant to acidic pH, any separation method known to those skilled in the art can be used, in particular by tangential filtration on ceramic membranes or organic membranes such as hollow polyethersulfone fibers . The thresholds of these filters can be chosen to separate molecules of molecular weight higher or lower than the targeted phycobiliproteins.
    According to a particular embodiment of the invention, the separation in step b) is made by tangential filtration. This stage makes it possible to concentrate and eliminate part of the proteins other than the phycobiliproteins, thereby increasing the purity rate of the final product.
    Step c) of drying / dehydration of phycobiliproteins resistant to acidic pH from the supernatant is carried out by any method of removing the solvent, water in this case, for example by evaporation at atmospheric pressure or under vacuum. Mention will be made in particular of atomization, lyophilization, zeodratation, drying by infrared, or drying by refraction window.
    In the event of evaporation by heating, the skilled person will be careful not to use too high temperatures which may cause denaturation of phycobiliproteins.
    It is possible, after recovery of the supernatant in step b), to recycle the phycobiliproteins contained in the precipitate. To do this, the residual phycobilliproteins are solubilized in an aqueous solution of acidic pH, about 6 or less, pH at which the impurities remain insoluble while the phycobiliproteins are soluble.
    These residual phycobiliproteins are then separated from the impurities and isolated by repeating steps b) and c) of the process. It is an iterative process that can be repeated as many times as necessary. When the conditions used for the process according to the invention allow preferential purification of the C-PCs, the residual phycobiliproteins are a C-PC / APC mixture enriched in APC.
    By recycling the precipitate, phycobiliproteins are then obtained comprising a C-PC / APC mixture whose molar ratio is less than 5, in particular less than 4, advantageously of the order of 3 to 0.1.
    By repeating steps a) to c) of the process, described above, it is possible, by an iterative process, on the one hand to exhaust the precipitate in C-PC, which can be added to the fractions previously obtained to enrich the content and on the other hand 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.
    It is then possible to obtain a mixture of APC comprising less than 5 mol% of CPC, preferably less than 1%, more preferably less than 0.1% of CPC, the percentages being expressed relative to the total sum of APC and ΟΡΟ.
    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 capable of being used as in the field of medical imaging, for example apart from these fluorescent properties
    The invention also relates to phycobiliproteins resistant to acidic pH, in particular phycocyanins, capable of being obtained by the purification process.
    The invention also relates to purified phycocyanins resistant to acidic pH comprising a C-PC / APC mixture whose molar ratio is at least 2.
    In particular, the invention relates to a phycocyanin resistant to acid pH which comprises 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 relative to the total sum of APC and C-PC.
    These C-PCs are known to a person skilled in the art and in particular defined above, in particular those in which the a-subunit of C-PC comprises the protein of SEQ ID NO 1 and the apoprotein of the β subunit of C-PC includes the protein of SEQ ID 2 or variants thereof.
    Advantageously, the variants according to the invention have a sequence identity of at least 83% for the α-subunits of C-PC, and of at least 82% for the p-subunits of C-PC.
    Preferably, the variants according to the invention have an identity of at least 90% for the a (SEQ ID NO 1) and β (SEQ ID NO 2) subunits.
    The invention also relates to a purified phycocyanin enriched in APC capable of being obtained by the process according to the invention.
    In particular, the invention relates to purified phycocyanin which comprises a mixture enriched in APC whose C-PC / APC molar ratio is less than 5, in particular 4, advantageously of the order of 3 to 0.1.
    According to a particular embodiment of the invention, the mixture enriched in APC has an APC / C-PC ratio of at least 5, preferably at least 10, more preferably at least 15.
    According to a more particular embodiment of the invention, phycocyanin consists essentially of APC, with at least 95% molar of APC and less than 5% molar of C-PC, preferably at least 99% molar of APC and less than 1 mol% of C-PC, the percentages being expressed relative to the total sum of APC and C-PC.
