US20180271119A1

US20180271119A1 - Acid composition comprising a phycocyanin

Info

Publication number: US20180271119A1
Application number: US15/762,659
Authority: US
Inventors: Olivier CAGNAC
Original assignee: Fermentalg SA
Current assignee: Fermentalg SA
Priority date: 2015-09-25
Filing date: 2016-09-22
Publication date: 2018-09-27
Also published as: WO2017050917A1; EP3353282A1; BR112018005928A2; FR3041653A1; JP2018529343A; CN108431202A; US11162126B2; US20180274002A1; WO2017050918A1; RU2739847C2; FR3041505B1; JP6976933B2; RU2018112989A3; JP2024010035A; CN108431202B; JP2018527940A; EP3353283A1; CN116349890B; CN116349890A; FR3041505A1

Abstract

The invention relates to an acid composition, particularly an acid food composition, comprising at least one phycocyanin resistant to pH acid.

Description

FIELD OF THE INVENTION

The present invention concerns an acidic composition, particularly an acidic food composition, comprising at least one acidic-pH-stable phycocyanin.

STATE OF THE ART

Phycocyanins are food colourings which give a blue colour to the products to which they are added. 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 blue pigment in foods.
That spirulina-derived phycocyanin, however, has the disadvantage of being unstable at acidic pH, below 5, leading to loss of colouring and to precipitation, which limit its use. In the best case, the loss of stability occurs around pH 4 (cf. technical specifications of the spirulina-derived phycocyanin Linablue®; http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html).
Consequently, there are many acidic food compositions, notably carbonated or noncarbonated beverages, for which spirulina-derived phycocyanin cannot be used as food colouring or for its antioxidant properties.
A need exists to identify new phycocyanins stable at acidic pH, particularly stable at acidic pH below 4. pH resistance or stability is measured herein as a less than 10% loss of colouring, after a minimum exposure of 10 minutes at acidic pH. pH stability can be measured by other methods, such as physical characterization of the phycocyanins within the acidic compositions as a function of time.

DISCLOSURE OF THE INVENTION

Thus, the present invention concerns an acidic composition, particularly an acidic food composition, which can comprise at least one acidic-pH-resistant phycocyanin. Advantageously, said phycocyanin can be a phycobiliprotein whose apoprotein can comprise at least the protein of SEQ ID NO 1 or SEQ ID 2 or a variant thereof.
According to the invention, the phycocyanin can in particular be a phycocyanin extracted from a Galdieriaceae, more particularly extracted from Galdieria.
The acidic composition according to the invention, particularly the acidic food composition, can be either solid or pasty, or liquid, in particular an optionally carbonated beverage.
By acidic composition, particularly by acidic food composition, is meant, according to the invention, a composition having a pH of 4 or less, advantageously having a pH of 2 to 4, more advantageously of 2.5 to 3.5.

