Acidic compositions comprising phycocyanin

Abstract

The present invention relates to acidic compositions, in particular acidic food compositions, comprising at least one phycocyanin that is resistant to pH acid.

Description

[0001] This application is a divisional application of an invention patent application with an application date of September 22, 2016, application number 201680055641.4, and invention name “Acidic composition containing phycocyanin”. Field of the Invention

[0002] The present invention relates to an acidic composition, in particular an acidic food composition, comprising at least one acidic-pH-stable phycocyanin. Prior art

[0003] Phycocyanin is a food coloring that imparts a blue color to the products to which they are added. Phycocyanin extracted from Spirulina is currently the only natural blue coloring approved by the US-FDA (FR File No.: 2013-19550). It is sold in liquid or powder form for use as a blue coloring in food.

[0004] However, phycocyanin derived from Spirulina has the disadvantage of being unstable at acidic pH (below 5), which results in loss of coloration and leads to precipitation, which limits its use. In the best case, loss of stability occurs around pH 4 (cf. phycocyanin derived from Spirulina). Technical specifications; http: / / www.dlt–spl.co.jp / business / en / spirulina / linablue.html).

[0005] Therefore, there are many acidic food compositions, especially carbonated or non-carbonated beverages, in which phycocyanin derived from Spirulina cannot be used as a food coloring or for its antioxidant properties.

[0006] There is a need to identify new phycocyanins that are stable at acidic pH, particularly at acidic pH below 4. pH resistance or stability is measured herein as less than 10% loss of coloration after a minimum exposure of 10 minutes at acidic pH. pH stability can be measured by other methods, such as physical characteristics of the phycocyanin in an acidic composition as a function of time. Summary of the invention

[0007] Therefore, the present invention relates to an acidic composition, in particular an acidic food composition, which may comprise at least one acidic pH resistant phycocyanin. Advantageously, the phycocyanin may be a phycobiliprotein, the apolipoprotein of which comprises at least the protein of SEQ ID NO 1 or SEQ ID 2 or a variant thereof.

[0008] According to the present invention, the phycocyanin may specifically be phycocyanin extracted from Galdieriaceae, more specifically phycocyanin extracted from Galdieria.

[0009] The acidic composition according to the invention, in particular the acidic food composition, may be in the form of a solid or paste or a liquid, in particular an optionally carbonated beverage.

[0010] The acidic composition, particularly the acidic food composition according to the present invention means a composition having a pH of 4 or less, preferably having a pH of 2 to 4, and more preferably having a pH of 2.5 to 3.5. DETAILED DESCRIPTION OF THE INVENTION

[0011] Phycocyanins and allophycocyanins are phycobiliproteins comprising α and β subunits consisting of an apolipoprotein covalently bound to a chromophore. The different phycocyanins are essentially distinguished by the sequence of their α and β-subunit apolipoproteins.

[0012] According to a specific embodiment of the present invention, the acidic composition, in particular the acidic food composition, comprises acidic-pH-resistant phycocyanin, wherein the α-subunit apolipoprotein of the acidic-pH-resistant phycocyanin comprises SEQ ID NO 1 (Accession No. YP_009051179.1) or a variant thereof, and the β-subunit apolipoprotein thereof comprises SEQ ID 2 (Accession No. YP_009051180.1) or a variant thereof.

[0013] According to a preferred embodiment of the present invention, the acidic composition, in particular the acidic food composition, comprises acidic-pH resistant phycocyanin, whose α-subunit apolipoprotein consists of SEQ ID NO 1 (Accession No. YP_009051179.1) or a variant thereof, and whose β-subunit apolipoprotein consists of SEQ ID 2 (Accession No. YP_009051180.1) or a variant thereof.

[0014] According to another embodiment of the present invention, the acidic composition, in particular the acidic food composition, may further comprise allophycocyanin in combination with phycocyanin.

[0015] Advantageously, the α-subunit apolipoprotein of allophycocyanin comprises SEQ ID NO 3 (Accession No. YP_009051103.1) or a variant thereof, and the β-subunit apolipoprotein comprises SEQ ID NO 4 (YP_009051104.1) or a variant thereof.

[0016] According to another preferred embodiment of the present invention, the α-subunit apolipoprotein of the allophycocyanin consists of SEQ ID NO 3 (Accession No. YP_009051103.1) or a variant thereof, and the β-subunit apolipoprotein consists of SEQ ID NO 4 (YP_009051104.1) or a variant thereof.

[0017] The amino acid composition of a protein can impart different properties to the protein depending on the amino acid composition. Among other things, the characteristics of a protein depend on its amino acid composition and its isoelectric point (pi). The isoelectric point is the pH of a solution at which a protein carries no net charge, or in other words the pH at which the molecule is electrically neutral, and the proteins tend to attract each other, aggregate, and precipitate. At pH above their isoelectric points, proteins tend to be negatively charged and repel each other.

