Improved stabilization of phycocyanin in acidic compositions

Abstract

The present technology generally relates to acidic compositions comprising stabilized phycocyanin and methods for obtaining such acidic compositions.

Description

[Technical field]

[0001] The present technology generally relates to acidic compositions comprising stabilized phycocyanin and methods for obtaining such acidic compositions. [Background technology]

[0002] Phycocyanin is a food coloring that imparts a blue color to products to which it is added. Phycocyanin extracted from spirulina is the only natural blue pigment currently approved by the FDA (FR Doc No:2013-19550). Phycocyanin is sold in liquid or powder form for use as a blue pigment in foods. The spirulina-derived phycocyanin, however, has the drawback of being unstable at acidic pH below 5, which results in lack of coloration and precipitation, which limits its applications. In the best case, the lack of stability occurs around pH 4 (see technical specifications of spirulina-derived phycocyanin Linablue®; http: / / www.dlt-spl.co.jp / business / en / spirulina / linablue.html). As a result, there are many acidic food compositions, especially carbonated and non-carbonated beverages, for which spirulina-derived phycocyanin cannot be used as a food colorant or for its antioxidant properties. In contrast to Spirulina-derived phycocyanin, it was previously demonstrated in WO 2017 / 050918, which is incorporated herein by reference, that phycocyanin extracted from Galdieria or Cyanidiophycae has the ability to tolerate acidic pH.

[0003] However, the use of phycocyanin in an industrial context in acidic beverages is much more complicated. Indeed, tests have shown that there is a significant loss of phycocyanin after heat treatment, such as during pasteurization, and subsequent accelerated aging tests, and that such loss is accompanied by a gradual change in color, including the appearance of a green hue. Depending on the conditions, these changes in hue can be total. This lack of stability can be problematic for the sale of the product. Sulfated polysaccharides such as λ-carrageenan have been previously disclosed for stabilizing phycocyanin derived from Spirulina. However, it is not appropriate to add large amounts of additives in food compositions. In view of this, there is a need to identify methods for stabilizing phycocyanin or the coloring provided by phycocyanin in acidic compositions, particularly in the context of acidic beverages. Summary of the Invention

[0004] According to various aspects, the present technology relates to an acidic composition comprising phycocyanin, wherein the phycocyanin is present in the composition in an amount ranging from about 0.001 to about 0.1% by weight. According to various aspects, the present technology relates to a carbonated beverage comprising an acidic composition as defined herein. According to various aspects, the present technology relates to a carbonated beverage comprising an acidic composition as defined herein. The present inventors have surprisingly found that small amounts of sulfated polysaccharides can be used to stabilize phycocyanin in acidic compositions. Thus, according to an embodiment, the present technology relates to an acidic composition comprising phycocyanin and at least one sulfated polysaccharide, wherein the phycocyanin and at least one sulfated polysaccharide are present in the composition in a ratio of about 0.20:1 to about 20:1, preferably about 0.25:1 to about 10:1, or about 0.3:1 to about 10:1, preferably about 1:1 to about 10:1, more preferably about 0.3:1 to about 0.6:1, about 0.9:1 to about 2.4:1, or about 0.3:1 to 1.5.

[0005] The present technology relates to an acidic composition comprising phycocyanin and at least one sulfated polysaccharide, wherein the phycocyanin and the at least one sulfated polysaccharide are present in the composition in a ratio of about 0.25:1 to about 1:1. In another embodiment, the present technology relates to an acidic composition comprising phycocyanin and at least one sulfated polysaccharide, wherein the phycocyanin and the at least one sulfated polysaccharide are present in the composition in a ratio of about 0.25:1, about 0.3:1, about 1:3, about 0.5:1, about 0.75:1, about 0.6:1, about 1:1, about 1.5:1, about 2:1, about 3:1, about 4:1, about 5:1, or about 6:1. According to various aspects, the present technology relates to an acidic composition comprising a Galdieria extract, wherein the Galdieria extract is present in the composition in an amount ranging from about 0.004% to about 0.4% by weight.

[0006] According to various aspects, the present technology relates to a food product comprising an acidic composition as defined herein. According to various aspects, the present technology relates to a food product comprising an acidic composition as defined herein. According to various embodiments, the present technology relates to a concentrated liquid composition comprising phycocyanin present in the concentrated composition in an amount ranging from about 50 mg / L to about 2500 mg / L. According to various aspects, the present technology relates to a food product comprising a concentrated liquid composition as defined herein. According to various aspects, the present technology relates to a food product comprising a concentrated liquid composition as defined herein.

[0007] Other aspects and features of the present disclosure will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments in combination with the accompanying drawings. All features of the embodiments described in this disclosure are not mutually exclusive and can be combined with each other. For example, elements of one embodiment can be utilized in other embodiments without further recitation. Detailed descriptions of specific embodiments are provided herein below with reference to the accompanying drawings as follows: [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a photograph showing the results of a thermal stability test of phycocyanin over a 4-hour period in a model beverage system containing 0.1%, 0.2%, or 0.4% Galdieria extract, all diluted to 0.1% after heating for visual comparison. [Figure 2A] FIG. 1 is a photograph showing the 8-week stability of phycocyanin in a model drink containing 0.2% Galdieria extract and a first series of electrolyte drinks, with or without vitamin C. [Figure 2B] FIG. 1 is a table of results after 8 weeks stability of phycocyanin in a model drink containing 0.2% Galdieria extract and a first series of electrolyte drinks with or without vitamin C. [Figure 3A] FIG. 1 is a photograph showing the 8-week stability of phycocyanin in a second series of model beverages containing 0.2% and 0.4% Galdieria extract, with or without vitamin C. [Figure 3B] FIG. 1 is a table of results after 8 weeks stability of phycocyanin in a second series of model beverages containing 0.2% and 0.4% Galdieria extract with or without vitamin C. [Figure 4] 1 is a photograph showing the results of a stability test for model beverages containing 0.01% and 0.02% Galdieria extract. [Diagram 5] 1 is a photograph showing the effect of High Temperature Short Time (HTST, also referred to in this application as Flash Pasteurization) pasteurization on the stability of a model beverage containing 0.01% Galdieria extract. [Figure 6] 1 is a photograph showing the effect of λ-carrageenan on the stability of a model beverage containing 0.01% Galdieria extract. [Figure 7] FIG. 1 shows a comparison of beverages containing Galdieria extract-carrageenan (5:1) to the commercially available beverages Gatorade® and Powerade® (dE CMC 1.99). [Figure 8] 1 is a graph showing the effect of HTST pasteurization on the dE CMC of beverages containing different ratios of Galdieria extract:λ-carrageenan. [Figure 9] 1 is a chart presenting the average dE CMC after HTST of different beverages of Example 10. [Figure 10] Photographs of beverages containing different contents of Galdieria extract (0.1% or 0.2% by weight relative to the total weight of the beverage) before (left bottle) and after (right bottle) HTST according to Example 10ter. [Figure 11] 1 is a graphical representation showing the mean dE CMC after light testing of different beverages according to Examples 10bis and 10ter. [Figure 12] Photographs of several beverages with different contents of Galdieria extract (0.1% or 0.2% by weight relative to the total weight of the beverage), with or without vitamin C, before (bottle on the left) and after (bottle on the right) light testing according to Examples 10bis and 10ter. [Figure 13] 1 is a chart providing the average dE CMC after accelerated stability testing of different beverages according to Example 10. [Figure 14] 1 is a photograph of several beverages containing different amounts of Galdieria extract (0.1% or 0.2% by weight relative to the total weight of the beverage) before (bottle on the left) and after (bottle on the right) an accelerated stability test according to Example 10. [Figure 15A] Photographs of beverages containing Galdieria extract and λ-carrageenan in a 5:1 ratio at different pH. [Figure 15B] 1 is a table of color measurements of beverages containing Galdieria extract and λ-carrageenan in a 5:1 ratio at different pH. [Figure 16A] Photographs of beverages containing Spirulina extract and lambda-carrageenan in a 5:1 ratio at different pHs. [Figure 16B] 1 is a table of color measurements of beverages containing Spirulina extract and λ-carrageenan in a 5:1 ratio at different pH. [Figure 17A]Photographs of beverages containing Galdieria extract and λ-carrageenan in a 1:1 ratio at different pH. [Figure 17B] 1 is a table of color measurements of beverages containing Galdieria extract and λ-carrageenan in a 1:1 ratio at different pH. [Figure 18A] Photographs of beverages containing Spirulina extract and lambda-carrageenan in a 1:1 ratio at different pHs. [Figure 18B] 1 is a table of color measurements of beverages containing Spirulina extract and λ-carrageenan in a 1:1 ratio at different pH. [Figure 19] 1 is a chart presenting the mean dE CMC after HTST of several beverages containing either Galdieria extract or Spirulina extract with different contents of λ-carrageenan and different pH. [Figure 20] 1 is a chart presenting the mean dE CMC after HTST of several beverages containing different contents of λ-carrageenan and either Galdieria extract or Spirulina extract with pH 3.0 and 4.0. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present technology will be described in more detail below. This description is not intended to be a detailed list of all the different ways in which the technology can be implemented or all the features that can be added to the present technology. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in view of this disclosure, and these modifications and additions do not depart from the present technology. Therefore, the following description is intended to illustrate some specific embodiments of the technology, and is not intended to exhaustively specify all permutations, combinations, and modifications thereof. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The recitation herein of numerical ranges by endpoints are intended to include all numbers subsumed within that range (e.g., recitation of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0010] The term "about" is used herein, either explicitly or implicitly, and all amounts given herein are intended to mean the values ​​actually given, as well as approximations to such given values ​​that would be reasonably inferred based on ordinary skill in the art, including equivalents and approximations resulting from experimental and / or measurement conditions for such given values. For example, the term "about" in the context of a given value or range means a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range. The expression "and / or" as used herein should be taken as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be taken as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth herein.