DETAILED DESCRIPTION OF THE INVENTION

Phycocyanins and allophycocyanins are phycobiliproteins comprising alpha and beta subunits composed of an apoprotein covalently bound to a chromophore. The different phycocyanins are distinguished essentially by the sequence of their alpha- and beta-subunit apoproteins.
According to a particular embodiment of the invention, the acidic composition, particularly the acidic food composition, comprises an acidic-pH-resistant phycocyanin whose α-subunit apoprotein comprises SEQ ID NO 1 (accession number YP_009051179.1) and whose β-subunit apoprotein comprises SEQ ID 2 (accession number YP_009051180.1) or variants thereof.
According to a preferred embodiment of the invention, the acidic composition, particularly the acidic food composition, comprises an acidic-pH-resistant phycocyanin whose α-subunit apoprotein consists of SEQ ID NO 1 (accession number YP_009051179.1) and whose β-subunit apoprotein consists of SEQ ID 2 (accession number YP_009051180.1) or variants thereof.
According to another embodiment of the invention, the acidic composition, particularly the acidic food composition, can further comprise an allophycocyanin combined with the phycocyanin.
Advantageously, the alpha-subunit apoprotein of said allophycocyanin comprises SEQ ID NO 3 (accession number YP_009051103.1) and the β-subunit apoprotein comprises SEQ ID NO 4 (YP_009051104.1) or variants thereof.
According to another preferred embodiment of the invention, the alpha-subunit apoprotein of said allophycocyanin consists of SEQ ID NO 3 (accession number YP_009051103.1) and the β-subunit apoprotein consists of SEQ ID NO 4 (YP_009051104.1) or variants thereof.
The amino acid composition of a protein can give said protein different properties according to said amino acid composition. The characteristics of a protein depend, among other things, on its amino acid composition and its isoelectric point (pI). The isoelectric point is the pH of the solution at which the protein carries no net charge or, in other words, the pH at which the molecule is electrically neutral and the proteins tend to attract one another, aggregate and precipitate. At a pH above their isoelectric point, the proteins tend to be negatively charged and to repel one another.
Comparative analysis of the isoelectric points of various proteins using the computational procedure 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) shows a certain correlation between the theoretical calculations and the acidic pH resistance observed experimentally.
Studies carried out by the Applicant indicate that the acidic pH resistance of a phycocyanin may be linked to the amino acid sequence of the a subunit of said phycocyanin. Furthermore, it is noted that within the amino acid sequence of the a subunit of the phycocyanin, the identity of the first 26 amino acids seem particularly important. It is particularly the case of phycocyanin obtained by culture of microalgae strains of the genera Cyanidioschyzon, Cyanidium or Galdieria, more particularly of strains Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae.
Thus, advantageously, the composition according to the invention can comprise at least one phycocyanin of which at least one apoprotein, particularly that of the a subunit, can have a low isoelectric point allowing better stability at acidic pH.
By low isoelectric point is meant an isoelectric point of 3 or less, preferentially of 2.5 or less, more preferentially of 2.2 or less.
Thus, more advantageously, the composition according to the invention can comprise at least one phycocyanin of which at least one apoprotein, particularly that of the α subunit, can have an isoelectric point of 3 or less, preferentially of 2.5 or less, more preferentially of 2.2 or less.
Thus, according to a particular embodiment of the invention, the acidic composition, particularly the acidic food composition, can comprise at least one phycocyanin whose α-subunit apoprotein can have a low isoelectric point, more particularly at least one phycocyanin whose α-subunit apoprotein can comprise SEQ ID NO 1, or a variant.
Thus, according to another particular embodiment of the invention, the acidic composition, particularly the acidic food composition, comprises at least one phycocyanin whose α-subunit apoprotein has a low isoelectric point, more particularly at least one phycocyanin whose α-subunit apoprotein consists of SEQ ID NO 1, or a variant.
By variant is meant, according to the invention, 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 NO 3 or SEQ NO 4, 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.
Advantageously, the variants according to the invention have at least 83% sequence identity with the α subunits of the phycocyanin, and at least 82% with the beta subunits of the phycocyanin.
Preferentially, the variants according to the invention have at least 90% identity with the α (SEQ ID NO 1) and β (SEQ ID NO 2) subunits.
Similarly, for the allophycocyanins, the variants advantageously have at least 89% sequence identity with the α subunits of the allophycocyanin, and at least 90% with the β subunits of the allophycocyanin.
Those skilled in the art know how to measure protein sequence identity using the common methods at their disposal, notably the BLASTP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Similarly, those skilled in the art know how to identify variants of said sequences and to verify that they retain the same structural properties by a simple stability test in acidic pH, for example by performing a test such as the test presented in example 3.
Those skilled in the art know that a polypeptide can be modified by substitution, insertion and/or deletion of at least one amino acid without substantially modifying the function thereof.
For example, the substitution of an amino acid at a given position by another chemically equivalent amino acid is a known example of sequence variation which does not substantially affect the properties of the protein.
These “conservative” substitutions can be defined as exchanges within the following groups of amino acids

- Ala, Ser, Thr, Pro, Gly
- Asp, Asn, Glu, Gln
- His, Arg, Lys
- Met, Leu, Ile, Val, Cys and
- Phe, Tyr, Trp

Thus, the variants of the apoproteins of the phycocyanins and/or allophycocyanins according to the invention can comprise a difference of 1 to 30 amino acids in relation to the corresponding reference sequence, particularly concerning the α and/or β subunits of the phycocyanin, insofar as the variant obtained retains the properties of the reference protein and the percent homologies/identities stated above.
More precisely according to the invention,

- the α-subunit apoprotein variants of the phycocyanins useful in the acidic compositions according to the invention, deriving from substitutions, insertions and/or deletions, can comprise a difference of 1 to 27 amino acids in relation to the corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percent identities stated above;
- the β-subunit apoprotein variants of the phycocyanins useful in the acidic compositions according to the invention, deriving from substitutions, insertions and/or deletions, can comprise a difference of 1 to 30 amino acids in relation to the corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percent identities stated above;
- the α-subunit apoprotein variants of the allophycocyanins useful in the acidic compositions according to the invention, deriving from substitutions, insertions or deletions, can comprise a difference of 1 to 24 amino acids in relation to the corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percent identities stated above;
- the β-subunit apoprotein variants of the allophycocyanins useful in the acidic compositions according to the invention, deriving from substitutions, insertions and/or deletions, can comprise a difference of 1 to 20 amino acids in relation to the corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percent identities stated above.