[0018] Comparative analysis of the isoelectric points of various proteins using a computer program described by Patrickios and Yamasaki (Polypeptide Amino Acid Composition and Isoelectric Point. II. Comparison between Experiment and Theory. Analytical Biochemistry. 231, 1, 1995: 82-91. 1995) showed a certain correlation between theoretical calculations and the acidic pH resistance actually observed.

[0019] Studies conducted by the applicant have shown that the acidic pH resistance of phycocyanins can be linked to the amino acid sequence of the alpha subunit of said phycocyanins. Furthermore, it is noteworthy that the identification of the first 26 amino acids in the amino acid sequence of the alpha subunit of phycocyanins appears to be particularly important. This is particularly the case for phycocyanins obtained by culturing microalgae strains of the genera Cyanidioschyzon, Cyanidium or Galdieria, more particularly Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzonmerolae.

[0020] Thus, preferably, the composition according to the invention may comprise at least one phycocyanin, wherein at least one apolipoprotein, in particular the alpha subunit thereof, may have a low isoelectric point allowing for better stability at acidic pH.

[0021] A low isoelectric point means an isoelectric point of 3 or less, preferably 2.5 or less, more preferably 2.2 or less.

[0022] Therefore, more preferably, the composition according to the present invention may comprise at least one phycocyanin, wherein at least one apolipoprotein, in particular the α subunit thereof, may have an isoelectric point of 3 or less, preferably 2.5 or less, more preferably 2.2 or less.

[0023] Therefore, according to a specific embodiment of the present invention, the acidic composition, in particular the acidic food composition, may comprise at least one phycocyanin, whose α-subunit apolipoprotein may have a low isoelectric point, and more specifically comprises at least one phycocyanin, whose α-subunit apolipoprotein may comprise SEQ ID NO 1, or a variant thereof.

[0024] Therefore, according to another specific embodiment of the present invention, the acidic composition, in particular the acidic food composition, comprises at least one phycocyanin, whose α-subunit apolipoprotein may have a low isoelectric point, and more specifically comprises at least one phycocyanin, whose α-subunit apolipoprotein may consist of SEQ ID NO 1 or a variant.

[0025] A variant according to the present invention means a protein sequence corresponding to a reference sequence, in which case the protein represented by SEQ ID NO 1 or SEQ ID NO 2 or SEQ NO 3 or SEQ NO 4 is modified by one or more substitutions, insertions or deletions of one or more amino acids of the reference sequence and has the same functional properties as the reference sequence.

[0026] Preferably, the variant according to the invention has at least 83% sequence identity with the alpha subunit of phycocyanin and at least 82% sequence identity with the beta subunit of phycocyanin.

[0027] Preferably, the variant according to the invention has at least 90% identity with the alpha (SEQ ID NO 1) and beta (SEQ ID NO 2) subunits.

[0028] Similarly, for allophycocyanin, the variant preferably has at least 89% sequence identity to the alpha subunit of allophycocyanin and at least 90% sequence identity to the beta subunit of allophycocyanin.

[0029] Those skilled in the art know how to measure protein sequence identity using conventional methods at their disposal, in particular the BLASTP program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0030] Similarly, the person skilled in the art knows how to identify variants of the sequence and confirm that they retain the same structural properties by simple stability experiments in acidic pH, for example by carrying out a test such as the one proposed in Example 3.

[0031] Those skilled in the art are aware that a polypeptide can be modified by substitution, insertion and / or deletion of at least one amino acid without substantially altering its function.

[0032] For example, it is known that substitution of an amino acid at a given position by another chemically equivalent amino acid is an example of a sequence change that does not substantially affect the properties of the protein.

[0033] These "conservative" substitutions can be defined as exchanges within the following amino acid groups:

[0034] -Ala, Ser, Thr, Pro, Gly

[0035] -Asp, Asn, Glu, Gln

[0036] -His, Arg, Lys

[0037] -Met, Leu, Ile, Val, Cys and

[0038] -Phe, Tyr, Trp

[0039] Thus, variants of the apolipoproteins of phycocyanin and / or allophycocyanin according to the invention may comprise from 1 to 30 amino acid differences relative to the corresponding reference sequence, in particular concerning the α and / or β subunit of phycocyanin, as long as the variants obtained retain the properties of the reference protein and the percentage homology / identity mentioned above.

[0040] More precisely according to the invention,

[0041] - The α-subunit apolipoprotein variants of phycocyanin used in the acidic composition according to the invention, derived from substitutions, insertions and / or deletions, may contain differences of 1 to 27 amino acids relative to the corresponding reference sequence, provided that the variants obtained retain the properties of the reference protein and the percentage identities mentioned above.