[0011] As used herein, the term "comprises" is used in its open-ended sense to mean that items following the word are included, but items not specifically mentioned are not excluded. As used herein, the expressions "wt %" and "%" are defined as the wt % of the component relative to the total weight of the composition, unless otherwise defined. In the sense of the present technology, the "stability" of phycocyanin means its ability to retain its color, brightness and transparency in an acidic environment, under long-term storage and / or heat treatment. Phycocyanin is considered to have improved or enhanced stability if less precipitation, less obscuration and / or less noticeable color change is observed after exposure to elevated temperatures and / or prolonged storage in the dark or light at ambient temperature. Precipitation and obscuration can be observed with the naked eye, and color change can be followed by spectrophototometric methods by examining the λmax value, or via colorimetry by determining the dE CMC.

[0012] Color is measured using a spectrophotometer for aqueous solutions and a DigiEye color measurement and imaging system for semi-solid and solid compositions. * a * b * The colorimeter measurements are based on the CIE L * a * b * But (CIELAB color space): L * Scale: Light vs. Dark, with smaller numbers (0-50) indicating dark and larger numbers (51-100) indicating light. a * Scale: Red vs. Green, positive numbers indicate red and negative numbers indicate green. b * Scale: Yellow vs. Blue, positive numbers indicate yellow and negative numbers indicate blue. It provides a numerical representation of colors based on the opponent color theory.

[0013]

number

[0014] In one embodiment, the present technology originates from experiments that showed that depending on the dose of phycocyanin used in the final product, the coloring effect was more or less pronounced. Counterintuitively, the more phycocyanin was present in the composition, the more instability was observed. On the other hand, when low concentrations were used, the color change was limited and sometimes barely visible to the human eye. It was also determined that the addition of carrageenan improved the thermal stability of phycocyanin. Generally, the color of phycocyanin becomes lighter when exposed to high temperature short time pasteurization (HTST pasteurization, also called flash pasteurization). It was further determined that the combination of phycocyanin and carrageenan allows for the creation of a color tone that could be used to replace Blue No. 1 in beverages. In particular, in a preferred embodiment; the acidic composition according to the present technology includes a 5:1 ratio of Galdieria phycocyanin-carrageenan (1.5:1 phycocyanin:carrageenan), resulting in a dE CMC of less than 2.5 between the Galdieria beverage and the Blue No. 1 beverage, thus providing a color that matches the color of the Blue No. 1-containing beverage.

[0015] It was further determined that when concentrated phycocyanin (e.g., a composition typically containing about 0.3% to about 10% phycocyanin) was heated under acidic conditions, a green extract was formed, and that this green extract could be used in food and beverage applications. Thus, the present technology also relates to a method for producing a green color based on phycocyanin extracted from algae or microalgae from the order Polytrichum ores, preferably from the genus Cyanidioschyzon, Cyanidium, or Galdieria, more preferably from the species Galdieria sulphuraria, Cyanidium caldarium, or Cyanidioschyzon merolae. In some embodiments, the phycocyanin useful in the present technology can be phycocyanin extracted from the family Galdieriaceae, in some examples from the genus Galdieria. In some instances, phycocyanin is a component of a Galdieria extract.

[0016] Typically, the colorant in the powder contains about 25% to about 30% phycocyanin to exhibit an absorbance of a 10% dilution of the colorant of E10 = 180. For illustrative purposes, 1 gram of Galdieria extract would contain about 0.25 to about 0.30 grams of phycocyanin mixed with at least one diluent, such as water, invert sugar, sucrose, and / or maltodextrin. In some embodiments, the phycocyanin of the present technology is extracted phycocyanin. Phycocyanin can be extracted from Galdieria genus using protocols known in the art, such as those described in WO2020 / 144331, which is incorporated herein by reference. In some embodiments, the phycocyanin of the present technology is extracted phycocyanin. In some embodiments, the phycocyanin of the present technology is isolated phycocyanin. As used herein, the terms "extracted" and "isolated" refer to compounds that have been isolated from a natural source (e.g., from the genus Galdieria) using, for example, chromatography, distillation, extraction, or similar techniques that result in a purity of greater than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 98% or greater than about 99%.

[0017] In some embodiments, the phycocyanin of the present technology is a purified phycocyanin, particularly a purified extract of phycocyanin. "Purified extract" refers to a crude extract in which phycocyanin is retained while a portion of its water-soluble components (proteins, ions, sugars) are removed by liquid separation methods such as, but not limited to, ultrafiltration, hollow fiber filtration, or ion exchange chromatography, which are methods known to those skilled in the art. In some embodiments, the Galdieria extract as used herein is a purified Galdieria extract. The total phycocyanin content, hereinafter referred to as "phycocyanin" for short, is the sum of C-phycocyanin and, if present, allophycocyanin.

[0018] In some embodiments, the "purified" compounds disclosed herein have a purity of greater than 70%. In some embodiments, the "purified" compounds disclosed herein have a purity of greater than 80%. In some embodiments, the "purified" compounds disclosed herein have a purity of greater than 90%. In some embodiments, the "purified" compounds disclosed herein have a purity of greater than 95%. In some embodiments, the "purified" compounds disclosed herein have a purity of greater than 98%.

[0019] Phycocyanins are phycobiliproteins containing α and β subunits composed of an apoprotein covalently linked to a chromophore. Different phycocyanins are essentially distinguished by the sequences of their α- and β-subunit apoproteins. According to certain embodiments, the acidic composition, particularly the acidic food composition, of the present technology comprises an acidic pH-tolerant phycocyanin whose α-subunit apoprotein comprises SEQ ID NO:1 (Accession No. YP_009051179.1) and whose β-subunit apoprotein comprises SEQ ID NO:2 (Accession No. YP_009051180.1) or a variant thereof. In some embodiments, the present technology relates to an acidic composition comprising phycocyanin. In some embodiments, the acidic composition comprises extracted, isolated or purified phycocyanin.

[0020] The acidic composition of the present technology can be an acidic food composition, which can be solid, pasty, or liquid. In some examples, the acidic food composition is a carbonated drink. In some other examples, the acidic food composition is a dairy product, such as, but not limited to, yogurt. An acidic composition, in particular an acidic food composition, according to the present technology means a composition having a pH of 4.0 or less, preferably from about 2.5 to about 3.5, preferably greater than about 3.0 and less than about 4.0. In some embodiments, the acidic composition, particularly the acidic food composition, comprises an acidic pH-resistant phycocyanin whose α-subunit apoprotein has the amino acid sequence shown in SEQ ID NO:1 (Accession No. YP_009051179.1) or a variant thereof, and whose β-subunit apoprotein has the amino acid sequence shown in SEQ ID NO:2 (Accession No. YP_009051180.1) or a variant thereof.

[0021] In some embodiments, the acidic compositions, particularly acidic food compositions, may further comprise allophycocyanin in combination with C-phycocyanin. In some examples, the α-subunit apoprotein of the allophycocyanin has the amino acid sequence set forth in SEQ ID NO: 3 (Accession No. YP_009051103.1) or a variant thereof, and the β-subunit apoprotein has the amino acid sequence set forth in SEQ ID NO: 4 (YP_009051104.1) or a variant thereof. In some embodiments, the α-subunit apoprotein of the allophycocyanin consists of the amino acid sequence set forth in SEQ ID NO:3 (Accession No. YP_009051103.1) or a variant thereof, and the β-subunit apoprotein consists of the amino acid sequence set forth in SEQ ID NO:4 (YP_009051104.1) or a variant thereof.

[0022] The properties of a protein depend, among other things, 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 molecules are electrically neutral and proteins tend to attract each other and aggregate and precipitate. At a pH above its isoelectric point, proteins become negatively charged and tend to repel each other. Comparative analysis of the isoelectric points of various proteins using a 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 theoretical calculations and experimentally observed acidic pH resistance.