    These APCs are known to those skilled in the art and in particular defined above, in particular those whose subunit a of said APC comprises SEQ ID NO 3 or variants thereof and the apoprotein of the β subunit of said APC comprises SEQ ID NO 4 or variants thereof.
    Advantageously, the variants according to the invention have a sequence identity of at least 83% for the α subunits of APC, and of at least 82% for the p subunits of APC.
    A person skilled in the art knows how to measure an identity of protein sequences according to the usual methods available to him, in particular the BLASTP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
    Likewise, 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 test of stability in acid pH, for example by carrying out a test like the test presented in Example 3. of application WO 2017/050918.
    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 modifying its function.
    For example, the substitution of an amino acid at a given position by another chemically equivalent amino acid is a known example of sequence variation which does not substantially affect the properties of the protein.
    These conservative substitutions can be defined as exchanges within the following amino acid groups
    - Ala, Ser, Thr, Pro, Gly
    - Asp, Asn, Glu, Gin
    - His, Arg, Lys
    - Met, Leu, Ile, Val, Cys and
    - Phe, Tyr, Trp
    Thus, the variants of the phycocyanin and / or allophycocyanin apoproteins according to the invention can comprise from 1 to 30 amino acids of difference in number compared to the so-called corresponding reference sequence, particularly as regards the subunits a and / or β of phycocyanin, insofar as the variant obtained retains the properties of the reference protein and the percentages of homology / identity set out above.
    More precisely according to the invention, for the variants of the apoproteins of the subunit to phycocyanins which can be used in the acid compositions according to the invention, derived from substitutions, insertions and / or deletions, they can comprise from 1 to 27 amino acids which differ from the so-called corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity set out above;
    for the variants of the apoproteins of the β subunit of the phycocyanins which can be used in the acid compositions according to the invention, derived from substitutions, insertions and / or deletions, they can comprise from 1 to 30 amino acids different from the so-called corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity set out above;
    for the apoprotein variants of the allophycocyanin subunit which can be used in the acid compositions according to the invention, derived from substitutions, insertions or deletions, they can comprise from 1 to 24 amino acids different from the sequence so-called corresponding reference, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity set out above;
    for the variants of the apoproteins of the β subunit of the allophycocyanins which can be used in the acid compositions according to the invention, derived from substitutions, insertions and / or deletions, they can comprise from 1 to 20 amino acids different from the so-called corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity set out above.
    Very particularly according to the invention, and whatever the reference sequence considered (subunit a and / or β of phycocyanin and / or subunit a and / or β of allophycocyanin) the variants of said subunits can advantageously comprise from 1 to 15 amino acids of difference, preferably from 1 to 10 amino acids of difference, in particular 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids of difference compared to the sequence called the corresponding reference, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity set out above.
    Preferably, the invention relates to C-PC in which the protein of the alpha subunit consists of the protein of SEQ ID 1 and the protein of the beta subunit consists of the protein of SEQ ID 2.
    According to another preferred embodiment, the invention relates to APC in which the protein of the alpha subunit consists of the protein of SEQ ID 3 and the protein of the beta subunit consists of the protein of SEQ ID 4.
    Phycobiliproteins are natural dyes mainly used for coloring food.
    The invention also relates to the use of phycobiliproteins resistant to acidic pH obtained by the process according to the invention and in particular phycocyanins resistant to acidic pH defined above as a color in a food product.
    The invention also relates to a composition, in particular a food composition, comprising phycobiliprotein resistant to acidic pH obtained by the process according to the invention and in particular phycocyanin resistant to acidic pH defined above.
    Such uses and compositions are known to those skilled in the art.
    Preferably, the food product or the food composition is an acid composition, as defined in application WO 2017/050918.
    By acid composition is meant according to the invention any composition comprising a mineral or organic acid and phycocyanin. This composition can be liquid, fluid or viscous, pasty or solid which has an acidic pH and in which phycocyanin resistant to acidic pH is incorporated.