[0042] - The β-subunit apolipoprotein variants of phycocyanin used in the acidic composition according to the invention, derived from substitutions, insertions and / or deletions, may contain differences of 1 to 30 amino acids relative to the corresponding reference sequence, as long as the variant obtained retains the properties of the reference protein and the percentage identities mentioned above.

[0043] - The α-subunit apolipoprotein variants of allophycocyanin used in the acidic composition according to the invention, resulting from substitutions, insertions and / or deletions, may contain from 1 to 24 amino acid differences relative to the corresponding reference sequence, provided that the variants obtained retain the properties of the reference protein and the percentage identities stated above.

[0044] - The β-subunit apolipoprotein variants of allophycocyanin used in the acidic composition according to the invention, derived from substitutions, insertions and / or deletions, may contain differences of 1 to 20 amino acids relative to the corresponding reference sequence, as long as the variants obtained retain the properties of the reference protein and the percentage identities mentioned above.

[0045] More specifically according to the present invention, and regardless of the reference sequence considered (phycocyanin alpha and / or beta subunit and / or allophycocyanin alpha and / or beta subunit), the variants of said subunits may preferably comprise 1 to 15 amino acid differences, preferably 1 to 10 amino acid differences, in particular 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acid differences relative to the corresponding reference sequence, as long as the variant obtained retains the properties of the reference protein and the percentage identities mentioned above.

[0046] According to the present invention, phycocyanin or its variants, alone or in admixture with allophycocyanin or its variants, for use in acidic compositions, especially acidic food compositions, can be obtained by culturing natural organisms that naturally express phycocyanin or its variants of interest or by culturing organisms that are genetically transformed to express phycocyanin or its variants of interest selected for their ability to produce said phycocyanin or its variants.

[0047] Exemplary native organisms that naturally express phycocyanin or variants thereof of interest for use in compositions according to the present invention include algae (or microalgae) of the order Cyanidiales.

[0048] The order Cyanidiales includes the families Cyanidiaceae and Galdieriaceae, which are themselves subdivided into the genera Cyanidioschyzon, Cyanidium and Galdieria, and members thereof include, inter alia, the species Cyanidioschyzonmerolae 10D, Cyanidioschyzon merolae DBV201, Cyanidiumcaldarium, Cyanidiumdaedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieriadaedala, Galdieria maxima, Galdieria partita and Galdieria sulphuraria. Galdieria sulphuraria (also known as Cyanidium caldarium) strain UTEX #2919 may be specifically mentioned.

[0049] Thus, according to an embodiment of the present invention, the acidic composition, in particular the acidic food composition, comprises acid-pH resistant phycocyanin from natural organisms such as algae or microalgae of the order Cyanidiales, in particular from the families Cyanidiaceae and Galdieriaceae.

[0050] More specifically according to the invention, the acidic composition, in particular the acidic food composition, comprises an acid-pH resistant phycocyanin from a natural organism belonging to the genera Cyanidioschyzon, Cyanidium, Galdieria, preferably a species selected from the genera Cyanidium and Galdieria.

[0051] Even more particularly according to the invention, the acidic composition, in particular the acidic food composition, comprises acid-pH resistant phycocyanin from a natural organism chosen 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 and Galdieria sulphuraria.

[0052] A preferred form of the acidic composition, in particular the acidic food composition, according to the present invention comprises acid-pH-resistant phycocyanin from natural microalgae such as Galdieria sulphuraria, Cyanidium caldarium or Cyanidioschyzon merolae. More preferably, the acid-pH-resistant phycocyanin is from natural microalgae selected from Galdieria sulphuraria and Cyanidium caldarium.

[0053] As examples of organisms transformed to express a phycocyanin or a variant thereof of interest selected for its ability to produce said phycocyanin or variant thereof, mention may be made of microorganisms transformed to express the apolipoprotein of SEQ ID NO 1 and / or SEQ ID NO 2 and / or SEQ ID NO 3 and / or SEQ ID NO 4, said microorganisms also comprising the biosynthetic pathways required for the production of the chromophore and its attachment to the apolipoprotein. Yeasts may in particular be mentioned as microorganisms that may be modified to produce phycocyanin and / or allophycocyanin for use in the food composition according to the invention.

[0054] A person skilled in the art will readily find in the prior art descriptions of methods for culturing natural and / or modified organisms that produce phycocyanin for use in the compositions according to the invention.

[0055] For example, the cultivation of Cyanidiaceae or Galdieriaceae of the order Cyanidiales known to those skilled in the art can be advantageously carried out in a mixed trophic mode, light being generally required for the biosynthesis of pigments.

[0056] Such industrial cultivation can be advantageously carried out in large volume (i.e., 1,000-liter, 10,000-liter, 20,000-liter, 100,000-liter) fermenters. The cultivation can be carried out under conditions known to those skilled in the art. It can be carried out in batch mode, in fed-batch mode or in continuous mode.