More particularly according to the invention, and regardless of the reference sequence considered (phycocyanin α and/or β subunit and/or allophycocyanin α and/or β subunit), the variants of said subunits can advantageously comprise a difference of 1 to 15 amino acids, preferably a difference of 1 to 10 amino acids, in particular a difference of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids in relation to the corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percent identities stated above.
According to the invention, the phycocyanin or variants thereof useful, alone or mixed with an allophycocyanin or variants thereof, in acidic compositions, particularly in acidic food compositions, can be obtained by culture of a natural organism naturally expressing the phycocyanin or the variant thereof of interest or by culture of an organism genetically transformed to express the phycocyanin or the variant thereof of interest selected for its capacity to produce said phycocyanin or variants thereof.
Exemplary natural organisms naturally expressing a phycocyanin useful in the compositions according to the invention or the variant thereof of interest include algae (or microalgae) of the order Cyanidiales.
The order Cyanidiales includes the families Cyanidiaceae and Galdieriaceae, themselves subdivided in the genera Cyanidioschyzon, Cyanidium and Galdieria, members of which include, among others, 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. Particular mention may be made of strain Galdieria sulphuraria (also called Cyanidium caldarium) UTEX#2919.
Thus, according to an embodiment of the invention, the acidic composition, particularly the acidic food composition, comprises an acidic-pH-resistant phycocyanin from natural organisms such as algae or microalgae of the order Cyanidiales, in particular from natural organisms of the families Cyanidiaceae or Galdieriaceae.
More particularly according to the invention, the acidic composition, particularly the acidic food composition, comprises an acidic-pH-resistant phycocyanin from natural organisms which belong to the genera Cyanidioschyzon, Cyanidium, Galdieria, advantageously selected from the species of the genera Cyanidium and Galdieria
Even more particularly according to the invention, the acidic composition, particularly the acidic food composition, comprises an acidic-pH-resistant phycocyanin from natural organisms selected from the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria.
The preferred form of the acidic composition, particularly the acidic food composition, according to the invention comprises an acidic-pH-resistant phycocyanin from a natural microalga such as Galdieria sulphuraria, Cyanidium caldarium or Cyanidioschyzon merolae. More preferentially, the acidic-pH-resistant phycocyanin comes from a natural microalga selected from Galdieria sulphuraria and Cyanidium caldarium.
By way of example of an organism transformed to express the phycocyanin or the variant thereof of interest selected for its capacity to produce said phycocyanin or variants thereof, mention may be made of 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 for the binding thereof to the apoprotein. Yeasts in particular may be mentioned as microorganisms that can be modified to produce the phycocyanin and/or allophycocyanin used in the food compositions according to the invention.
Those skilled in the art will have no difficulty in finding in the prior art the description of the methods for culturing natural and/or modified organisms that can produce a phycocyanin useful in the compositions according to the invention.
For example, the culture of Cyanidiaceae or Galdieriaceae, of the order Cyanidiales, well-known to those skilled in the art, can advantageously be carried out in mixotrophic mode, light usually being necessary for the biosynthesis of pigments.
Such an industrial culture can advantageously be carried out in large-volume (i.e., 1,000-litre, 10,000-litre, 20,000-litre, 100,000-litre) fermenters. The culture can be carried out under the conditions known to those skilled in the art. It can be carried out in batch mode, in fed-batch mode or in continuous mode.
The phycocyanin useful in the compositions according to the invention can more particularly be extracted from biomass obtained by culture of an alga of the order Cyanidiales, as defined above, cultivated in mixotrophic mode with light having a wavelength of 400 nm to 550 nm, advantageously of 420 nm to 500 nm, preferentially of 430 to 480 nm, more preferentially of about 455 nm. It can be “white” light having a broad spectrum comprising light of said wavelength. It can also advantageously be narrow spectrum light consisting of said wavelength.
Such a method for industrial preparation of Cyanidiales biomass in mixotrophic mode, and the biomass thus obtained, are in particular described in patent application FR 15 59072 filed on 25 Sep. 2015, the contents of which are incorporated herein by reference.
The object of the invention is to provide a composition in which the phycocyanin is stable at acidic pH. By acidic composition is meant, according to the invention, any composition comprising a mineral or organic acid and a phycocyanin. Said composition can be liquid, fluid or viscous, pasty or solid, which has an acidic pH and into which an acidic-pH-resistant phycocyanin is incorporated.
For the aqueous liquid compositions, the pH is measured in the usual manner. For the non-aqueous liquid compositions or for the pasty or solid compositions, the pH is measured after dissolution of the composition in a sufficient amount of water to dissolve the soluble compounds contained therein, including the mineral or organic acids and the phycocyanin.