    For aqueous liquid compositions, the pH is measured in the usual manner. For non-aqueous liquid compositions or for pasty or solid compositions, the pH is measured after dissolving the composition in an amount of water sufficient to dissolve the soluble compounds it contains, including mineral or organic acids and phycocyanin.
    Advantageously, the composition according to the invention is an aqueous liquid composition, optionally in the form of a gel, or a pasty or solid composition intended to be dissolved in an aqueous solution or in a solid or pasty composition comprising water. According to another advantageous embodiment of the invention, the acid composition, pasty or solid composition intended to be used and / or stored in a humid environment.
    The mineral or organic acids capable of being used in the compositions according to the invention are well known to those skilled in the art. Among the mineral acids, mention will be made in particular of carbonic, phosphoric, hydrochloric, sulfuric, perchloric, sulphonic and nitric acids. Among the organic acids, mention will be made in particular of citric, lactic, malic, tartaric, succinic acids, advantageously citric acid.
    By acidic food composition according to the invention means any composition intended to be ingested by humans or animals which falls within the preceding definition. Neutraceutical acid compositions should be considered as falling within the definition of acidic food compositions within the meaning of the invention.
    The acidic food compositions according to the invention are well known to those skilled in the art. They may include a vehicle which may include structural constituents associated with active compounds identified with regard to their nutritional benefits or even for their properties beneficial to the health of humans or animals. The acidic food composition according to the invention can also comprise food additives such as texturing agents, flavoring agents, preserving agents, all constituents 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 benefits.
    The acidic food composition according to the invention can be ready for use or else 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.
    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.
    As regards the acid compositions other than foodstuffs according to the invention, they can be, inter alia, pharmaceutical, veterinary or cosmetic and further comprise any additives and / or active agents known and used in this type of composition.
    In a solid, liquid or pasty acid composition according to the invention, phycocyanin can be incorporated for example in the form of a powder. Said acid composition, particularly said acid food composition may then be in any usual known form such as creams, gels, foams, pastes, etc.. Particularly for a solid food composition, mention may in particular be made of cakes or cookies, foods dry to cook, powders to be diluted, gelatinous solid or jelly compositions, foams etc.
    According to the invention, said liquid acid composition may be an aqueous composition in which the phycocyanin is dissolved. It can be in the form of a ready-to-use composition or as a liquid concentrate to be diluted, in particular for its ingestion or to be added to a solid food either for its preparation or for its ingestion, for example a liquid composition concentrated coating or topping which will be placed on a cake to give it its color. Among these concentrated compositions, there may be mentioned syrups, alcoholic or not.
    The liquid acid composition according to the invention may be of 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 customary for the preparation of liquid food compositions.
    According to a particular embodiment of the invention, the liquid food composition can be an acid drink, carbonated or not. These include in particular sodas, juices, sports drinks, exercise drinks, recovery drinks, etc. The compositions of these drinks are well known to those skilled in the art and may include, in particular, sugars, mineral salts, food additives, dissolved gas, etc. The drink according to the invention is a usual acid drink in which the colorant usually used has been replaced in whole or in part by a phycocyanin resistant to an acid pH according to the invention.
    According to the invention, the phycocyanin content in the compositions according to the invention may be in accordance with the uses of a person skilled in the art.
    For example, when phycocyanin is used to color the acid composition, then the content of phycocyanin in said composition may be in accordance with the uses of a person skilled in the art as regards coloring.
    In a liquid acid composition within the meaning of 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.
    In a ready-to-use liquid composition of the beverage type, the phycocyanin content can generally be between 25 mg / l and 300 mg / L, preferably between 50 mg / L and 100 mg / L.
    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.
    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.
    EXAMPLES
    Example 1. Purification by acid precipitation on fresh cell.