[0057] The phycocyanin used in the composition according to the invention may more particularly be extracted from a biomass obtained by cultivating an algae of the order Cyanidiales as defined above, in mixotrophic mode and with light having a wavelength ranging from 400 nm to 550 nm, preferably from 420 nm to 500 nm, preferably from 430 to 480 nm, more preferably of about 455 nm. It may be "white" light with a broad spectrum comprising light of said wavelengths. It may also advantageously be light of a narrow spectrum consisting of said wavelengths.

[0058] Such a process for the industrial preparation of Cyanidiales biomass in mixotrophic mode and the biomass thus obtained are notably described in patent application FR 15 59072 filed on September 25, 2015, the content of which is incorporated herein by reference.

[0059] The object of the present invention is to provide a composition in which phycocyanin is stable at acidic pH. According to the present invention, an acidic composition means any composition comprising an inorganic acid or an organic acid and phycocyanin. The 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.

[0060] 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 a sufficient amount of water to dissolve the soluble compounds contained therein (including inorganic or organic acids and phycocyanin).

[0061] Preferably, the composition according to the invention is an aqueous liquid composition, optionally in the form of a gel, or a paste or solid composition designed to be dissolved in an aqueous solution or a solid or paste composition comprising water. According to another preferred embodiment of the invention, the acidic composition paste or solid composition is intended to be used in a humid environment and / or stored in a humid environment.

[0062] The inorganic or organic acids used in the compositions according to the invention are well known to those skilled in the art. Exemplary inorganic acids include in particular carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulfonic acid and nitric acid. Exemplary organic acids include in particular citric acid, lactic acid, malic acid, tartaric acid, succinic acid, preferably citric acid.

[0063] An acidic food composition according to the invention is taken to mean any composition designed to be ingested by humans or animals and falling within the previous definition.Nutritive acidic compositions have to be considered to fall within the definition of acidic food compositions in the context of the present invention.

[0064] Acidic food compositions according to the present invention are well known to those skilled in the art. They may comprise carriers which may comprise structural components relevant to the active compound identified for its nutritional supply or its health properties that are of benefit to humans or animals. Acidic food compositions according to the present invention may also comprise food additives such as deforming agents, flavoring agents, preservatives or any component well known to those skilled in the art. Carriers may comprise water and / or protein and / or fat and / or fiber and / or sugar. The components of carriers may only have structural properties, but they are usually well known for their nutritional supply.

[0065] The acidic composition according to the present invention may be ready-to-use or added to a solid, pasty or liquid preparation in the form of a food additive to prepare an edible food.

[0066] For food compositions, the acid will preferably be selected from the list of acidulants approved for use in foods, in particular carbonic acid, phosphoric acid, citric acid, malic acid, tartaric acid and lactic acid, more in particular citric acid.

[0067] Furthermore, relating to the non-food acidic compositions according to the invention, they may be pharmaceutical, veterinary or cosmetic compositions and further comprise any additives and / or active agents known and used in such compositions.

[0068] In the solid, liquid or pasty acidic composition according to the present invention, phycocyanin can be incorporated, for example, in the form of a powder. The acidic composition, in particular the acidic food composition, can therefore be in any known conventional form, such as a cream, gel, foam, paste, etc. Exemplary solid food compositions include cakes or biscuits, dry foods for cooking, soluble powders, gelatinous solid compositions (jelly), foams, etc.

[0069] According to the invention, the liquid acidic 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 in the form of a liquid concentrate for dilution, in particular to be ingested or added to solid foods for their preparation or for their ingestion, such as a concentrated liquid "topping" composition applied to cakes to give them their color. Among these concentrated compositions, mention may be made of syrups, which optionally contain alcohol.

[0070] The liquid acidic composition according to the present invention may be of modified viscosity and optionally contain additives such as viscosity agents, gelling agents and other constituent additives known to those skilled in the art and generally used in the preparation of liquid food compositions.

[0071] According to a particular embodiment of the invention, the liquid food composition may optionally be a carbonated acidic beverage. Mention may be made in particular of sodas, fruit juices, sports drinks, energy drinks, recovery drinks, etc. The composition of these beverages is well known to those skilled in the art and may in particular contain sugars, mineral salts, food additives, dissolved gases, etc. The beverage according to the invention is a conventional acidic beverage in which the colorants commonly employed have been replaced in whole or in part by the acid-pH resistant phycocyanin according to the invention.

[0072] According to the present invention, the content of phycocyanin in the composition according to the present invention may be consistent with the practice of those skilled in the art.

[0073] For example, when phycocyanin is used to color an acidic composition, the amount of phycocyanin in the composition can be consistent with the practice of those skilled in the art regarding coloring.

