Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • 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
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/185—Vegetable proteins
• • 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
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
  • A23L2/52—Adding ingredients
  • A23L2/58—Colouring agents
• • 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
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
• • 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
  • A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • 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
  • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
  • C12P17/165—Heterorings having nitrogen atoms as the only ring heteroatoms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the present invention relates to an acidic composition, particularly an acidic food composition, comprising at least one stable phycocyanin at acidic pH.
• Phycocyanins are dietary dyes that give products in which they are incorporated a blue color.
• Phycocyanin extracted from spirulina is today the only natural blue pigment approved by the US-FDA (FR Doc No: 2013-19550). It is sold in liquid form, or in powder form for use as a blue pigment in food.
• this phycocyanin extracted from spirulina has the disadvantage of being unstable at acid pH, less than 5, resulting in loss of color and precipitation which limit its use. In the best case, the loss of stability will occur around a pH of 4 (see the technical specifications of phycocyanin extracted from Linablue® spirulina http://www.dlt-spl.co.jp/business/en/ spirulina / linablue.html).
• acidic food compositions including carbonated or non-carbonated beverages, for which phycocyanin extracted from spirulina can be used, both as a food coloring agent and for its antioxidant properties.
• PH stability can be measured by other methods such as physical characterization of phycocyanins within acidic compositions as a function of time.
• the present invention relates to an acid composition, particularly an acidic food composition, which may comprise at least one phycocyanin resistant to acidic pH.
• said phycocyanin may be a phycobiliprotein whose apoprotein may comprise at least the protein of SEQ ID NO 1 or SEQ ID No. 2 or a variant thereof.
• the phycocyanin may be in particular a phycocyanin extracted from a Galdieriaceae, more particularly extracted from Galdieria.
• the acid composition according to the invention, particularly the acidic food composition may be either solid or pasty, or liquid, in particular a carbonated beverage or not.
• acidic composition particularly acidic food composition is meant according to the invention, a composition of pH less than or equal to 4, preferably pH ranging from 2 to 4, more preferably from about 2.5 to 3.5.
• Phycocyanins and allophycocyanins are phycobilliproteins comprising alpha and beta subunits composed of an apoprotein covalently bound to a chromophore.
• the different phycocyanins are essentially distinguished by the sequence of their apoproteins alpha and beta subunits.
• the acidic composition comprises a phycocyanin resistant to acidic pHs, the apoprotein of the ⁇ -subunit comprising SEQ ID NO 1 (accession number YP_009051 179.1) and the apoprotein of the ⁇ subunit comprises SEQ ID 2 (accession number YP_009051 180.1) or variants thereof.
• the acidic composition particularly the acidic food composition, comprises a phycocyanin resistant to acidic pH, the apoprotein of the ⁇ -subunit consisting of SEQ ID No. 1 (accession number YP_009051 179.1) and the apoprotein of the ⁇ subunit consists of SEQ ID 2 (accession number YP_009051 180.1) or variants thereof.
• the acid composition may further comprise an allophycocyanin associated with phycocyanin.
• the apoprotein of the alpha subunit of said allophycocyanin comprises SEQ ID NO 3 (accession number YP_009051 103.1) and the apoprotein of the ⁇ subunit comprises SEQ ID NO 4 (YP_009051 104.1). or variants thereof.
• the apoprotein of the alpha subunit of said allophycocyanin consists of SEQ ID NO 3 (accession number YP_009051 103.1) and the apoprotein of the ⁇ subunit is constituted of SEQ ID NO: 4 (YP_009051 104.1) or variants thereof.
• the amino acid composition of a protein may impart to said protein different properties according to said amino acid composition.
• the characteristics of a protein depend inter alia on its amino acid composition and the value from its isoelectric point (pl).
• the isoelectric point is the pH value of the solution from which the net charge of the protein is zero or, in other words, the pH for which the molecule is electrically neutral and the proteins tend to attract, aggregate and precipitate. At a pH above their isoelectric point the proteins tend to be negatively charged and repel.
• composition according to the invention may comprise at least one phycocyanin of which at least one apoprotein, particularly that the ⁇ -subunit, may have a low isoelectric point allowing a better stability at acidic pH.
