EP3601318A2 - Aufreinigung von phycobiliproteinen - Google Patents

Aufreinigung von phycobiliproteinen

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

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Classifications

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

Definitions

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

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EP18714772.3A 2017-03-30 2018-03-30 Aufreinigung von phycobiliproteinen Pending EP3601318A2 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR1752674A FR3064635B1 (fr) 2017-03-30 2017-03-30 Purification des phycobiliproteines
PCT/EP2018/058294 WO2018178334A2 (fr) 2017-03-30 2018-03-30 Purification des phycobiliproteines

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EP3601318A2 true EP3601318A2 (de) 2020-02-05

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FR3091640B1 (fr) 2019-01-11 2021-06-11 Fermentalg Procédé de purification de phycocyanines
FR3091703B1 (fr) 2019-01-11 2021-02-12 Fermentalg Procédé d’extraction de phycocyanines
FR3092586A1 (fr) * 2019-02-08 2020-08-14 Fermentalg Procédé optimisé d’exploitation industrielle d’algues rouges unicellulaires
BE1027115B1 (fr) * 2019-03-12 2020-10-12 B Blue Nutraceuticals S A Composition liquide comprenant de la phycocyanine
EP3939437A1 (de) * 2020-07-17 2022-01-19 GNT Group B.V. Zusammensetzung mit spirulina-extrakt
AU2022334888A1 (en) * 2021-08-24 2024-03-07 The Williamson Group, Llc Improved stabilization of phycocyanins in acidic compositions
WO2023085922A1 (en) * 2021-11-15 2023-05-19 Ful Foods B.V. A method of producing a composition, an water soluble extract composition and a non-water soluble extract composition, each composition being a phycobiliprotein containing micro-organism-based composition
JPWO2023112407A1 (de) * 2021-12-13 2023-06-22
FR3130842A1 (fr) 2021-12-22 2023-06-23 CarbonWorks Procede de captation des phytotoxines dans un reacteur biologique

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FR2863615A1 (fr) * 2003-12-12 2005-06-17 Minard Florence Procede de photo-stabilisation de phycobiliproteines dans un extrait aqueux, compositions contenant des phycobiliproteines stabilisees et utilisation de phycobiliproteines stabilisees
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WO2016099261A1 (en) * 2014-12-16 2016-06-23 Rijksuniversiteit Groningen Natural blue photopigments, methods for producing them and to uses thereof as colorant.
FR3041653B1 (fr) * 2015-09-25 2017-12-29 Fermentalg Procede de culture d'algues, particulierement d'algues rouges unicellulaires (arus)
JP6681065B2 (ja) * 2016-01-14 2020-04-15 学校法人明治大学 食品、食品の加熱処理方法、フィコシアニン含有物の製造方法、及び食品の製造方法

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CA3057780A1 (en) 2018-10-04
IL269540A (en) 2019-11-28
KR20190141135A (ko) 2019-12-23
FR3064635B1 (fr) 2021-07-23
JP2020512334A (ja) 2020-04-23
WO2018178334A3 (fr) 2018-11-22
AU2018246351B2 (en) 2023-12-21
MX2019011554A (es) 2019-11-28
JP2023062127A (ja) 2023-05-02
JP7313283B2 (ja) 2023-07-24
AU2024201833A1 (en) 2024-05-30
BR112019020420A2 (pt) 2020-06-09
CN110461866A (zh) 2019-11-15
WO2018178334A2 (fr) 2018-10-04
US20200115423A1 (en) 2020-04-16
RU2019132193A3 (de) 2021-11-26
FR3064635A1 (fr) 2018-10-05
IL269540B1 (en) 2024-09-01
AU2018246351A1 (en) 2019-10-10
RU2019132193A (ru) 2021-04-30
CN118440167A (zh) 2024-08-06

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