[0023] Studies carried out by the applicant show that the acidic pH resistance of phycocyanins can be linked to the amino acid sequence of the α-subunit of said phycocyanins. Furthermore, it is noted that within the amino acid sequence of the α-subunit of phycocyanins, the identity of the first 26 amino acids appears to be particularly important. This is especially true in the case of phycocyanins obtained by culturing microalgae strains of the genera Cyanidioschizon, Cyanidium or Galdieria, more particularly the strains Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschizon melorae. In some embodiments, the composition of the present technology can include at least one phycocyanin, of which at least one apoprotein, particularly the α-subunit, can have a low isoelectric point that allows for better stability at acidic pH. Low isoelectric point means an isoelectric point of 3 or less, preferentially 2.5 or less, more preferentially 2.2 or less. In some embodiments, the composition of the present technology can include at least one phycocyanin, of which at least one apoprotein, particularly the α-subunit, can have an isoelectric point of 3 or less, preferentially 2.5 or less, more preferentially 2.2 or less.

[0024] The acidic composition, particularly the acidic food composition, may comprise at least one phycocyanin whose α-subunit apoprotein may have a low isoelectric point, more particularly at least one phycocyanin whose α-subunit apoprotein may comprise the amino acid sequence shown in SEQ ID NO:1, or a variant thereof. In some embodiments, the acidic composition, particularly the acidic food composition, comprises at least one phycocyanin whose α-subunit apoprotein may have a low isoelectric point, more particularly at least one phycocyanin whose α-subunit apoprotein consists of the amino acid sequence shown in SEQ ID NO:1, or a variant thereof. As used herein, the term "mutant" refers to a protein sequence corresponding to a reference sequence, in this case a protein represented by SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:4, which has been 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 the reference sequence, in particular the same stability in acidic environments.

[0025] In some examples, the variants of the present technology have at least about 83% sequence identity with the α-subunit of phycocyanin and at least about 82% sequence identity with the β-subunit of phycocyanin. In some other examples, the variants of the present technology have at least about 90% identity with the α (SEQ ID NO: 1) and β (SEQ ID NO: 2) subunits, preferably at least about 95%, 96%, 97%, 98%, 99% identity with the α (SEQ ID NO: 1) and β (SEQ ID NO: 2) subunits. Similarly, with respect to allophycocyanin, the variant has at least about 89% sequence identity with the α subunit of allophycocyanin and at least about 90% sequence identity with the β subunit of allophycocyanin. Those skilled in the art know how to determine protein sequence identity using the general methods at their disposal, in particular the BLASTP program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0026] The identity of variants encompassed by the present technology can be determined using methods known in the art, such as by verifying that the variants retain the same structural and functional properties as the original amino acid sequence, by performing stability tests at acidic pH, such as those provided in the examples of this application. The polypeptide of the present technology can be modified by substitution, insertion and / or deletion of at least one amino acid without substantially changing its function.For example, the substitution of an amino acid at a given position with another chemically equivalent amino acid is a known example of a sequence change that does not substantially affect the properties of a protein.These "conservative" substitutions can be defined as exchanges within the following amino acid groups: i) Ala, Ser, Thr, Pro, Gly; ii) Asp, Asn, Glu, Gln; iii) His, Arg, Lys; iv) Met, Leu, Ile, Val, Cys; and v) Phe, Tyr, Trp.

[0027] Thus, variants of C-phycocyanin and / or allophycocyanin apoproteins according to the present technology can contain 1 to 30 amino acid differences from the corresponding reference sequence. In some examples, variants of C-phycocyanin and / or allophycocyanin apoproteins according to the present technology can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid differences from the corresponding reference sequence. In some examples, variants of the α and / or β subunits of phycocyanin retain the properties of the reference protein and the homology / identity percentages described above.

[0028] In some embodiments, phycocyanin α-subunit apoprotein variants useful in the acidic compositions of the present technology derived from substitutions, insertions and / or deletions can contain between 1 and 27 amino acid differences relative to the corresponding reference sequence, so long as the resulting variant retains the properties of the reference protein and the percent identity set forth above. In some embodiments, the α-subunit apoprotein variants of phycocyanin useful in the acidic compositions of the present technology derived from substitutions, insertions and / or deletions of the reference sequences and the β-subunit apoprotein variants of phycocyanin useful in the acidic compositions according to the present technology can contain 1 to 30 amino acid differences from the corresponding reference sequences, so long as the resulting variants retain the properties of the reference proteins and the percent identity set forth above.

[0029] In some embodiments, the C-phycocyanin α-subunit apoprotein variants useful in the acidic compositions of the present technology derived from substitutions, insertions and / or deletions, and the allophycocyanin α-subunit apoprotein variants useful in the acidic compositions according to the present technology, can contain from 1 to 24 amino acid differences relative to the corresponding reference sequence, so long as the resulting variant retains the properties of the reference protein and the percent identity set forth above. In some embodiments, the α-subunit apoprotein variants of C-phycocyanin useful in the acidic compositions of the present technology derived from substitutions, insertions and / or deletions, and the β-subunit apoprotein variants of allophycocyanin useful in the acidic compositions according to the present technology, can contain between 1 and 20 amino acid differences relative to the corresponding reference sequence, so long as the resulting variants retain the properties of the reference protein and the percent identity as stated above. Regardless of the reference sequence considered (phycocyanin α and / or β subunits and / or allophycocyanin α and / or β subunits), variants of said subunits may contain between 1 and 15 amino acid differences, preferably between 1 and 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 with respect to the corresponding reference sequence, as long as the resulting variant retains the properties of the reference protein and the percent identity stated above.

[0030] In some embodiments, C-phycocyanin or variants thereof useful in acidic compositions, particularly acidic food compositions, alone or in combination with allophycocyanin or variants thereof, can be obtained by culturing naturally occurring organisms which naturally express C-phycocyanin or its variant of interest, or by culturing organisms which have been genetically transformed to express C-phycocyanin or its variant of interest and which have been selected for their ability to produce said C-phycocyanin or variants thereof. Examples of naturally occurring organisms which naturally express C-phycocyanin or its variant of interest useful in compositions according to the present technology include algae or microalgae of the order Polytrichum edulis. The Cyanidiales include the families Cyanidiaceae and Galdieriaceae, which are themselves subdivided into the genera Cyanidioschizon, Cyanidium and Galdieria, whose members include, among others, the species Cyanidioschizon merolae 10D, Cyanidium merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria phlegrea, Galdieria spp. Particular mention may be made of the strain Galdieria sulphuraria (also called Cyanidium caldarium).

[0031] Thus, according to an embodiment of the present technology, the acidic composition, particularly the acidic food composition, comprises an acidic pH-resistant phycocyanin derived from a natural organism, such as an alga or microalga of the order Polytrichumales, particularly from a natural organism of the family Polytrichumaceae or Galdieriaceae. In some examples, the acidic composition, particularly the acidic food composition, comprises an acidic pH-resistant phycocyanin derived from a natural organism belonging to the genera Cyanidioschizon, Cyanidium, and Galdieria, advantageously selected from the species of Cyanidium and Galdieria. In some other examples, the acidic composition, particularly the acidic food composition, comprises an acidic pH tolerant phycocyanin derived from a natural organism selected from the species Cyanidioschizon melorae 10D, Cyanidioschizon melorae DBV201, Cyanidium caldarium, Cyanidium daedarum, Cyanidium maximum, Cyanidium partitum, Cyanidium lumpens, Galdieria daedara, Galdieria maxima, Galdieria partita, Galdieria phlegrea, and Galdieria sulphuraria. In some further examples, the acidic food composition according to the present technology comprises an acidic pH-tolerant phycocyanin derived from natural microalgae such as Galdieria sulphuraria, Cyanidium caldarium or Cyanidioschyzon melorae. More preferentially, the acidic pH-tolerant phycocyanin is derived from natural microalgae selected from Galdieria sulphuraria and Cyanidium caldarium.

[0032] As an example of an organism 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 a microorganism transformed to express the apoproteins of SEQ ID NO:1 and / or SEQ ID NO:2 and / or SEQ ID NO:3 and / or SEQ ID NO:4, which also contain the biosynthetic pathways necessary for the production of the chromophore and for its binding to the apoprotein. As a microorganism that can be modified to produce C-phycocyanin and / or allophycocyanin used in the food composition according to the present technology, mention may be made in particular of yeast. Methods for culturing natural and / or modified organisms capable of producing phycocyanins useful in the composition according to the present technology are known in the art. For example, the cultivation of the family Pycocyanaceae or the family Galdieriaceae of the order Pycocyanales, known to those skilled in the art, can be carried out in a mixotrophic manner, with light usually being required for the biosynthesis of the pigments. Such industrial cultivation can be carried out in large scale (i.e. 1,000 liters, 10,000 liters, 20,000 liters, 100,000 liters) fermentors. The cultivation can be carried out under conditions known to those skilled in the art. The cultivation can be carried out in batch, fed-batch or continuous mode.