Advantageously, the composition according to the invention is an aqueous liquid composition, optionally in gel form, or a pasty or solid composition designed to be dissolved in an aqueous solution or in a solid or pasty composition comprising water. According to another advantageous embodiment of the invention, the acidic composition pasty or solid composition intended to be employed and/or stored in a humid environment.
The mineral or organic acids useful in the compositions according to the invention are well-known to those skilled in the art. Exemplary mineral acids include in particular carbonic, phosphoric, hydrochloric, sulphuric, perchloric, sulphonic and nitric acids. Exemplary organic acids include in particular citric, lactic, malic, tartaric, succinic acids, advantageously citric acid.
By acidic food composition is meant, according to the invention, any composition designed to be ingested by humans or animals and which falls within the preceding definition. Nutraceutical acidic compositions must be regarded as falling within the definition of the acidic food compositions within the context of the invention.
The acidic food compositions according to the invention are well-known to those skilled in the art. They can comprise a carrier which can comprise structural components associated with active compounds identified for their nutritive supply or for their health properties which benefit humans or animals. The acidic food composition according to the invention can also comprise food additives such as texturing agents, flavouring agents, preservatives, or any components well-known to those skilled in the art. The carrier can comprise water and/or proteins and/or fats and/or fibre and/or sugars. The components of the carrier may have only structural properties, but they are generally known for their nutritive supply.
The acidic food composition according to the invention can be ready-to-use or in the form of a food additive to be added to a solid, pasty or liquid preparation in order to prepare the edible food.
For the food compositions, the acid will preferably be selected from the list of acidifiers authorized for foods, in particular carbonic, phosphoric, citric, malic, tartaric and lactic acids, more particularly citric acid.
Concerning the non-food acidic compositions according to the invention, they can be, among other things, pharmaceutical, veterinary or cosmetic compositions and further comprise any additives and/or active agents known and used in such compositions.
In a solid, liquid or pasty acidic composition according to the invention, the phycocyanin can be incorporated, for example, in powder form. Said acidic composition, particularly said acidic food composition, may thus be in any known conventional form such as creams, gels, foams, pastes, etc. Exemplary solid food compositions include cakes or biscuits, dry food for cooking, soluble powders, gelatinous solid compositions (jelly), foams etc.
According to the invention, said liquid acidic composition can be an aqueous composition into which the phycocyanin is dissolved. It can be in the form of a ready-to-use composition or a liquid concentrate for dilution, notably to be ingested or to be added to a solid food either for its preparation or for its ingestion, for example a concentrated liquid “topping” composition to be applied to a cake to give it colour. Among these concentrated compositions, mention may be made of syrups, optionally containing alcohol.
The liquid acidic composition according to the invention can be of varying viscosity and optionally comprise additives such as viscosity agents, gelling agents, and other structuring additives known to those skilled in the art and typical for the preparation of liquid food compositions.
According to a particular embodiment of the invention, the liquid food composition can be an optionally carbonated acidic beverage. Particular mention may be made of sodas, juices, sports drinks, energy drinks, recovery drinks, etc. The compositions of these beverages are well-known to those skilled in the art and can notably comprise sugars, mineral salts, food additives, dissolved gas, etc. The beverage according to the invention is a conventional acidic beverage in which the colouring usually employed has been wholly or partly replaced by an acidic-pH-resistant phycocyanin according to the invention.
According to the invention, the phycocyanin content in the compositions according to the invention can be consistent with the practices of those skilled in the art.
For example, when the phycocyanin will be used to colour the acidic composition, then the phycocyanin content in said composition can be consistent with the practice of those skilled in the art as regards colouring.
In a liquid acidic composition within the context of the invention, the phycocyanin content can be from 2.5 mg/L to 2,500 mg/L, preferentially from 25 mg/L to 300 mg/L.
In a liquid composition of the ready-to-use beverage type, the phycocyanin content can generally be from 25 mg/L to 300 mg/L, preferentially from 50 mg/L to 100 mg/L.
In a concentrated liquid composition for dilution before use, such as a syrup, the phycocyanin content can generally be from 250 mg/L to 2,500 mg/L, preferentially from 500 mg/L to 1,000 mg/L.
In a solid composition, the phycocyanin content can generally be from 0.01 mg/g to 10 mg/g, preferentially from 0.1 mg/g to 5.0 mg/g, more preferentially from 0.25 mg/g to 2.5 mg/g.
One of the advantages of the invention resides, as can be seen in the following examples, in the fact that the colouring provided by the acidic-pH-resistant phycocyanins is more stable over time.
Other aspects and features of the invention will become apparent from reading the examples and figures.