• low isoelectric point is meant an isoelectric point of a value less than or equal to 3, preferably less than or equal to 2.5, more preferably less than or equal to 2.2.
• composition according to the invention may comprise at least one phycocyanin of which at least one apoprotein, particularly that of the ⁇ -subunit, may have an isoelectric point less than or equal to 3, preferably less than or equal to 2.5, plus preferably less than or equal to 2.2.
• the acid composition may comprise at least one phycocyanin, the apoprotein of which may have a low isoelectric point, in particular at least one phycocyanin of which the apoprotein of the? -subunit may comprise SEQ ID NO: 1, or a variant.
• the acid composition particularly the acidic food composition, comprises at least one phycocyanin whose apoprotein of the ⁇ -subunit has a low isoelectric point, very particularly at least one phycocyanin whose ⁇ -subunit apoprotein consists of SEQ ID No. 1, or a variant.
• a protein sequence corresponding to a reference sequence in this case the protein represented by SEQ ID NO 1 or SEQ ID NO 2, or SEQ N03, or SEQ N04, modified by one or more substitutions, insertions or deletions of one or more amino acids of the reference sequence and which has the same functional properties as said reference sequence.
• the variants according to the invention have a sequence identity of at least 83% for phycocyanin ⁇ subunits, and at least 82% for beta subunits of phycocyanin.
• 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 variants advantageously have a sequence identity of at least 89% for the ⁇ subunits of allophycocyanin, and at least 90% for the ⁇ subunits of the allophycocyanin.
• 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 ⁇ phycocyanin, insofar as 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 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;
• 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 may comprise from 1 to 24 amino acids of difference relative to the so-called corresponding 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 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 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 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 acids amines 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 phycocyanin or its variants which can be used, alone or as a mixture with an allophycocyanin or its variants, in acidic compositions, particularly in acidic food compositions, can be obtained by culturing a natural organism naturally expressing phycocyanin. or its desired variant or by culturing a genetically transformed organism to express phycocyanin or its selected desired variant on its ability to produce said phycocyanin or its variants.
• the order Cyanidiales includes the families Cyanidiaceae or Galdieriaceae, themselves subdivided into the genera Cyanidioschyzon, Cyanidium or Galdieria, to which belong inter alia the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldahum, Cyanidium daedalum, Cyanidium maximum , Cyanidium partitum. Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria. In particular, the strain Galdieria sulphuraria (also called Cyanidium caldarium) UTEX # 2919 is mentioned.
• the acidic composition particularly the acidic food composition, comprises a phycocyanin resistant to acidic pH which comes from natural organisms such as algae or microalgae of the order of cyanide, especially from of natural organisms belonging to the families of Cyanidiaceae or Galdieriaceae.
• the acid composition particularly the acidic food composition, comprises a phycocyanin resistant to acidic pH from natural organisms which belong to the genera Cyanidioschyzon, Cyanidium, Galdieria, advantageously chosen from the species of the genera Cyanidium and Galdieria
• the acidic composition comprises a phycocyanin resistant to acidic pH from natural organisms chosen from Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum , Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria.
• natural organisms chosen from Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum , Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria.
• the preferred form of the acidic composition, particularly the acidic food composition, according to the invention comprises phycocyanin resistant to acidic pH from a natural microalga such as Galdieria sulphuraria, Cyanidium caldarium or Cyanidioschyzon merolae. More preferably, phycocyanin resistant to acidic pH comes from a natural microalga selected from Galdieria sulphuraria and Cyanidium caldarium.
• a transformed organism for expressing the selected phycocyanin or its selected variant on its ability to produce said phycocyanin or its variants may be mentioned a microorganism transformed so as to express the apoprotein of SEQ ID NO 1 and / or SEQ ID NO 2 and / or SEQ ID NO 3 and / or SEQ ID NO 4, said microorganism also comprising the biosynthetic pathways necessary for the production of the chromophore and its binding to the apoprotein.
• yeasts as microorganisms may be modified to produce phycocyanin and / or allophycocyanin used in food compositions according to the invention.
• the culture of Cyanidiaceae or Galdieriaceae, belonging to the order of Cyanidiales, well known to those skilled in the art can advantageously be performed in mixotrophic mode, the light being generally necessary for the biosynthesis of pigments.