[0074] In the liquid acidic composition in the context of the present invention, the phycocyanin content may be 2.5 mg / L to 2,500 mg / L, preferably 25 mg / L to 300 mg / L.

[0075] In a ready-to-use beverage type liquid composition, the phycocyanin content may generally be 25 mg / L to 300 mg / L, preferably 50 mg / L to 100 mg / L.

[0076] In concentrated liquid compositions for dilution before use, such as syrups, the phycocyanin content may generally be 250 mg / L to 2,500 mg / L, preferably 500 mg / L to 1,000 mg / L.

[0077] In the solid composition, the phycocyanin content may generally be 0.01 mg / g to 10 mg / g, preferably 0.1 mg / g to 5.0 mg / g, and more preferably 0.25 mg / g to 2.5 mg / g.

[0078] As can be seen in the following examples, one of the advantages of the present invention is that the coloration provided by the acid-pH resistant phycocyanin is more stable over time.

[0079] Other aspects and features of the present invention will become more apparent from a review of the embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Amino acid sequences of Galdieria sulphuraria phycocyanin and allophycocyanin apolipoproteins are described having the following:

[0081] SEQ ID NO 1: YP_009051179.1: phycocyanin alpha subunit;

[0082] SEQ ID NO 2: YP_009051180.1: phycocyanin beta subunit;

[0083] SEQ ID NO 3: YP_009051103.1: allophycocyanin alpha subunit; SEQ ID NO 4: YP_009051104.1: allophycocyanin beta subunit;

[0084] Figure 2 Presented relative to phycocyanin from Spirulina Stability curves as a function of pH of phycocyanin extracted from Galdieria sulphuraria (UTEX 2919) and Cyanidioschyzon merolae (ACUF 199) whose apolipoprotein sequence consists of SEQ ID NO 1 or a variant thereof.

[0085] Phycocyanin from Galdieria sulphuraria (UTEX #2919)

[0086] Phycocyanin from Cyanidioschyzon merolae (ACUF 199)

[0087] (––o*––):Lina Phycocyanin from Spirulina platensis (Arthrospira platensis) (data obtained from the website http: / / www.dlt-spl.co.jp / business / en / spirulina / linablue.html), showing the pH value at which precipitation of phycocyanin occurs.

[0088] Figure 3 Shows the color change of an acidic beverage containing phycocyanin over time. W0, W2, W4 and W6: Weeks 0, 2, 4 and 6. Example

[0089] Example 1: Production and extraction of phycocyanin from Galdieria sulphuraria UTEX #2919

[0090] Materials and Methods Strain: Galdieria sulphuraria (also known as Cyanidium caldarium) UTEX #2919 medium

[0091] Mixed nutrition: 30 g / L glycerol, 8 g / L (NH4)2SO4, 1 g / L KH2PO4, 716 mg / L MgSO4, 44 mg / L CaCl2, 3 mL / L Fe–EDTA stock solution (6.9 g / L FeSO4 and 9.3 g / L EDTA–Na2) and 4 mL / L trace metal solution (3.09 g / L EDTA–Na2; 0.080 g / L CuSO4, 5H2O; 2.860 g / L H3BO3; 0.040 g / L NaVO3, 4H2O; 1.820 g / L MnCl2; 0.040 g / L CoCl2, 6H2O; 0.220 g / L ZnSO4, 7H2O; 0.017 g / L Na2SeO3; 0.030 g / L (NH4)6Mo7O 24 ,4H2O).

[0092] Culture conditions:

[0093] The culture was carried out in a 1- to 2-L working volume reactor with a computer-controlled automation system. The culture pH was regulated by adding base (14% ammonia solution (wNH3 / w)) and / or acid (4N sulfuric acid solution). The culture temperature was set to 37°C. The culture was irradiated by a baffle equipped with a white LED or blue LED system (455 nm) in a similar manner to that described in patent WO 2014 / 174182. The cell growth was tracked at different times by measuring the absorbance at 800 nm. The dry mass was measured by filtration.

[0094] The performance characteristics of the cultures at the end of growth are summarized in Table 1 below:

[0095]

[0096] Measurement of intracellular phycocyanin content per gram of dry matter was performed using the extraction and assay described by Moon and colleagues [Moon et al., Korean J. Chem. Eng., 2014, 1–6], while using water instead of phosphate buffer.

[0097] Example 2: Extraction of Phycocyanin

[0098] Galdieria sulphuraria (UTEX#2919) and / or Cyanidioscyizon merolae (ACUF199) strains were cultured under the conditions of Example 1.

[0099] Phycocyanin was then extracted according to a modification of the protocol described by Moon et al., 2014 (op. cit.) The modification consisted in replacing the phosphate buffer used to dissolve the phycocyanin with demineralized water.