[0033] Phycocyanin useful in the composition according to the present technology can be extracted from a biomass obtained by culturing algae of the order Polytrichum communes as defined above, cultivated in mixotrophic mode with light having a wavelength of about 400 nm to about 550 nm, or about 420 nm to about 500 nm, or about 430 to about 480 nm, or about 455 nm. This can be a "white" light with a broad spectrum including said wavelengths of light. It can also be a narrow spectrum consisting of said wavelengths. Such a method for the industrial preparation of Polytrichum communes in mixotrophic mode, and the biomass thus obtained, is described in particular in patent application FR 15 59072, the contents of which are incorporated herein by reference.

[0034] The objective of the present technology is to provide an acidic composition in which phycocyanin exhibits improved or enhanced stability at acidic pH. By acidic composition is meant, according to the present technology, any composition comprising a mineral or organic acid and phycocyanin. The composition may be a fluid or viscous liquid, a paste or a solid having an acidic pH and into which an acidic pH-resistant phycocyanin is incorporated. "Improved" or "enhanced" stability at acidic pH means that the color intensity is better maintained upon heat and / or storage treatment of the solution containing phycocyanin. The enhancement can be measured via spectrophototometric methods following the λmax value or via colorimetric methods following the dE CMC.

[0035] For aqueous liquid compositions, pH is measured in the usual manner. For non-aqueous liquid compositions or for pasty or solid compositions, pH is measured after dissolving the composition in a sufficient amount of water to dissolve the soluble compounds contained therein, including mineral or organic acids and phycocyanin. Examples of mineral acids that can be used in the compositions of the present technology include, but are not limited to, carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulfonic acid and nitric acid. Other examples include organic acids such as citric acid, lactic acid, malic acid, tartaric acid, and succinic acid. In some embodiments, the composition according to the present technology is an aqueous liquid composition, which may be in the form of a gel, or a pasty or solid composition designed for dissolution in an aqueous solution or a solid or pasty composition containing water. According to other embodiments of the present technology, the acidic composition is a pasty or solid composition intended to be utilized and / or stored in a moist environment.

[0036] An acidic food composition, according to the present technology, means any composition designed for consumption by humans or animals and falling within the scope of the preceding definition. Nutraceutical acidic compositions must be considered to fall within the definition of an acidic food composition within the context of the present technology. The acidic food composition according to the present technology is well known to those skilled in the art. The acidic food composition may comprise a carrier that may comprise structural components associated with active compounds identified for their nutritional supply or for their health properties that benefit humans or animals. The acidic food composition according to the present technology may also comprise food additives such as texturizing agents, flavoring agents, preservatives, or any components known to those skilled in the art. The carrier may comprise water and / or protein and / or lipid and / or fiber and / or sugar. The components of the carrier may only have structural properties, but are generally known for their nutritional supply.

[0037] The acidic food composition according to the present technology can be ready to use or in the form of a food additive that is intended to be added to a solid, pasty or liquid preparation to prepare an edible food product. For food compositions, the acid may be selected from the list of oxidizing agents approved for food, in particular carbonic acid, phosphoric acid, citric acid, malic acid, tartaric acid, lactic acid, and citric acid. As regards the non-food acidic compositions according to the present technology, they may be, inter alia, pharmaceutical, veterinary or cosmetic compositions and may further comprise any additive and / or active agent known and used in such compositions.

[0038] In the solid, liquid or pasty acidic compositions according to the present technology, phycocyanin can be incorporated, for example, in powder form. The acidic compositions, in particular the acidic food compositions, can be in any known conventional form, i.e. creams, gels, foams, pastes, etc. Examples of solid food compositions include cakes or biscuits, dry foods for cooking, soluble powders, gelatinous solid compositions (jelly), foams, etc. According to the present technology, the liquid acidic composition may be an aqueous composition in which phycocyanin is dissolved. The liquid acidic composition may be in the form of a ready-to-use composition or a liquid concentrate for dilution, in particular intended to be ingested or added to solid foods, either for their preparation or for their consumption, such as a concentrated liquid "topping" composition, intended to be applied to cakes to give them their color. Among these concentrated compositions, mention may be made of syrups, which may contain alcohol.

[0039] Liquid acidic compositions according to the present technology can be of various viscosities and can include additives such as thickeners, gelling agents, and other structuring additives known to those skilled in the art and typical for the preparation of liquid food compositions. According to one embodiment of the present technology, the liquid food composition is a carbonated acidic beverage. Particular mention may be made of soda, juice, sports drinks, energy drinks, recovery drinks, etc. The composition of these beverages is well known to those skilled in the art and may include, among others, sugars, inorganic salts, food additives, dissolved gases, etc. The beverage according to the present technology is a conventional acidic beverage in which the normally used coloring agents are completely or partially replaced by the acidic pH-resistant phycocyanin according to the present technology.

[0040] We have found that, counterintuitively, the more phycocyanin is concentrated in a composition, the less stable it is and therefore the more the color of the final product varies. Therefore, to obtain a more colorful product, it is recommended to reduce the phycocyanin, especially C-phycocyanin, content, in other words, to reduce the amount of coloring agent to increase the color of the product. In some embodiments, phycocyanin is present in the acidic composition of the present technology in an amount that allows for improved stabilization of phycocyanin, also referred to herein below as "low content". In some examples, phycocyanin is present in the acidic composition in an amount ranging from about 0.001 to about 0.1% by weight, or from about 0.001 to about 0.05% by weight, or from about 0.001 to about 0.025% by weight, or from about 0.0025 to about 0.010% by weight, or from about 0.0025 to about 0.005% by weight. In some examples, phycocyanin is present in the acidic composition in an amount ranging from about 2.5 mg / L to about 2500 mg / L, from about 25 mg / L to about 300 mg / L, or from about 50 mg / L to about 100 mg / L. In some implementations of these embodiments, the acidic composition is a liquid acidic composition.

[0041] In embodiments in which the acidic composition is a ready-to-use beverage, the phycocyanin content may range from about 25 mg / L to about 300 mg / L, or from about 50 mg / L to about 100 mg / L. In embodiments in which the acidic composition is a concentrated liquid composition for dilution before use (e.g., a syrup), the phycocyanin content may range from about 50 mg / L to about 2500 mg / L, or from about 500 mg / L to about 1000 mg / L. In embodiments in which the acidic composition is a solid composition, the phycocyanin content may range from about 0.01 mg / g to about 10 mg / g, or from about 0.1 mg / g to about 5.0 mg / g, or from about 0.25 mg / g to about 2.5 mg / g. In some embodiments, the Galdieria extract is present in the acidic composition of the present technology in an amount that allows for improved stability of phycocyanin. In some examples, the Galdieria extract is present in the acidic composition in an amount ranging from about 0.004 to about 0.4% by weight, or from about 0.004 to about 0.2% by weight, or from about 0.004 to about 0.1% by weight, or from about 0.01 to about 0.04% by weight, or from about 0.01 to about 0.02% by weight. In some examples, the Galdieria extract is present in the acidic composition in an amount ranging from about 10 mg / L to about 10 g / L, from about 100 mg / L to about 1200 mg / L, or from about 200 mg / L to about 400 mg / L. In some implementations of these embodiments, the acidic composition is a liquid acidic composition.

[0042] In embodiments where the acidic composition is a ready-to-use beverage, the Galdieria extract content can range from about 100 mg / L to about 1200 mg / L, or from about 200 mg / L to about 400 mg / L. In embodiments where the acidic composition is a concentrated liquid composition (e.g., a syrup) for dilution before use, the Galdieria extract content can range from about 200 mg / L to about 10 g / L, or from about 2000 mg / L to about 4000 mg / L. In embodiments where the acidic composition is a solid composition, the Galdieria extract content can range from about 0.04 mg / g to about 40 mg / g, or from about 0.4 mg / g to about 20 mg / g, or from about 1 mg / g to about 10 mg / g.

[0043] In some embodiments, the composition of the present technology further comprises one or more sulfated polysaccharides. In some examples, the one or more sulfated polysaccharides are polysulfated carrageenans. Carrageenans are derived from seaweed and are very flexible molecules that can form spiral-type helical structures. Carrageenans are characterized as linear polysaccharides that contain repeating galactose units. Carrageenans are classified by their degree of sulfation. Examples of monosulfated carrageenans are k-carrageenan, g-carrageenan, and o-carrageenan. Examples of disulfated carrageenans are i-carrageenan, d-carrageenan, m-carrageenan, and q-carrageenan. Examples of trisulfated carrageenans are l-carrageenan and v-carrageenan.