    A Galdieria sulphuraria cell fermentation must, centrifuged then rinsed with an equivalent volume of water, undergoes mechanical grinding in order to release the phycobiliproteins in an aqueous phase at pH 6. The ground material is acidified in steps of 0.5 pH unit per addition of citric acid. At each stage, a sample of the mixture is taken and then centrifuged for 10 min at 11,000 g. The supernatant containing the phycobiliproteins is removed and the purity index measured by making the absorbance ratio at 618 nm over the absorbance at 280 nm with a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).
    It is clear that the lower the pH, the higher the purity index (Figure 1). This increase in the purity index reflects a drop in contaminating proteins in the supernatant, while C-PC, for the most part, remains in the supernatant. Due to its resistance to acidic pH, there is no significant loss of the C-PC content in the supernatant (Figure 2). Surprisingly, allophycocyanin (APC) completely disappears from the supernatant for pH values below 5, to end up in the pellet with the other precipitated proteins and cellular debris (Figure 3). In this pellet there is also C-PC and APC with a higher APC content (Figure 3).
    Acidification also makes it possible to obtain a better separation of the liquid and solid phases and to obtain a pellet of cellular debris and proteins which is more compact and easier to separate from the aqueous phase.
    Example 2. Purification by acid precipitation on lyophilized cell and rehydrated.
    A Galdieria sulphuraria cell fermentation must, centrifuged and then rinsed with an equivalent volume of water, undergoes mechanical grinding in order to release the phycobiliproteins in an aqueous phase at pH 6. The ground material is then lyophilized. The lyophilized dry matter is suspended in a volume of water equivalent to the initial volume of the must and then acidified in steps of 0.5 pH unit by adding citric acid. At each stage, a sample of the mixture is taken and then centrifuged for 10 min at 11,000 g. The supernatant containing the phycobiliproteins is removed and the purity index measured by making the absorbance ratio at 618 nm over the absorbance at 280 nm with a spectrophotometer (Amersham Biosciences Ultra Spec 2100 Pro).
    As previously described in Example 1, we can see an increase in the purity index correlated with a drop in pH (Figure 4). In this case too, acidification also makes it possible to obtain a better separation of the liquid and solid phases and to obtain a pellet of cellular debris and of proteins more compact and easier to separate from the aqueous phase. Similar to what was observed in Example 1, the APC is found in the pellet and not in the aqueous phase (Figure 5) for pHs below 5. For pHs between 6 and 5, the amount of APC decreases in the supernatant as a function of the drop in pH.
    Example 3. Purification and concentration of C-PC by tangential filtration.
    The crude extract after acid precipitation and centrifugation is filtered on a tangential hollow fiber type filtration module. Filtration through this mesh eliminates part of the proteins other than C-PC and thus increases the purity index (Figure 6). The purity index that can be reached by this method approaches the values normally obtained by much more complex methods involving biphasic extractions, or precipitation with ammonium sulfate, see even chromatographic methods (Soresen et al., 2013 ; Cruz de Jesûs et al., 2006). In parallel with the purification, this filtration stage makes it possible to remove the water from the C-PC extract (Figure 7), and to facilitate the drying of the product thereafter.
    REFERENCES
    - Moon, Myounghoon, Sanjiv K Mishra, Chul Woong Kim, William I Suh, Min S Park, and Ji-Won Yang. “Isolation and Characterization of Thermostable Phycocyanin from Galdieria Sulfuraria,” Korean journal of Chemical engineering, 31 (2014): 16.
    - Verônica Cruz de Jesûs, Gabriel Alfonso Gutiérrez-Rebolledo, Marcela Hernândez-Ortega, Lourdes Valadez-Carmona, Angélica Mojica-Villegas, Gabriela Gutiérrez-Salmeân and Germân Chamorro-Cevallos. Methods for
    Extraction, Isolation and Purification of C-phycocyanin: 50 years of Research in Review. (2016) Int J Food Nutr Soi 3 (3): 1-10.