[0100] An extract (also called "phycocyanin extract" or "crude extract") is thus obtained, which contains, in addition to the phycocyanin of interest, other water-soluble proteins. The phycocyanin extract may have several possible properties depending on the extraction and / or purification methods used. For example, the crude extract will contain, in addition to the phycocyanin, a higher amount of water-soluble proteins than is found in the purified extract. Purified extract means a crude extract from which a portion of the water-soluble proteins has been removed while retaining the phycocyanin by methods known to those skilled in the art, such as ultrafiltration, hollow fiber filtration or ion exchange chromatography.

[0101] The purity index is traditionally expressed by calculating the ratio of the absorbance of the solution at 618 nm (specific absorbance of phycocyanin) to the absorbance of the solution at 280 nm, the specific absorbance of aromatic amino acids giving an idea of ​​the total protein level. The lower the ratio, the higher the amount of protein other than phycocyanin in the solution.

[0102] Use from Laboratory Tangential flow filtration system was used to purify the crude extract.

[0103]

[0104] Table 2. Purity index measurements of phycocyanin extracts before and after purification

[0105] Example 3: Stability Study of Phycocyanin Extracted from Galdieria sulphuraria (UTEX #2919) and Cyanidioschyzon merolae (ACUF199) Strains and Spirulina (Arthrospira) platensis as a Function of pH

[0106] Galdieria sulphuraria (UTEX#2919) and Cyanidioschyzonmerolae (ACUF199) strains were cultured under the conditions of Example 1.

[0107] By acquiring commercial products from DIC Lifetec Co., Ltd. (Tokyo, Japan) The data of phycocyanin extracted from Spirulina platensis (http: / / www.dlt–spl.co.jp / business / en / spirulina / linablue.html) were used as a reference for the test.

[0108] These tests were performed with phycocyanin prepared in Example 2 and having a purity index of 2.12. Measurement of the blue color was accomplished by measuring the absorbance at 618 nm using an Ultraspeed 2100pro spectrophotometer (Amersham). Percent color loss was calculated relative to the absorbance measurement of the sample under reference conditions (pH 6).

[0109] For the resistance test under acidic conditions, the pH was gradually lowered by adding 5% citric acid solution (Sigma 251275) to the phycocyanin preparation. For each pH value, a sample of the phycocyanin solution was collected and its absorbance was measured at 618 nm 10 minutes after lowering the pH to the desired value.

[0110] exist Figure 2 The results of these tests are presented in .

[0111] Compared with the pH-resistance of Spirulina, the pH-resistance of The extracted phycocyanin showed very good pH-resistance, with less than 10% pigment loss until pH 2.75 (its coloration at pH 3 was 98.25%, at pH 2.75 was 92.4%), with the loss increasing at pH 2.5 (79% at pH 2.5; 75% at pH 2.25; 46% at pH 2).

[0112] Compared with the pH-resistance of Spirulina, the pH-resistance of Cyanidioschyzon merolae (ACUF 199) The phycocyanin extracted from the strain showed good pH-resistance, with coloration of more than 90% at pH 3, 70% at pH 2.75, 40% at pH 2.5, 23% at pH 2.25, and 20% at pH 2.

[0113] Phycocyanin extracted from Spirulina platensis [(––o*––)] strain showed that its coloration at pH 4 was only 90%, at pH 3.8 it was 80%, at pH 3.6 it was 60%, and at pH 3.4 it was 38%. Phycocyanin extracted from Spirulina platensis began to precipitate at pH 3.8.

[0114] In conclusion, Galdieria sulphuraria or Cyanidioschyzon merolae phycocyanin is more resistant to acidic pH than phycocyanin extracted from Spirulina.

[0115] Example 4 - Comparative analysis of apolipoprotein sequences of phycocyanin and allophycocyanin from different microalgae and measurement of their isoelectric points

[0116] In order to identify possible reasons for the increased resistance to acidic pH, the sequences of the α and β subunits of phycocyanin and allophycocyanin produced by various microorganisms, in particular microalgae, were compared using the BLASTP program widely used by those skilled in the art, based on public sequences accessible in databases (see accession numbers). A comparison was made with respect to the apolipoprotein sequence corresponding to the subunits of the Galdieria sulphuraria strain.

[0117] In parallel, the isoelectric points of the α and β subunits of the phycocyanin and allophycocyanin sequences were determined by the computer program described by Patrickios and Yamasaki (1995).

[0118] Phycocyanin and allophycocyanin produced by a strain of Galdieria sulphuraria were used as reference for these studies.

[0119] result

[0120] The results of sequence comparison and pi calculation are presented in Tables 3 and 4 below:

[0121]

[0122]

[0123] Table 3. Comparison of α and β apolipoprotein sequences of phycocyanin from various organisms. The isoelectric point of each protein is shown (Patrickios and Yamasaki, 1995).