[0044] Polysulfated carrageenan is a carrageenan that contains at least two sulfate groups, preferably two or three sulfate groups per disaccharide unit.The examples of polysulfated carrageenan that can be used in the present technology include, but are not limited to, i-carrageenan, d-carrageenan, m-carrageenan, 0-carrageenan, l-carrageenan and v-carrageenan, the hydrolysis product of i-carrageenan, the hydrolysis product of d-carrageenan, the hydrolysis product of m-carrageenan, the hydrolysis product of 0-carrageenan, the hydrolysis product of l-carrageenan and the hydrolysis product of v-carrageenan; i-carrageenan and l-carrageenan and their hydrolysis products are more preferred, and l-carrageenan and hydrolyzed l-carrageenan are most preferred. In some embodiments, the carrageenan content of the composition of the present technology ranges from about 0.006% to about 0.6% by weight, or from about 0.02% to about 0.5% by weight, or from 0.03% to about 0.4% by weight, or from about 0.05% to about 0.3% by weight, or from about 0.005% to about 0.1% by weight, or from about 0.001% to about 0.1% by weight, or from about 0.05% to about 0.1% by weight.

[0045] In some embodiments, the mass ratio of phycocyanin extract to carrageenan present in the acidic composition of the present technology depends on the water content and pH of the composition. At high water contents of at least about 45% by weight and up to about 95% by weight, the mass ratio between phycocyanin extract (phycocyanin extract containing about 25% to about 30% phycocyanin) and carrageenan (phycocyanin extract:carrageenan) ranges from about 1:1 to about 10:1, or from about 1:1 to about 5:1, or from about 1:1 to about 4:1, or from about 1:1 to about 3:1, or from about 1:1 to about 2:1. In some embodiments, the pH of the composition at this high water content ranges from about 2.0 to about 3.7, or from about 2.2 to about 3.5, or from about 2.3 to about 3.3. At low water content of at least 15 and less than 45% by weight water, the mass ratio between the phycocyanin extract (phycocyanin extract containing about 25% to about 30% phycocyanin) and the carrageenan is in the range of 1:1.5, preferably 1:1 to 10:1, at a pH of at least about 2 and less than about 2.5. The mass ratio between the phycocyanin and the carrageenan is in the range of about 1:2, preferably 1:1 to about 10:1, at a pH of at least about 2.5 and less than about 2.9, and the mass ratio between the phycocyanin extract and the carrageenan is in the range of about 1:1 to about 10:1, at a pH of at least about 2.9 and less than about 4. The phycocyanin and the carrageenan are preferably dissolved in the liquid raw material. Dissolved means when the phycocyanin and the carrageenan remain in the aqueous solution. The solution is visually clear and does not show any precipitate or suspended particles.

[0046] It is known that phycocyanin composition affects color. When the pH is decreased and the equilibrium of C-phycocyanin shifts towards the monomer, the color shifts from dark blue to blue-green (Buchweitz, 2016). Generally, at pH values ​​below 3.9, the equilibrium of C-phycocyanin shifts towards the monomer. Color performance is measured by colorimetry (CIE L * a * b * and h value, see above) and the sum of the blue absorbance. Without being bound by any theory, it is hypothesized that in acidic conditions, negatively charged polysulfated carrageenans, e.g., λ- and ι-carrageenans, form complexes with positively charged phycocyanin, preventing aggregation and stabilizing color. However, a clear color shift is observed with the addition of polysulfated carrageenans, e.g., ι- and λ-carrageenans, in acidic conditions, especially at pH 2.6-4.0. The addition of carrageenans results in a brighter (higher L * ) and less blue / more yellow (higher b * ) to shift the color tone.

[0047] A standard method for phycocyanin determination to calculate native phycocyanin content (mg / mL) from photometric measurements was established by Yoshikawa & Belay (2008). The method relies on absorbance measurements at 620 nm and 650 nm and the extinction coefficients of C-phycocyanin and allophycocyanin at these wavelengths at pH 6.0. The addition of polysulfated carrageenan changes the shape of the phycocyanin peak, and in some cases the peak maximum shifts from 620 nm to 660-670 nm. Calculation of phycocyanin content using only absorbance measurements at 620 nm and 650 nm would underestimate the phycocyanin content for solutions containing polysulfated carrageenan. Thus, the sum of blue absorbance measurements at A620nm-A750nm, A650nm-A750nm, and A667nm-A750nm is calculated and used to determine the magnitude of color retention. The total phycocyanin content in Spirulina extracts is calculated using the method of Yoshikawa & Belay (2008) at pH 6.0 listed below.

[0048]

number

[0049] To determine the amount of C-phycocyanin to add to the composition, the dosage level of the Galdieria extract is multiplied by the total C-phycocyanin content in the Galdieria extract. In some embodiments, the phycocyanin content of the liquid composition is from about 0.001% to about 0.1% by weight, or from about 0.003% to about 0.5% by weight, or from about 0.007% to about 0.4% by weight, or from about 0.01% to about 0.4% by weight. A difficulty with phycocyanin containing Spirulina extracts is application to acidified aqueous systems. Spirulina proteins (including phycocyanin) precipitate out of solution when the pH is about 2.7 to about 6.0. Precipitation may occur immediately or after several weeks, depending on factors such as solution viscosity, ionic strength, order of addition, temperature, and pH. Aggregation / precipitation is assessed visually and may be responsible for discoloration. High absorbance at 750 nm after acidification and / or processing may indicate a strong potential for protein aggregation / precipitation.

[0050] In some embodiments, acidic compositions according to the technology have a pH of less than 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, and more preferably, the pH is 2.0 or greater. Particularly advantageous is the combination of a low content of phycocyanin from the genus Galdieria with at least one sulfated polysaccharide, in particular λ-carrageenan, in a ratio of 0.3:1 to 0.6:1 at a pH above 3.0 and below 4.0.

[0051] In some embodiments, the compositions of the present technology include additional ingredients such as, for example, sweeteners, stabilizers, chelating agents, acids, proteins, salts, flavors, vitamins, minerals, colorants, thickeners, and preservatives. The composition of the present technology may have a blue color due to the phycocyanin content, but other colors based on blue can be obtained by mixing other pigments in the composition. The composition can contain other pigments such as safflower, anthocyanins, carotenoids, betanins, annatto, lycopene, curcumin, and chlorophyll. Pigments can be added to the blue phycocyanin-containing composition to blend into other colors, such as green, for example, with safflower.

[0052] Examples of sweeteners are invert sugar, sucrose, high fructose corn syrup, fructose, glucose, trehalose, lactose, honey, agave, stevia, sucralose, aspartame, neotame, acesulfame potassium, monk fruit, or saccharin. Examples of preservatives are salt, sorbic acid, benzoic acid, natamycin, nisin, and sulfite. Examples of salts are ionic compounds that can be formed with positively charged cations such as calcium, magnesium, potassium, sodium, or copper, and salt-forming anions such as acetate, carbonate, chloride, citrate, oxide, phosphate, sorbate, benzoate, hexametaphosphate, or sulfate. Examples of acids are phosphoric acid, citric acid, sulfuric acid, malic acid, adipic acid, tartaric acid, or sodium acid pyrophosphate. Examples of thickening agents are gelatin, starch, pectin, konjac, or agar.

[0053] The compositions according to the technology can contain chelators, sometimes also referred to as chelating agents. Chelators are binding agents that affect the chemical and / or physical state of the molecules / atoms to which they are bound by forming chelates. Chelators can improve color retention and have been found to act synergistically with polysulfated carrageenan. The chelating agents can be synthetic and natural compounds, and are in particular selected from the group of ethylenediaminetetraacetic acid and / or its Na, K, Ca salts (EDTA), L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA), sodium hexametaphosphate, glutathione, metallotheionein, 2,3-dimerapto-1-propanesulfonic acid, chlorella, garlic, coriander, selenium, milk thistle, vitamin C, vitamin E, citrate, grape seed extract, quercetin, and lipoic acid. Preferably, the chelating agent is selected from the group of ethylenediaminetetraacetic acid and / or its Na, K, Ca salts (EDTA) and L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA). EDTA is considered to be ethylenediaminetetraacetic acid together with its Na, K, Ca salts, such as calcium disodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, and tripotassium ethylenediaminetetraacetate. Calcium disodium ethylenediaminetetraacetate is CaNa 2 Disodium ethylenediaminetetraacetate (EDTA) is 2 Tetrasodium ethylenediaminetetraacetate (EDTA) is 4 Dipotassium ethylenediaminetetraacetate (EDTA) is 2 EDTA is abbreviated as tripotassium ethylenediaminetetraacetate (K). 3It is abbreviated as EDTA. More preferably, the chelating agent is vitamin C and / or EDTA. The chelating agent may be present in the composition in an amount ranging from about 1 to about 2000 ppm. 1 ppm of the chelating agent should be understood as 0.0001% by weight; for example, 30 ppm of EDTA is 0.003% by weight. The EDTA may be present in the composition in an amount ranging from about 10 to about 300 ppm, or (por) about 15 to about 200 ppm, or about 20 to about 100 ppm.