DESCRIPTION OF THE FIGURES

FIG. 1 describes the amino acid sequences of the Galdieria sulphuraria phycocyanin and allophycocyanin apoproteins with:

- SEQ ID NO 1: YP_009051179.1: Phycocyanin α subunit;
- SEQ ID NO 2: YP_009051180.1: Phycocyanin β subunit;
- SEQ ID NO 3: YP_009051103.1: Allophycocyanin α subunit;
- SEQ ID NO 4: YP_009051104.1: Allophycocyanin β subunit;

FIG. 2 presents the stability curve as a function of pH of the phycocyanins extracted from Galdieria sulphuraria (UTEX2919) and Cyanidioschyzon merolae (ACUF 199) whose apoprotein sequence consists of SEQ ID NO 1 or a variant, in relation to the stability curve of the spirulina-derived phycocyanin Linablue®.

- (-▪-): Phycocyanin from Galdieria sulphuraria (UTEX#2919)
- (•••X•••): Phycocyanin from Cyanidioschyzon merolae (ACUF 199)
- (-∘*-): Lina Blue®: 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 values at which precipitation of the phycocyanin appears.

FIG. 3 represents colour change over time of an acidic beverage comprising phycocyanin. W0, W2, W4 and W6:

week

0, 2, 4 and 6.

EXAMPLES

Example 1: Production and Extraction of Phycocyanin from Galdieria sulphuraria UTEX#2919

Materials and Methods

Strain: Galdieria sulphuraria (Also Called Cyanidium caldarium) UTEX#2919

Culture Medium

Mixotrophy: 30 g/L glycerol, 8 g/L (NH₄)₂SO₄, 1 g/L KH₂PO₄, 716 mg/L MgSO₄, 44 mg/L CaCl₂, 3 mL/L of Fe-EDTA stock solution (6.9 g/L FeSO₄and 9.3 g/L EDTA-Na₂) and 4 mL/L of 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)₆Mo₇O₂₄, 4H₂O).

Culture Conditions:

The cultures are carried out in 1- to 2-L-useful-volume reactors with computer-controlled automated systems. Culture pH is regulated by adding base (14% ammonia solution (wNH₃/w)) and/or acid (4 N sulphuric acid solution). Culture temperature is set to 37° C. The culture is illuminated by baffles equipped with a system of white LEDs or blue LEDs (455 nm) in a way similar to that described in patent WO 2014/174182. Tracking of cell growth is carried out at different times by measuring absorbance at 800 nm. And a measurement of the dry mass is carried out by filtration.
The performance characteristics of the culture at the end of growth are summarized in the following Table 1:

	TABLE 1

	Mixotrophy

	Time (h)	160
	Dry biomass (g/L)	20
	Phycocyanin content (mg/g dry biomass)	40

Measurements of intracellular phycocyanin content per gram of dry matter were carried out using the extraction and assay method described by Moon and colleagues [Moon et al., Korean J. Chem. Eng., 2014, 1-6] while replacing the phosphate buffer with water.

Example 2: Extraction of Phycocyanins

Strains Galdieria sulphuraria (UTEX#2919) and/or Cyanidioscyizon merolae (ACUF199) were cultivated under the conditions of example 1.
Phycocyanin is then extracted according to a modification of the protocol described by Moon et al., 2014 (op. cit.). Said modification consists in replacing the phosphate buffer used to solubilize phycocyanin with demineralized water.
An extract (also called “phycocyanin extract” or “crude extract”) which comprises, in addition to the phycocyanin of interest, other water-soluble proteins, is thus obtained. The phycocyanin extract can have several possible qualities depending on the method of extraction and/or purification used. For example, a crude extract will contain a higher amount of water-soluble proteins, other than phycocyanin, than that found in a purified extract. By purified extract is meant a crude extract of which a portion of the water-soluble proteins have been removed by ultrafiltration, hollow-fibre filtration, or ion-exchange chromatography, methods known to those skilled in the art, while retaining the phycocyanin.
Purity index is traditionally expressed by calculating the ratio of the absorbance of the solution at 618 nm (specific absorbance of phycocyanin) to that at 280 nm, the specific absorbance of aromatic amino acids giving an idea of the total protein level. The lower this ratio, the higher the amount of proteins other than phycocyanin in the solution.
The crude extract was purified using the KrosFlo® tangential flow filtration system from Spectrum® Labs.

TABLE 2

Purity index measurement of a phycocyanin extract before and after
purification.