• Such an industrial culture can advantageously be carried out in large volume fermenters of the 1000 liter, 10,000 liter, 20,000 liter, 100,000 liter type.
• the culture can be carried out according to the conditions known to those skilled in the art. It can be performed in batch mode called “batch”, in semi-continuous mode said "fed batch” or in continuous mode.
• the phycocyanin that can be used in the compositions according to the invention can more particularly be extracted from a biomass obtained by cultivating an alga of the order of cyanidiales as defined previously cultivated in a mixotrophy with a light of wavelength between 400 nm and 550 nm, advantageously between 420 nm and 500 nm, preferably between 430 and 480 nm, more preferably about 455 nm. It may be a so-called "white" light, broad spectrum comprising the light of said wavelength. It can also be advantageously a so-called narrow spectrum light consisting of said wavelength.
• the object of the invention is to provide a composition in which the phycocycanine is stable at acidic pH.
• 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 solid or pasty composition comprising 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 furthermore include any additives and / or active agents known and used in this type 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 to cook, the powders to be diluted, the 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, 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 deposited on a cake to bring its color.
• 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 drink according to the invention is a usual acidic beverage in which the 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 phycocyanin content 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.
• Figure 1 depicts the amino acid sequences of the apoproteins of phycocyanin and allophycocyanin of Galdieria sulphuraria with:
• SEQ ID NO. 1 YP_009051 179.1: ⁇ -subunit Phycocyanin;
• SEQ ID NO: 2 YP_009051 180.1: ⁇ subunit of Phycocyanin;
• SEQ ID NO: 3 YP_009051 103.1 A subunit of Allophycocyanin
• SEQ ID NO: 4 YP_009051 104.1 ⁇ Subunit of Allophycocyanin
• Figure 2 shows the pH stability curve of phycocyanins extracted from Galderia sulphuraria (UTEX2919) and Cyanidioschyzon merolae (ACUF 199) whose apoprotein sequence consists of SEQ ID NO 1 or a variant, on which is reported the stability curve of phycocyanin extracted from Linablue® spirulina. (- ⁇ -) Galdieria sulphuraria Phycocyanin (UTEX # 2919)
• Lina Blue® Phycocyanin from Spirulina platensis (Arthrospira platensis) (data obtained from the website http: //www.dlt- spl.co.jp/business/en/spirulina/linablue.html), with indication of the pH at which precipitation of Phycocyanin appears.
• Figure 3 shows the evolution of the color of an acidic beverage comprising phycocyanin over time.
• SO, S2, S4 and S6 week 0, 2, 4, and 6 EXAMPLES
• Galdieria sulphuraria also called Cyanidium caldarium
• the cultures are carried out in reactors of 1 to 2 L of useful volume with dedicated automata and supervision by computer station.
• the pH of the culture is regulated via the addition of base (14% ammonia solution (WNH3 / W) and / or acid (4N sulfuric acid solution) .
• the culture temperature is set at 37 ° C.
• the culture is illuminated by pale counters equipped with a white LED system or blue LED (455 nm) in a manner similar to that described in patent WO 2014/174182.
• the monitoring of the growth of the cells is carried out at different times by absorbance measurement at 800 nm and a measurement of the dry mass is carried out by filtration.
• the phycocyanin is then extracted according to a modification of the protocol described by
• This modification consists of replacing the phosphate buffer used to solubilize phycocyanin with demineralized water.
• An extract (also called “phycocyanin extract” or “crude extract”) is then obtained which comprises, in addition to the desired phycocyanin, other water-soluble proteins.
• phycocyanin extract also called “phycocyanin extract” or “crude extract”
• a crude extract will contain a water-soluble amount of protein, other than phycocyanin, greater than that found in a purified extract.
• Purified extract comprises a crude extract of which a portion of the water-soluble proteins have been removed by ultrafiltration, hollow-fiber filtration or ion-exchange chromatography methods known to those skilled in the art, while retaining phycocyanin.