[0124]

[0125]

[0126] Table 4. Comparison of α and β apolipoprotein sequences of allophycocyanin from various organisms. The isoelectric point of each protein is shown (Patrickios and Yamasaki, 1995).

[0127] Sequence and isoelectric point comparisons showed that the α subunit of the phycocyanin most resistant to acidic pH had an isoelectric point below 3 and a higher percentage identity with the sequence of Galdieria sulphuraria.

[0128] Example 5: Stability in beverages over time

[0129] The stability test of phycocyanin extracted from UTEX #2919 strain in acidic medium over time was conducted by adding phycocyanin to a lemonade beverage (pH 2.95; as described in Example 6: Beverage 1). After adding 0.025‰ of a phycocyanin apolipoprotein sequence consisting of a variant of SEQ ID NO 1, the beverage was exposed to a day / night cycle (16h / 8h) using artificial light for 6 weeks at room temperature.

[0130] Samples were routinely taken over time to measure absorbance at 618 nm. Aliquots were taken at week 0 (W0), 2 (W2), 4 (W4) and 6 (W6) and the absorbance at 618 nm was measured with an Ultraspeed 2100pro spectrophotometer (Amersham) to define the 100% coloration point. The remaining color was expressed as a percentage of the initial reference value.

[0131] Figure 3 The results of this experiment are shown. Notably, the stable color is lost over time during long-term exposure to light. However, after 6 weeks of exposure, more than 60% of the color is retained.

[0132] Example 6: Example of an acidic beverage in liquid form containing phycocyanin

[0133] The beverage containing phycocyanin may have the following composition:

[0134] Drink 1-Soda Drink:

[0135]

[0136] The pH of this drink is 2.95

[0137] Drink 2 – Health Drink for Athletes:

[0138]

[0139] The pH of this drink is 3.5

[0140] Example 6: Acidic beverage in soluble powder form containing phycocyanin

[0141]

[0142] The powder thus prepared (75 to 110 g) can be dissolved in 1 L of water to obtain a blue colored acidic beverage.

[0143] Example 7: Solid acidic composition containing phycocyanin: Acidic candy:

[0144]

[0145]

[0146] References

[0147] -Moon et al.,Korean J.Chem.Eng.,2014,1–6

[0148] -Patrickios et Yamasaki,Polypeptide Amino Acid Composition andIsoelectricPoint.II.Comparison between Experiment and Theory.Analytical

[0149] Biochemistry.231,1,1995:82–91

[0150] -FR Doc No:2013–19550

[0151] -WO 2014 / 174182

[0152] -FR 15 59072filed on 25September 2015

[0153] -http: / / www.dlt–spl.co.jp / business / en / spirulina / linablue.html

[0154] -http: / / blast.ncbi.nlm.nih.gov / Blast.cgi

[0155] More specifically, the present application provides the following:

[0156] 1. An acidic composition, in particular an acidic food composition, comprising at least one inorganic acid or organic acid and at least one acid-pH resistant phycocyanin.

[0157] 2. The composition according to item 1, characterized in that the phycocyanin is a phycobiliprotein, and its apolipoprotein comprises a protein of SEQ ID NO 1 or SEQ ID 2 or a variant thereof.

[0158] 3. A composition according to item 2, characterized in that the α-subunit apolipoprotein of the phycocyanin comprises SEQ ID NO 1 or a variant thereof, and the β-subunit of the phycocyanin comprises SEQ ID 2 or a variant thereof.

[0159] 4. A composition according to item 3, characterized in that the α-subunit apolipoprotein consists of SEQ ID NO 1 or a variant thereof, and the β-subunit consists of SEQ ID 2 or a variant thereof.

[0160] 5. The composition according to any one of items 1 to 4, characterized in that it further comprises allophycocyanin in combination with the phycocyanin.

[0161] 6. The composition according to item 5, characterized in that the α-subunit apolipoprotein of the allophycocyanin comprises SEQ ID NO 3 or a variant thereof, and the β-subunit of the allophycocyanin comprises SEQ ID NO 4 or a variant thereof.

[0162] 7. The composition according to item 6, characterized in that the allophycocyanin α-subunit apolipoprotein consists of SEQ ID NO 3 or a variant thereof, and the β-subunit apolipoprotein consists of SEQ ID NO 4 or a variant thereof.

[0163] 8. The composition according to any one of items 1 to 7, characterized in that the phycocyanin is phycocyanin extracted from algae (or microalgae) of the order Cyanidiales.

[0164] 9. The composition according to any one of items 1 to 8, characterized in that the phycocyanin is phycocyanin extracted from algae (or microalgae) of the Cyanidiaceae family or the Galdieriaceae family.

[0165] 10. The composition according to any one of items 1 to 9, characterized in that the phycocyanin is phycocyanin extracted from algae (or microalgae) of the genus Cyanidioschyzon, Cyanidium or Galdieria.