	Purity index
	Abs 618 nm/Abs 280 nm

	Crude phycocyanin extract	0.54
	Purified extract	2.12

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

Strains Galdieria sulphuraria (UTEX#2919) and Cyanidioschyzon merolae (ACUF199) were cultivated under the conditions of example 1.
The tests are carried out by taking as reference the data of the commercial product LineBlue® (http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html) from the company DIC Lifetec Co., Ltd. (Tokyo, Japan), which is a phycocyanin extracted from Spirulina (Arthrospira) platensis.
These tests were carried out with the phycocyanins prepared in example 2 and having a purity index of 2.12. Measurement of blue colour is done by measuring absorbance at 618 nm with an ultraspec 2100 pro spectrophotometer (Amersham). Percent colour loss is calculated relative to the absorbance measurement of the sample under the reference conditions (pH 6).
For the resistance test under acidic conditions, the pH is gradually lowered by adding 5% citric acid solution (Sigma 251275) to the phycocyanin preparation. For each pH value, a sample of the phycocyanin solution is collected and its absorbance measured at 618 nm, 10 minutes after lowering the pH to the desired value.
The results of these tests are presented in FIG. 2:
Phycocyanin extracted from Galdieria sulphuraria (UTEX#2919) [(-▪-)] exhibits very good pH-resistance compared with that of Spirulina, with less than 10% loss of pigmentation up to pH 2.75 (98.25% of its colouring at pH 3, 92.4% at pH 2.75), the loss increasing as of pH 2.5 (79% at pH 2.5; 75% at pH 2.25; 46% at pH 2).
Phycocyanin extracted from strain Cyanidioschyzon merolae (ACUF 199) [(•••X•••)] exhibits good pH-resistance compared with that of Spirulina, with more than 90% of its colouring at pH 3, 70% at pH 2.75; 40% at pH 2.5; 23% at pH 2.25; 20% at pH 2.
Phycocyanin extracted from strain Spirulina platensis [(-∘*-)] exhibits only 90% of its colouring at pH 4, 80% at pH 3.8; 60% at pH 3.6; 38% at pH 3.4. Phycocyanin extracted from Spirulina platensis starts to precipitate as of pH 3.8.
In conclusion, Galdieria sulphuraria or Cyanidioschyzon merolae phycocyanins are more resistant to acidic pH than that extracted from Spirulina.

Example 4—Comparative Analysis of Apoprotein Sequences of Phycocyanin and Allophycocyanin from Different Microalgae and Measurement of the Isoelectric Points Thereof

In order to identify a possible cause for the increase in resistance to acidic pH, a comparison of the sequences of the α and β subunits of phycocyanins and allophycocyanins produced by various microorganisms, particularly microalgae, was performed using the BLASTP program, the use of which is well-known to those skilled in the art, on the basis of published sequences accessible in databases (see accession number). The comparison is made in relation to the apoprotein sequence of the corresponding subunit of strain Galdieria sulphuraria.
At the same time, the isoelectric point of the α- and β-subunit apoproteins of phycocyanins and allophycocyanins whose sequence comparison was carried out was determined by the computational procedure described by Patrickios and Yamasaki (1995).
Phycocyanin and allophycocyanin produced by strain Galdieria sulphuraria serve as the reference in these studies.
Results
The results of the sequence comparisons and the pI calculations are presented in tables 3 and 4 below:

TABLE 3

Comparisons of the alpha and beta apoprotein sequences of phycocyanin
from various organisms. The isoelectric point of each protein is shown
(Patrickios and Yamasaki, 1995).

	aa/aa (% Identity)	pI

α subunit of Phycocyanin from (accession #)
Galdieria sulphuraria (YP_009051179.1)	162/162 (100%)	2.115
Cyanidium caldarium (P00306.3)	159/162 (98%)	2.1
Cyanidioschyzon merolae (NP_848986.1)	135/162 (83%)	2.129
Arthrospira maxima CS328 (EDZ96896.1)	119/162 (73%)	3.657
Spirulina platensis (P72509.2)	119/162 (73%)	3.657
Arthrospira jenneri fz (AEV40872.1)	118/162 (73%)	3.657
Cyanobacterium stanieri PCC 7202	123/162 (76%)	3.327
(AFZ46322.1)
Halothece sp. PCC 7418 (WP_015227201.1)	130/162 (80%)	3.786
Geitlerinema sp. PCC 7407	120/162 (74%)	3.936
(WP_015173541.1)
Nostoc sp. PCC 7120 (WP_010994705.1)	130/162 (80%)	3.978
β subunit of Phycocyanin from (# accession)
Galdieria sulphuraria (YP_009051180.1)	171/171 (100%)	4.112
Cyanidium caldarium (AAB34027.2)	169/171 (99%)	4.112
Cyanidioschyzon merolae (NP 848987.1)	141/171 (82%)	4.112
Arthrospira maxima CS328 (EDZ96897.1)	130/171 (76%)	3.966
Spirulina platensis (1HA7 B)	130/170 (76%)	3.966
Arthrospira jenneri fz (AEV40871.1)	130/171 (76%)	4.062
Cyanobacterium stanieri PCC 7202	135/171 (79%)	3.966
(AFZ46321.1)
Halothece sp. PCC 7418 (WP 015227202.1)	133/171 (78%)	4.074
Geitlerinema sp. PCC 7407	138/171 (81%)	4.12
(WP 015173542.1)
Nostoc sp. PCC 7120 (BAB72486.1)	141/170 (83%)	4.159