• the purity index is conventionally expressed by making the absorbance ratio of the solution at 618 nm (specific absorbance of phycocyanin) to that of the specific 280 nm absorbance value of the aromatic amino acids giving an idea of the rate of global proteins. The lower the ratio, the more protein other than phycocyanin in solution.
• the crude extract was purified using the KrosFlow tangential flow filtration system from Spectrum-Labs. Purity Index
• Galdieria sulphuraria (UTEX # 2919) and Cyanidioschizon merolae (ACUF199) and Spirulina (Arthrospira) platensis as a function of pH
• the Galdieria sulphuraria (UTEX # 2919) and Cyanidioschizon merolae (ACUF199) strains were cultured under the conditions of Example 1.
• the pH is lowered gradually by adding a 5% citric acid solution (Sigma 251275) to the phycocyanin preparation.
• a sample of the phycocyanin solution is taken and its absorbance measured at 618 nm, 10 minutes after the pH drop to the desired value.
• Phycocyanin extracted from Galdieria sulphuraria [(- ⁇ -)] has a very good pH resistance compared to that of Spirulina, with less than 10% loss of pigmentation up to pH 2.75 (98.25 % of its color at pH 3, 92.4% at pH 2.75), the loss becoming greater from pH 2.5 (79% at pH 2.5, 75% at pH 2.25, 46% at pH 2).
• [(••• X ⁇ )] has good pH resistance compared to that of Spirulina, with still more than 90% of its coloration at pH 3, 70% at pH 2.75; 40% at pH 2.5; 23% at pH 2.25; 20% at pH 2.
• the phycocyanin extracted from the strain Spirulina platensis [(-o * -)] has only 90% of its coloration at pH 4.80% at pH 3.8; 60% at pH 3.6; 38% at pH 3.4.
• Phycocyanin extracted from Spirulina platensis begins to precipitate from pH 3.8.
• the phycocyanins of Galdieria sulphuraria or Cyanidioschyzon merolae are more resistant to acidic pH than those extracted from Spirulina.
• the value of the isoelectric point of the apoproteins of the ⁇ and ⁇ subunits of the phycocyanins and allophycocyanins whose sequence comparison was performed was determined by the calculation method described by Patrickios and Yamasaki (1995).
• Galdieria sulphuraria (YP_009051 179.1) 162/162 (100%) 2.1 15
• Galdieria sulphuraria (YP_009051 180.1) 171/171 (100%) 4.1 12
• Halothece sp. PCC 7418 (WP 015227202.1) 133/171 (78%) 4.074
• Allophycocyanin A subunit (accession) aa / aa (% ldentity) pi
• Arthrospira jenneri (AEV40869.1) 131/161 (81%) 4.008
• Arthrospira platensis (CAA65141.1) 131/161 (81%) 4.054
• Halothece sp. PCC 7418 (WP_015226049.1) 133/161 (83%) 3.724
• Galdieria sulphuraria (YP_009051 104.1) 161/161 (100%) 3.502
• Arthrospira jenneri (AEV40870.1) 138/161 (86%) 3.502
• Nostoc sp. PCC 7120 (WP_010994199.1) 138/160 (86%) 3.657
• the phycocyanin stability test extracted from the UTEX strain # 2919, in an acidic medium over time was carried out by adding the phycocyanin in a lemonade-type beverage (as described in Example 6: drink 1) of pH 2.95. After addition of 0.025% o phycocyanin whose apoprotein sequence consists of a variant of SEQ ID NO 1, the beverage was exposed to a day / night cycle (16/8 hour) with artificial light for 6 hours. weeks, at room temperature.
• Figure 3 shows the results of this experiment. There is a steady loss of color over time with lasting exposure to light. However after 6 weeks of exposure there is still more than 60% of the color.
• Example 6 Examples of Acidic Drinks in Liquid Form Comprising Phycocyanin
• Drinks containing phycocyanin may have the following composition: Drink 1 - Soda drink:
• the pH of this drink is 2.95
• the pH of this drink is pH 3.5
• Example 6 Acid Drink in the form of a powder to be dissolved, comprising phycocyanin
• the powder (75 to 110 g) thus prepared can be dissolved in 1 L of water to obtain an acidic beverage colored blue.
• Example 7 Solid acid composition comprising phycocyanin: acid confectionery:

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