[0054] The EDTA and phycocyanin (such as phycocyanin from a Galdieria extract) are preferably present in the composition of the present technology in a mass ratio of about 4:1 to about 1:200, or about 2:1 to about 1:125, or about 1:1 to about 1:75. Compositions are typically transported and stored prior to consumption, and thermal processes, preservatives, water activity, or any combination of the above are used to ensure that these compositions do not spoil and are safe for consumption. A significant blue discoloration is observed during high temperature short time processing (210°C for 6 seconds) and when preservatives are used for solutions that do not contain carrageenan.

[0055] The composition can be obtained according to a process comprising the steps of: a) adding and dissolving polysulfated carrageenan in water and mixing until the polysulfated carrageenan is dissolved by visual observation; b) adding the phycocyanin-containing Galdieria extract and mixing until dissolved at a pH of at least 5, preferably between 5 and 10; c) adding an acid or oxidizing component; d) optionally adding sweeteners, flavors, vitamins, minerals, salts, buffers, or other beverage ingredients; e) optionally adding a chelating agent such as EDTA; f) optionally adding other pigments such as anthocyanins; g) optionally adding additives such as preservatives; h) treating the mixture of a)-h) by thermally treating the liquid to at least 65°C; or cold filling without thermal treatment, or a combination of the two. It is important that the phycocyanin-containing Galdieria extract is added to the dissolved polysulfated carrageenan, which may be in the presence of other ingredients, at a pH of 5.0 or higher. A list of compounds that can destabilize phycocyanin includes, but is not limited to, acids, anthocyanins, safflower, and alcohol. Acids can be used to prepare hydrolyzed carrageenan, but the charge of the hydrolyzed carrageenan should be neutralized to a pH of at least 5 before mixing with phycocyanin.

[0056] Ingredients that do not destabilize phycocyanin can be added during or before step a) of the process. Polysulfated carrageenan can be dry mixed with non-acidic carbohydrates such as sucrose or maltodextrin, for example, to improve dissolution. Step c) (addition of acid or acidifying ingredient) can be performed before or after steps d), e), or f). Step h) is preferably performed after steps a)-g). If present, preservatives (step g) are preferably added before or during step a), due to the solubility limitations of the preservatives. Carrageenan is a linear polysaccharide containing repeating sulfated galactose units. The glycosyl bonds between the galactose units are subject to hydrolysis by heat and acid. Hydrolyzed polysulfated carrageenan has also been found to stabilize phycocyanin, however, the charge of the hydrolyzed polysulfated carrageenan must be neutralized to a pH above 5 prior to complexation with phycocyanin. Both hydrolyzed and undegraded polysulfated carrageenan must have a pH above 5 prior to the addition of phycocyanin to prevent precipitation. Preferably, the pH of the solution containing the polysulfated carrageenan is between 5 and 10, more preferably between 5.5 and 9.9, prior to the addition of phycocyanin.

[0057] The present technology also relates to a method for producing a green color comprising heating phycocyanin in an aqueous solution at a temperature of about 30° C. to about 90° C. and a pH of about 3.6 or less than 3.6, wherein the phycocyanin is extracted from algae or microalgae of the order Polytrichum algae, preferably from the genus Cyanidioschizon, Cyanidium, or Galdieria, more preferably from the species Galdieria sulphuraria, Cyanidium caldarium, or Cyanidioschizon merolae. In a preferred embodiment, the aqueous solution contains about 0.3% to about 10% phycocyanin, more preferably about 0.1% to about 1% phycocyanin.

[0058] In a particular embodiment, the method for producing a green color is characterized in that the heating temperature is from about 30° C. to about 90° C., particularly about 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98 7°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C or higher. More preferably, the heating temperature is about 40°C to about 90°C, particularly about 50°C to about 85°C, and even more preferably about 60°C to about 80°C.

[0059] In a particular embodiment, the method for producing a green color is characterized in that the pH of the aqueous solution is preferably between about 2.0 and 4.0, more preferably around 3.0. According to a particular embodiment, the method for producing a green color comprises the use of phycocyanin, which is preferably an acidic pH-tolerant phycocyanin having the amino acid sequence, structure and functional properties as described herein. The heating step of the method for producing the green color may have a duration comprised between about 1 minute and about 480 minutes, preferably between about 60 minutes and about 360 minutes, and more preferably between about 120 minutes and about 300 minutes. It should be noted that the higher the temperature, the shorter the duration required. In a particularly preferred embodiment, the method for producing the green color includes a heating step at a temperature of about 70° C. to about 80° C. for a period of about 200 minutes to about 280 minutes. The present technology also relates to the green color obtained by this method, which is therefore also called green phycocyanin, and which has a hue angle of about 120° to 190°.

[0060] In another embodiment, the present technology relates to a method for stabilizing the green color obtained from phycocyanin by the above method, comprising a step of mixing said color with at least one chelator selected from the group of ethylenediaminetetraacetic acid and / or its Na, K, Ca salts (EDTA), L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA), sodium hexametaphosphate, glutathione, metallotheionein, 2,3-dimerapto-1-propanesulfonic acid, chlorella, garlic, coriander, selenium, milk thistle, vitamin C, vitamin E, citrate, grape seed extract, quercetin, and lipoic acid, preferably vitamin C and / or EDTA. It should be noted that in the art, those skilled in the art are aware that vitamin C is a compound that can destabilize phycocyanin. Therefore, it is particularly surprising that in the present technology, the inventors use vitamin C as a stabilizing agent.

[0061] Preferably, the method for stabilizing the green color obtained from phycocyanin by the above method further comprises the step of adding at least one sulfated polysaccharide as described above. The present technology relates to a stabilized green color obtained from phycocyanin by the method as described above. Preferably, the stabilized green color obtained is stable under heat and / or light exposure. In certain embodiments, the green color according to the technology, stabilized or not, can be in either solid, pasty, or liquid form. The present technology also relates to products comprising the green color obtained from phycocyanin by the above-described production method or the stabilized green color obtained from phycocyanin by the above-described stabilization method.

[0062] In some embodiments, the acidic compositions, food products and / or concentrated liquids according to the technology contain or consist of phycocyanin under the green form. According to a preferred embodiment, the green colour has been subjected to sterilisation, such as flash sterilisation. According to another aspect, the present technology relates to a method for improving the thermal stability and / or photostability of phycocyanin, comprising the steps of: a) adding and dissolving at least one sulfated polysaccharide in water; b) adding phycocyanin at a pH of at least 5.0, preferably at a pH of 5.0 to 10.0, and mixing until dissolved. With regard to this method, in some embodiments, the composition of the present technology comprises one or more sulfated polysaccharides as described above. For this method, the phycocyanin is preferably an acid pH tolerant phycocyanin having the amino acid sequence, structure and functional properties as described herein. For this method, the phycocyanin may be a green phycocyanin according to the art. It has been surprisingly observed that at higher concentrations a green shift occurs. Thus, the present invention relates to compositions comprising at least 0.1% phycocyanin, such as at least 0.2%, at least 0.3%, at least 0.5%, at least 0.6%, at least 1%, at least 5%, at least 10%, preferably about 0.1% to about 10% phycocyanin, more preferably about 0.1% to about 1% phycocyanin. In a preferred embodiment, the phycocyanin is Galdieria phycocyanin. The composition can have a basic, neutral or acidic pH.

[0063] Preferably, the composition is an acidic composition, ie, having a pH of less than 4.0, preferably between 2.0 and 4.0. It is noted that the technology relates to all possible combinations of features described herein, with the combinations of features present in the claims being particularly preferred.Therefore, it will be understood that all combinations of features related to the compositions according to the technology, all combinations of features related to the processes according to the technology, and all combinations of features related to the compositions according to the technology, as well as features related to the processes according to the technology, are described herein. One of the advantages of this technology lies in the fact that the coloration provided by the acidic pH-resistant phycocyanin is more stable over time, as can be seen in the examples below. Other aspects and features of the present technology will become apparent from a study of the examples and drawings. EXAMPLES

[0064] Example 1 Production of phycocyanin from Galdieria sulphuraria Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX#2919. Batch culture medium: 30g / L glycerol, 8g / L (NH 4 ) 2 SO 4 , 1g / L KH 2 PO 4 , 716mg / L MgSO 4 , 44mg / L CaCl 2 , 3 mL / L Fe-EDTA stock solution (6.9 g / L FeSO 4 and 9.3g / L EDTA-Na 2 ) and 4mL / L of trace metal solution (3.09g / L EDTA-Na 2 ;0.080g / L CuSO 4 ,5H 2 O: 2.860 g / LH 3 BO 3 ;1.820g / L MnCl 2 ;0.220g / L ZnSO 4 ,7H 2 O).