TABLE 4

Comparisons of the alpha and beta apoprotein sequences of
allophycocyanin from various organisms. The isoelectric point of
each protein is shown (Patrickios and Yamasaki, 1995).

	aa/aa (% Identity)	pI

α subunit of Allophycocyanin from
(accession #)
Galdieria sulphuraria (YP_009051103.1)	161/161 (100%)	3.9
Cyanidium caldarium (AAA20110.1)	158/161 (98%)	3.932
Cyanidioschyzon merolae (NP_849064.1)	144/161 (89%)	3.989
Arthrospira jenneri (AEV40869.1)	131/161 (81%)	4.008
Arthrospira platensis (CAA65141.1)	131/161 (81%)	4.054
Cyanobacterium stanieri PCC 7202	125/161 (78%)	3.997
(AFZ46138.1)
Halothece sp. PCC 7418	133/161 (83%)	3.724
(WP_015226049.1)
Geitlerinema sp. PCC 7407	137/161 (85%)	4.054
(WP_015172402.1)
Nostoc sp. PCC 7120 (WP_010994198.1)	130/161 (81%)	4.041
β subunit of Allophycocyanin from
(accession #)
Galdieria sulphuraria (YP_009051104.1)	161/161 (100%)	3.502
Cyanidium caldarium (AAB01577.1)	158/161 (98%)	3.657
Cyanidioschyzon merolae (NP_849065.1)	145/161 (90%)	3.298
Arthrospira jenneri (AEV40870.1)	138/161 (86%)	3.502
Arthrospira platensis (BAA19986.1)	141/161 (88%)	3.532
Cyanobacterium stanieri PCC 7202	141/161 (88%)	3.532
(AFZ46566.1)
Halothece sp. PCC 7418	138/161 (86%)	3.502
(WP_015226048.1)
Geitlerinema sp. PCC 7407	140/161 (87%)	3.502
(WP_015172403.1)
Nostoc sp. PCC 7120 (WP_010994199.1)	138/160 (86%)	3.657

The sequence and isoelectric point comparisons show that the α subunits of the phycocyanins most resistant to acidic pH have an isoelectric point below 3 and a higher percent identity with the sequence of Galdieria sulphuraria.

Example 5: Stability Over Time in a Beverage

The stability test in acid medium over time for the phycocyanin extracted from strain UTEX#2919 was carried out by adding the phycocyanin to a lemonade beverage (pH 2.95; as described in example 6: beverage 1). After adding 0.025‰ of phycocyanin the apoprotein sequence of which consists of variant of SEQ ID NO 1, the beverage was exposed to a day/night cycle (16 h/8 h) with artificial light for 6 weeks, at room temperature.
Regular samples are taken over time in order to measure the absorbance at 618 nm. An aliquot is taken at weeks 0 (W0), 2 (W2), 4 (W4) and 6 (W6), and the absorbance at 618 nm measured with the ultraspec 2100 pro spectrophotometer (Amersham) in order to define the 100% colour point. Residual colour is expressed as a percentage of the initial reference value.
FIG. 3 shows the results of this experiment. Steady colour loss over time during long-term exposure to light is noted. However, after 6 weeks of exposure more than 60% of the colour remains.