    - Laila Sorensen, Andrea Hantke, Niels T. Eriksen. Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria. J Soi Food Agric. 2013 Sep; 93 (12): 2933-8.
    - Eriksen NT. Production of phycocyanin - a pigment with applications in biology, biotechnology, foods and medicine. Microbiol Biotechnol app.
    2008 Aug; 80 (1): 114.
    权利要求:
    Claims (17)
    [1" id="c-fr-0001]
    1. Process for the purification of phycobiliproteins resistant to acid pH from a crude extract of phycobiliproteins resistant to acid pH originating from cells of microorganisms producing phycobiliproteins, characterized in that it comprises the steps of
    a) adjustment of the pH of the raw extract of phycobiliproteins resistant to acidic pHs to a pH below 6 so as to precipitate organic materials other than phycobiliproteins resistant to acidic pHs,
    b) recovery of the supernatant comprising phycobiliproteins resistant to acid pH and
    c) isolation of phycobiliproteins resistant to acidic pH from the supernatant.
    [2" id="c-fr-0002]
    2. Method according to claim 1, characterized in that the phycobiliprotein is phycocyanin resistant to acid pH.
    [3" id="c-fr-0003]
    3. Method according to one of claims 1 or 2, characterized in that the phycocyanin comprises a mixture of c-phycocyanin (C-PC) and of allophycocyanin (APC), the C-PC / APC molar ratio being at least minus 5.
    [4" id="c-fr-0004]
    4. Method according to one of claims 1 to 3, characterized in that the pH of the culture medium in step a) is adjusted to a pH below 5.
    [5" id="c-fr-0005]
    5. Method according to claim 4, characterized in that the phycobiliprotein comprises at least 95% of C-PC resistant to acidic pH and less than 5 mol% of APC, the percentages being expressed relative to the total sum of APC and C-PC.
    [6" id="c-fr-0006]
    6. Method according to one of claims 1 to 5, characterized in that the recovery of the supernatant is made by filtration.
    [7" id="c-fr-0007]
    7. Method according to one of claims 1 to 6, characterized in that the phycobiliprotein-producing microorganism is chosen from strains of microalgae of the genera Cyanidioschyzon, Cyanidium or Galdieria.
    [8" id="c-fr-0008]
    8. Method according to claim 7, characterized in that the microalga is chosen from the strains Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae.
    [9" id="c-fr-0009]
    9. Method according to one of claims 1 to 8, characterized in that the phycobiliproteins contained in the pellet are dissolved in an aqueous solution of acid pH and separated from the impurities and isolated by repeating steps b) and c).
    [10" id="c-fr-0010]
    10. Phycobiliprotein resistant to acid pH obtained by the process according to one of claims 1 to 9.
    5
    [11" id="c-fr-0011]
    11. Phycocyanin resistant to acidic pH comprising a mixture of cphycocyanin (C-PC) and of allophycocyanin (APC), characterized in that the C-PC / APC molar ratio is at least 5.
    [12" id="c-fr-0012]
    12. Phycocyanin according to claim 11, characterized in that it comprises less than 5 mol% of APC.
    10
    [13" id="c-fr-0013]
    13. Phycocyanin according to one of claims 11 or 12, characterized in that the apoprotein of the α-subunit of C-PC comprises SEQ ID NO 1 and the apoprotein of the β subunit of C -PC includes SEQ ID 2 or variants thereof.
    [14" id="c-fr-0014]
    14. Use of a phycobiliprotein according to claim 10 or of a
    [15" id="c-fr-0015]
    15 phycocyanin according to 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 one of claims 11 to 13.
    20
    [16" id="c-fr-0016]
    16. Phycocyanin comprising a mixture of allophycocyanin (APC) and c-phycocyanin (C-PC) whose C-PC / APC molar ratio is less than 5.
    [17" id="c-fr-0017]
    17. Phycocyanin according to claim 16, characterized in that the apoprotein of the AP subunit a of the APC comprises SEQ ID NO 3 and the apoprotein of the β subunit of the APC comprises the SEQ ID 4 or variants thereof.