[0166] 11. A composition according to any one of items 1 to 10, characterized in that the phycocyanin is phycocyanin extracted from algae (or microalgae) of the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidiumcaldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, preferably from algae (or microalgae) of the species Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae.

[0167] 12. The composition according to any one of items 1 to 11, characterized in that it has a pH of 4 or less.

[0168] 13. The composition according to any one of items 1 to 11, characterized in that it has a pH of 2 to 4.

[0169] 14. A composition according to any one of items 1 to 11, characterised in that it has a pH of about 2.5 to 3.5.

[0170] 15. The composition according to any one of items 1 to 14, characterized in that the α-subunit apolipoprotein of the phycocyanin has an isoelectric point lower than 3.

[0171] 16. The composition according to any one of items 1 to 14, characterized in that the α-subunit apolipoprotein of the phycocyanin has an isoelectric point lower than 2.5.

[0172] 17. The composition according to any one of items 1 to 14, characterized in that the α-subunit apolipoprotein of the phycocyanin has an isoelectric point lower than 2.2.

[0173] 18. The composition according to any one of items 1 to 17, characterized in that it is solid.

[0174] 19. The composition according to claim 18, characterized in that the phycocyanin content is 0.25 mg / g to 2.5 mg / g.

[0175] 20. The composition according to any one of items 1 to 17, characterized in that it is liquid.

[0176] 21. The composition according to claim 15, characterized in that the phycocyanin content is 2.5 mg / L to 2,500 mg / L.

[0177] 22. A carbonated or non-carbonated beverage comprising a liquid acidic composition according to one of items 20 or 21.

Claims

1. An acidic food composition having a pH range of 2.5 to 4 and comprising at least one inorganic acid or organic acid and at least one acid-pH resistant phycocyanin, wherein the acid-pH resistant phycocyanin is stable at a pH of 4 or less, and the acid-pH resistant phycocyanin is selected from Cyanidioschyzon merolae Phycocyanin extracted from algae.

2. The composition according to claim 1, characterized in that It further comprises allophycocyanin in combination with the phycocyanin.

3. The composition according to claim 1 or 2, characterized in that The composition has a pH of 2.5 to 3.

5.

4. The composition according to claim 1 or 2, characterized in that The α-subunit apolipoprotein of the phycocyanin has an isoelectric point below 3.

5. The composition according to claim 1 or 2, characterized in that The α-subunit apolipoprotein of the phycocyanin has an isoelectric point below 2.

5.

6. The composition according to claim 1 or 2, characterized in that The α-subunit apolipoprotein of the phycocyanin has an isoelectric point below 2.

2.

7. The composition according to claim 1 or 2, characterized in that The α-subunit apolipoprotein of the phycocyanin has an isoelectric point of 2.

129.

8. The composition according to claim 1 or 2, characterized in that The β-subunit apolipoprotein of the phycocyanin has an isoelectric point of 4.

112.

9. The composition according to claim 2, characterized in that The α-subunit apolipoprotein of the allophycocyanin has an isoelectric point of 3.

989.

10. The composition according to claim 2, characterized in that The β-subunit apolipoprotein of allophycocyanin has an isoelectric point of 3.

298.

11. The composition according to claim 1 or 2, characterized in that The composition is solid.

12. The composition according to claim 11, characterized in that The phycocyanin content is 0.01 mg / g to 10 mg / g.

13. The composition according to claim 11, characterized in that The phycocyanin content is 0.1 mg / g to 5 mg / g.

14. The composition according to claim 11, characterized in that The phycocyanin content is 0.25 mg / g to 2.5 mg / g.

15. The composition according to claim 1 or 2, characterized in that The composition is a liquid.

16. The composition according to claim 15, characterized in that The phycocyanin content is 2.5 mg / L to 2500 mg / L.

17. The composition according to claim 15, characterized in that The phycocyanin content is 25 mg / L to 300 mg / L.

18. The composition according to claim 15, characterized in that The phycocyanin content is 50 mg / L to 100 mg / L.

19. The composition according to claim 1, characterized in that The inorganic acid is selected from carbonic acid, phosphoric acid and hydrochloric acid.

20. The composition according to claim 1, characterized in that The organic acid is selected from the group consisting of citric acid, lactic acid, malic acid, tartaric acid and succinic acid.

21. A carbonated or non-carbonated beverage comprising the composition according to claim 15.

22. The carbonated or non-carbonated beverage according to claim 21, characterized in that The acidic composition is a liquid composition for dilution before use, and the content of phycocyanin in the liquid composition is 250 mg / L to 2500 mg / L.

23. The carbonated or non-carbonated beverage according to claim 22, characterized in that The content of phycocyanin in the composition is 500 mg / L to 1000 mg / L.

Images