[0065] Culture conditions: Culture was performed at 1 ml using a computer-controlled automated system in a fed-batch mode as described in patent application WO2020161280, which is incorporated herein by reference. 3The cultivation is carried out in a volumetric reactor. For the feed medium, the amount of carbon source is adjusted according to the targeted dry mass of the cultivation. All other elements of the medium are added taking into account the proportions used for the batch medium defined in the examples. The culture pH is adjusted with a base (14% ammonia solution (wNH 3 The incubation temperature is set at 37° C. The cell growth is followed at different time points by measuring the absorbance at 800 nm and the dry mass is measured by filtration. The performance characteristics of the culture at the end of the growth are summarized in Table 1 below.

[0066] [Table 1] Measurement of intracellular phycocyanin content per gram of dry matter was performed using extraction according to Modeste et al., Toxicology Research and Applications VOL. 3, 1 - 13, 2019, and was measured using the method described by Yoshikawa and Beal, JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 3, 2008, replacing the phosphate buffer with Tris-Cl buffer.

[0067] Example 2 Extraction of phycocyanin Strains Galdieria sulphuraria (UTEX#2919) and / or Cyanidioschyzon melorae (ACUF199) were cultivated under the conditions of Example 1. Phycocyanin was then extracted according to the protocols described in WO 2020 / 161280 and WO 2018 / 17833, both of which are incorporated herein by reference. An extract was obtained that contained other water-soluble proteins in addition to the phycocyanin of interest (also called "phycocyanin extract" or "crude extract"). Phycocyanin extracts can have several possible qualities depending on the extraction and / or purification method used. For example, a crude extract will contain a higher amount of water-soluble proteins other than phycocyanin than those found in a purified extract. The 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, which is the specific absorbance of aromatic amino acids that represents the total protein level. The lower this ratio, the greater the amount of non-phycocyanin proteins in the solution. The crude extract was purified using a KrosFlo® tangential flow filtration system from Spectrum® Labs. Table 2 shows the purity index measurements of the phycocyanin extract before and after purification.

[0068] [Table 2]

[0069] Example 3 Stability of phycocyanin in beverages over a 4-hour period Purified phycocyanin extracts from Galdieria as outlined in Examples 1 and 2 were used to perform stability studies (4 hours at 43°C) in a model beverage (water, 7% sucrose, citric acid pH 3) where different concentrations of Galdieria extract (Galdieria extract containing approximately 25% phycocyanin) were added in the amounts of 0.4% (i.e., 0.10% phycocyanin), 0.2% (i.e., 0.05% phycocyanin), and 0.1% (i.e., 0.025% phycocyanin) of the total mass of the beverage. Stability was evaluated by measuring the color difference using dE CMC values ​​between the negative control (maintained at 4°C) and the 43°C treated samples. Model beverages were prepared with three different Galdieria extract concentrations: 0.1%, 0.2%, and 0.4%, i.e., 0.03%, 0.06%, and 0.10% by weight of phycocyanin, along with 7% by weight granulated sugar, and adjusted to pH 3.0 with 50% citric acid solution. The samples were placed in a 43°C oven for 4 hours and measured on a colorimeter to obtain the dE CMC values. If the dE CMC is greater than 2, the color difference is detectable by the human eye.

[0070] The results of this experiment are presented in Figure 1, showing all three concentrations diluted to 0.1% after heat treatment for visual comparison. The drink on the far left was 0.1% w / w Galdieria extract and had an average dE CMC of 1.9 after heat treatment. The drink in the middle was 0.2% w / w Galdieria extract and had an average dE CMC of 5.475 after heat treatment. The drink on the far right was 0.4% w / w Galdieria extract and had an average dE CMC of 16.45 after heat treatment. These results indicate that the more phycocyanin present in the composition, the less stable the product and / or coloration.

[0071] Example 4 Phycocyanin stability over 8 weeks in a first series of beverages The model beverages were tested for 8 week stability. The model beverages were formulated as presented in Table 3. Table 4 shows the electrolyte content for the indicated model beverages, and Table 5 shows the electrolyte mixture formulation for the indicated model beverages. The Galdieria extract contains about 30% phycocyanin.

[0072] [Table 3]

[0073] [Table 4]

[0074] [Table 5] The reference beverages were sterilized using HTST sterilization (BottomLine sterilizer, Advantage Engineering) at 75°C for 30 seconds, followed by cooling in an ice bath. After cooling, the beverages were stored at various temperatures, including 4°C (which served as the control), 32°C in the dark, and 25°C in the dark and light. The beverages were removed for observation and photographed at 8 weeks. Figures 2A and 2B show the results of stability tests on model beverages and electrolyte beverages containing 0.2% Galderia extract. Significant changes during storage of beverages containing Galderia at 0.2%. It can be noted that the decrease in hue angle during storage indicates a shift in hue from blue to green, thus the blue color shows a green nuance as storage time increases, and that higher storage temperatures result in a greater shift to green.

[0075] Example 5 Phycocyanin stability over 8 weeks in a second series of beverages The model beverages were tested for stability over a period of 8 weeks. The model beverages were formulated as presented in Table 6. Table 7 shows the electrolyte mixture formulations for the model beverages shown. The Galdieria extract contains approximately 30% phycocyanin.

[0076] [Table 6]

[0077] [Table 7] 3A and 3B show the results of a stability study on a model beverage containing 0.2% and 0.4% Galdieria extract. The addition of vitamin C reduced the darkening (L) compared to no vitamin C addition.* The apparent difference in color between those with and without vitamin C is believed to be due to a darkening effect. The addition of vitamin C shows a slight protective effect on the color based on Galdieria phycocyanin.

[0078] Example 6 Stability of phycocyanin in beverages over a four week period in the absence of vitamin C The stability of phycocyanin in beverages in the absence of vitamin C was evaluated. Model beverages were formulated as presented in Table 8. Table 7 above shows the electrolyte mixture formulations for the model beverages shown. The Galdieria extract contains approximately 30% phycocyanin. [Table 8]

[0079] The reference beverages were pasteurized using HTST pasteurization (BottomLine pasteurizer, Advantage Engineering) at 75°C for 30 seconds, followed by cooling in an ice bath. After cooling, the beverages were stored at various temperatures, including 4°C (which served as the control), 32°C in the dark, and 25°C in the dark and light. The beverages were removed for observation and photographed at 4 weeks. Figures 4A and 4B show the results of the stability test for the model beverages containing 0.01% and 0.02% Galdieria extract. The hue angle remained almost constant for all storage conditions after 4 weeks for 0.02% and 0.01% Galdieria, in other words, no significant change in the color of the beverage was observed when Galdieria was used at concentrations of 0.01% and 0.02%. At dE CMC values ​​below 3, the color change was barely visible. The mechanism causing the instability is not really understood, but is related to the acid pH. The more acidic the pH, the more unstable the phycocyanin.

[0080] Example 7 Stability of Galdieria extracts in combination with λ-carrageenan The effect of λ-carrageenan on the stability of Galdieria extract in a model beverage was evaluated. A model beverage (control) was made by dissolving 12% by weight of granulated sugar and 0.05% Galdieria extract powder (Galdieria extract contains about 30% phycocyanin) in deionized water, i.e., 0.015% phycocyanin compared to the total weight of the beverage, or 0.01% Galdieria extract powder, i.e., 0.003% phycocyanin compared to the total weight of the beverage. λ-carrageenan powder was added to the model beverage in different concentrations to obtain a phycocyanin:carrageenan ratio of 1.5:1. The pH of the beverage was then adjusted to 3 with 50% citric acid. The preparations were tested for color change through flash pasteurization. The model beverage and the beverage containing carrageenan were placed in 20 mL scintillation vials and immersed in a 80°C water bath for 4 minutes. The vials were then immediately immersed in ice water, whereupon the contents were measured on a colorimeter. Each beverage vial was placed in an 80°C bath for 4 minutes, followed by an ice bath for 4 minutes to simulate HTST sterilization. The dE CMC was then measured to determine color loss. A dE CMC greater than 2 is within the visible range. The presence of λ-carrageenan in the acidic beverage resulted in better heat stability and a hue similar to Blue No. 1.

[0081] Figures 5 and 6 show the discoloration that occurs due to flash pasteurization. Each photograph has an unheated vial on the left and a heated vial on the right. Greater discoloration can be seen for the control photograph (left) than for the Galdieria phycocyanin:carrageenan photograph (right). This confirms that λ-carrageenan helps stabilize Galdieria and protect the color in heated conditions. The Galdieria 0.05% model beverage control (left) has a dE CMC of 5.0 after HTST pasteurization, while the Galdieria:carrageenan (right) showing a 1.5:1 ratio (0.0015% Galdieria phycocyanin:0.01% carrageenan) has a dE CMC of 3.4 after HTST pasteurization. These results indicate that higher concentrations of Galdieria result in a less stable product, and also that λ-carrageenan has a protective effect on phycocyanin during heat treatment.