Example 6: Examples of Acidic Beverages in Liquid Form Comprising Phycocyanin

Beverages containing phycocyanin can have the following composition:

Beverage 1—Soda Beverage:

Carbonated water qs 1 L

Sugars 70 g

Citric acid 1.5 g

Natural organic flavour 4 to 8 g

Phycocyanin extract obtained in example 2 qs 100 mg of Phycocyanin

The pH of this beverage is 2.95

Beverage 2—Health Drink for Athletes:


Spring water	qs 1	L
Glucose	20	g
Fructose	10	g
Citric acid	2.7	g
Sodium citrate	1.87	g
Potassium citrate	0.327	g
Magnesium chloride	2.5	mg
Calcium chloride
	3	mg
Natural organic flavour	4	g
Phycocyanin extract obtained in example 2	qs 150	mg of Phycocyanin

The pH of this beverage is 3.5

Example 6: Acidic Beverage in Soluble Powder Form Comprising Phycocyanin

Sugars 70 to 100 g

Citric acid 1 to 1.5 g

Natural organic flavour 4 to 8 g

Phycocyanin extract obtained in qs 250 mg of Phycocyanin

example 2

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

Example 7: Solid Acidic Composition Comprising Phycocyanin: Acidic Sweet


Water:	qs 1	kg
Sugars:	800	g
Citric acid:	1 to 2	g
Phycocyanin extract obtained in	qs 2,500	mg of Phycocyanin
example 2

REFERENCES

Moon et al., Korean J. Chem. Eng., 2014, 1-6
Patrickios et Yamasaki, Polypeptide Amino Acid Composition and Isoelectric Point. II. Comparison between Experiment and Theory. Analytical Biochemistry. 231, 1, 1995: 82-91
FR Doc No: 2013-19550
WO 2014/174182
FR 15 59072 filed on 25 Sep. 2015
http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html
http://blast.ncbi.nlm.nih.gov/Blast.cgi

Claims

1. An acidic composition, comprising at least one mineral or organic acid and at least one acidic-pH-resistant phycocyanin, wherein.

2. The acidic composition according to claim 1, wherein said acidic-pH-resistant phycocyanin is a phycobiliprotein the apoprotein of which comprises the protein of SEQ ID NO 1 or SEQ ID 2 or a variant thereof.

3. The acidic composition according to claim 2, wherein the α-subunit apoprotein of said phycocyanin comprises SEQ ID NO 1 and the β-subunit apoprotein of said phycocyanin comprises SEQ ID 2 or variants thereof.

4. The acidic composition according to claim 3, wherein said α-subunit apoprotein consists of SEQ ID NO 1 and the β-subunit apoprotein consists of SEQ ID or variants thereof.

5. The acidic composition according to claim 1, wherein it further comprises an allophycocyanin combined with the phycocyanin.

6. The acidic composition according to claim 5, wherein the α-subunit apoprotein of said allophycocyanin comprises SEQ ID NO 3 or variants thereof and the β-subunit apoprotein of said allophycocyanin comprises SEQ ID NO 4 or variants thereof.

7. The acidic composition according to claim 6, wherein the allophycocyanin α-subunit apoprotein consists of SEQ ID NO 3 or variants thereof and the β-subunit apoprotein consists of SEQ ID NO 4 or variants thereof.

8. The acidic composition according to claim 1, wherein the acidic-pH-resistant phycocyanin is a phycocyanin extracted from an alga (or microalga) of the order Cyanidiales.

9. The acidic composition according to claim 8, wherein the phycocyanin is a phycocyanin extracted from an alga (or microalga) of the family Cyanidiaceae or the family Galdieriaceae.

10. The acidic composition according to claim 9, wherein the phycocyanin is a phycocyanin extracted from an alga (or microalga) of the genera Cyanidioschyzon, Cyanidium or Galdieria.

11. The acidic composition according to claim 10, wherein the phycocyanin is a phycocyanin extracted from an alga (or microalga) selected among the group consisting in 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, preferentially the species Galdieria sulphuraria, Cyanidium caldarium, and Cyanidioschyzon merolae.

12. The acidic composition according to claim 1, wherein it has a pH of 4 or less.

13. The acidic composition according to claim 1, wherein it has a pH of 2 to 4.

14. The acidic composition according to claim 1, wherein it has a pH of about 2.5 to 3.5.

15. The acidic composition according to claim 1, wherein the α-subunit apoprotein of said phycocyanin has an isoelectric point below 3.

16. The acidic composition according to claim 1, wherein the α-subunit apoprotein of said phycocyanin has an isoelectric point below 2.5.

17. The acidic composition according to claim 1, wherein the α-subunit apoprotein of said phycocyanin has an isoelectric point below 2.2.

18. The acidic composition according to claim 1, wherein it is solid.

19. The acidic composition according to claim 18, wherein the phycocyanin content is from 0.25 mg/g to 2.5 mg/g.

20. The acidic composition according to claim 1, wherein it is liquid.

21. The acidic composition according to claim 20, wherein the phycocyanin content is from 2.5 mg/L to 2,500 mg/L.

22. (canceled)

23. The acidic composition according to claim 1, wherein it is an acidic food composition.

24. The acidic composition according to claim 20, wherein it is a carbonated or non-carbonated beverage.