    1/4
    2.30
    Purity (A618 / A280)
    6 5.5 5 4.5 4 3.5
    PH
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    同族专利:
    公开号 | 公开日
    RU2019132193A|2021-04-30|
    RU2019132193A3|2021-11-26|
    CA3057780A1|2018-10-04|
    AU2018246351A1|2019-10-10|
    BR112019020420A2|2020-06-09|
    FR3064635B1|2021-07-23|
    EP3601318A2|2020-02-05|
    US20200115423A1|2020-04-16|
    IL269540D0|2019-11-28|
    JP2020512334A|2020-04-23|
    MX2019011554A|2019-11-28|
    WO2018178334A2|2018-10-04|
    KR20190141135A|2019-12-23|
    CN110461866A|2019-11-15|
    WO2018178334A3|2018-11-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2005065697A1|2003-12-12|2005-07-21|Florence Minard|Method for the photostabilisation of phycobiliproteins in an aqueous extract, compositions containing stabilised phycobiliproteins and use of stabilised phycobiliproteins|
    WO2015090697A1|2013-12-18|2015-06-25|Basf Se|Stabilized phycocyanin for blue color|
    WO2016099261A1|2014-12-16|2016-06-23|Rijksuniversiteit Groningen|Natural blue photopigments, methods for producing them and to uses thereof as colorant.|
    WO2017050918A1|2015-09-25|2017-03-30|Fermentalg|Acid composition comprising a phycocyanin|WO2019228947A1|2018-05-31|2019-12-05|Fermentalg|Method for cultivating unicellular red algaeon a mixture of substrates|
    FR3091640B1|2019-01-11|2021-06-11|Fermentalg|PHYCOCYANIN PURIFICATION PROCESS|
    FR3091703B1|2019-01-11|2021-02-12|Fermentalg|PHYCOCYANIN EXTRACTION PROCESS|
    FR3092586A1|2019-02-08|2020-08-14|Fermentalg|OPTIMIZED PROCESS FOR INDUSTRIAL EXPLOITATION OF SINGLE-CELLS RED ALGAE|
    BE1027115B1|2019-03-12|2020-10-12|B Blue Nutraceuticals S A|LIQUID COMPOSITION INCLUDING PHYCOCYANIN|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1752674|2017-03-30|
    FR1752674A|FR3064635B1|2017-03-30|2017-03-30|PURIFICATION OF PHYCOBILIPROTEINS|FR1752674A| FR3064635B1|2017-03-30|2017-03-30|PURIFICATION OF PHYCOBILIPROTEINS|
    MX2019011554A| MX2019011554A|2017-03-30|2018-03-30|Purification of phycobiliproteins.|
    PCT/EP2018/058294| WO2018178334A2|2017-03-30|2018-03-30|Purification of phycobiliproteins|
    RU2019132193A| RU2019132193A3|2017-03-30|2018-03-30|
    CA3057780A| CA3057780A1|2017-03-30|2018-03-30|Purification of phycobiliproteins|
    BR112019020420A| BR112019020420A2|2017-03-30|2018-03-30|purification of phycobiliproteins|
    CN201880022959.1A| CN110461866A|2017-03-30|2018-03-30|The purifying of phycobniliprotein|
    AU2018246351A| AU2018246351A1|2017-03-30|2018-03-30|Purification of phycobiliproteins|
    US16/497,510| US20200115423A1|2017-03-30|2018-03-30|Purification of phycobiliproteins|
    EP18714772.3A| EP3601318A2|2017-03-30|2018-03-30|Purification of phycobiliproteins|
    JP2019552498A| JP2020512334A|2017-03-30|2018-03-30|Purification of phycobiliprotein|
    KR1020197029453A| KR20190141135A|2017-03-30|2018-03-30|Purification of Picobiliprotein|
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