[0082] FIG. 7 shows a comparison of the Galdieria phycocyanin:carrageenan (1.5:1) containing beverage to the commercial beverages Gatorade® and Powerade® (dE CMC 1.99). The vial on the left shows the Galdieria phycocyanin:carrageenan model beverage at a 1.5:1 ratio (0.05% Galdieria extract, 0.025% carrageenan) compared to the middle vial containing blue Powerade and the right vial containing blue Gatorade. The dE CMC measured between the Galdieria beverage and the Powerade and Gatorade beverages is 1.99, which is within the visible range. This low deCMC value indicates a color match of the Galdieria phycocyanin:carrageenan (1.5:1) beverage to the blue No. 1 containing beverage.

[0083] Example 8 Stability of different ratios of phycocyanin:carrageenan upon flash pasteurization A model beverage (control) was made by dissolving 12% by weight of granulated sugar and various concentrations of extract powder in deionized water and adjusting the pH to 3.0 with 50% citric acid solution. Test beverages were prepared by adding λ-carrageenan powder to the model beverage at different concentrations, followed by further adjusting the beverage to a pH of 3.0 with 50% citric acid. The applied concentrations of phycocyanin and carrageenan are presented in Table 9 below.

[0084] [Table 9] The formulations were tested for color change through flash pasteurization. The model beverage and the beverage containing carrageenan were placed in 20 mL scintillation vials and immersed in an 80°C water bath for 4 minutes. The vials were then immediately immersed in ice water, whereupon the contents were measured on a colorimeter. Figure 8 shows the HTST pasteurization of beverages with different ratios of Galdieria phycocyanin: λ-carrageenan. dE CMC values ​​of less than 2 were found for ratios of 0.3:1 and 0.6:1, indicating that the ideal ratio of Galdieria: λ-carrageenan is from about 0.6:1 to about 0.3:1.

[0085] Example 9 Stability of Galdieria extract combined with λ-carrageenan upon flash pasteurization A model beverage without carrageenan (control) was prepared by dissolving granulated sugar in deionized water, adding Galdieria powder at a concentration of 0.05%, i.e., 0.015% phycocyanin compared to the total mass of the composition, and adjusting the pH of the composition to 3.0 with 50% citric acid solution. The test beverage was made by mixing Galdieria powder and λ-carrageenan in a 5:1 ratio and dissolving granulated sugar to achieve 12% by mass in deionized water. The pH was then adjusted to 3.0 with 50% citric acid. The preparations were tested for color change through flash pasteurization. The model beverage and the beverage containing carrageenan were placed in 20 mL scintillation vials and immersed in a 80°C water bath for 4 minutes. The vials were then immediately immersed in ice water, after which the contents were measured on a colorimeter. Table 10 provides the dE CMC results. The results show that the model beverage carrageenan has a protective effect on color during heat treatment.The model beverage without carrageenan has an average dE CMC of 4.37, which is outside the visible range as can be seen in the left photo, and the beverage containing carrageenan has an average dE CMC of 1.225, which is within the visible range as can be seen in the left photo.This further proves the protective effect of λ-carrageenan in phycocyanin beverage under heat treatment.

[0086] [Table 10]

[0087] Example 10 Preparation of a green concentrate for use in beverages A green concentrate was prepared for use in beverages containing Galderia extract. The beverage was prepared as follows: 1% by weight of Galderia extract (powder form, 0.30% by weight of phycocyanin) based on the total weight of the beverage was dissolved in DI water, adjusted to pH 3.0 with 50% citric acid, and placed in a 75°C bath for 4 hours to obtain a green liquid. This green liquid was then added to a model beverage system at a concentration of 2.5% with 12% sugar. Experiment: Test the stability of green beverage in 43°C oven for accelerated shelf life stability. Green beverage is placed in 43°C oven and tested after 3 and 4 days. Table 11 shows the green beverage in accelerated shelf life condition. After 3 and 4 days in 43°C oven, green beverage is outside the visible range with dE CMC of 3.22 and 4.07 respectively. In some cases, carrageenan can be added to the composition after the composition undergoes green color shift.

[0088] [Table 11]

[0089] (Example 10bis) Stability of a green beverage prepared with Galdieria phycocyanin and vitamin C The green Galdieria concentrate is prepared as follows: 1. Dilute 1% Galdieria powder in deionized water; 2. Adjust pH to 3.0 with citric acid; 3. Heat the composition obtained in step 2 in a 75° C. water bath for 4 hours. Several samples are then prepared using the green extract already obtained so as to contain: 12% by weight of crystalline sucrose; and - 0.1% and 0.2% by weight of green Galdieria concentrate or 0.02% by weight of powdered blue Galdieria extract; and - Optionally, 300 ppm of Vitamin C. The color hues obtained between the standard blue Galdieria extract and the green Galdieria extract are shown in Table 12.

[0090] [Table 12] Each sample was tested using Suntest XLS + The samples were tested for dE CMC on a ColorQuest colorimeter before and after 2 hours of light treatment at 100° C. The dE CMC for the samples that underwent light treatment are presented in Figures 11 and 12. The results show that Vitamin C had a protective effect in the light treatment.

[0091] (Example 10ter) Stability of green beverages prepared with Galdieria phycocyanin A green Galdieria concentrate is prepared as already described in Example 10bis. Several samples are then prepared using the green extract already obtained, so as to contain: 12% by weight of crystalline sucrose; and - 0.1% and 0.2% by weight of green Galdieria concentrate or 0.02% by weight of powdered blue Galdieria extract. Each sample is tested for dE CMC on a ColorQuest colorimeter before and after the following treatments: HTST sterilization (80°C, 5 min); Light treatment: Suntest XLS + 2 hours in; and Accelerated heat stability (4 days, 40°C oven).

[0092] The dE CMC for the samples that underwent HTST is presented in Figures 9 and 10. In view of these results, the green beverage is more stable in HTST than the blue one (control). The dE CMC for the samples that underwent light treatment is presented in Figures 11 and 12. The results show that the green beverage is more stable in light than the blue one (control). The dE CMC for the samples that underwent accelerated stability treatment is presented in Figures 13 and 14. A relatively low concentration of green in the beverage system demonstrates that it is more stable than the blue control beverage. An inverse relationship is seen between the usage rate and the accelerated thermal stability. In view of the above, the green Galdieria concentrate beverage is stable under heat and light.

[0093] Example 11 Stability of various phycocyanin extracts in combination with λ-carrageenan under different pH The beverages were made with 12% sugar and the pH was adjusted with 50% citric acid solution. The color content was 0.06% Galdieria and Spirulina extracts, i.e. 0.018% by weight of phycocyanin compared to the total weight of the beverage, and λ-carrageenan was added at 0.01% and 0.06%. The "model" or "control" beverages have no carrageenan added. The "as is" beverages are beverages prepared without the addition of citric acid. The individual concentrations of phycocyanin and carrageenan applied are presented in Table 13 below.

[0094] [Table 13] The formulations were tested for color change through HTST sterilization. The model beverage and the beverage containing carrageenan were placed in 20 mL scintillation vials and immersed in an 80° C. water bath for 4 minutes. The following Figures 15A-18B show carrageenan beverages at various pH levels before heat treatment. Both Galdieria and Spirulina begin to turn green in the carrageenan system as the pH decreases, but the color is different between the Galdieria carrageenan beverage and the Spirulina carrageenan beverage, with Galdieria having a yellower color than Spirulina. Even before heat treatment, the Spirulina control beverage precipitated and was cloudy below pH 3.5, while the Galdieria control was clear and did not precipitate.

[0095] The dE CMC was measured on a ColorQuest colorimeter and is reported in FIG. 19. These results show that the beverages containing carrageenan are more heat stable than the control beverages that do not contain carrageenan. However, the Spirulina and Galdieria beverages behave differently in the presence of carrageenan at various starting pH levels. For example, at pH 3.2, the 0.3:1 Galdieria beverage is more stable than the Spirulina beverage, while the reverse is true for the "as is" sample at pH 4.3. These results indicate that the beverage containing both Galdieria extract and carrageenan is more heat stable than the beverage containing both Spirulina extract and carrageenan.

[0096] Furthermore, it can be concluded that when the pH is above 3.0 and below 4.0, the acidic composition containing Galdieria phycocyanin and carrageenan is even more stable under heat treatment than that containing Spirulina phycocyanin.

[0097] Example 12 Stability of different ratios of phycocyanin:carrageenan upon flash pasteurization The beverages were made with 12% sugar and the pH was adjusted to 3.0 and 4.0 with 50% citric acid solution. The color content was 0.06% Galdieria and Spirulina extracts, i.e. 0.018% by weight of phycocyanin compared to the total weight of the beverage, and λ-carrageenan was added at 0.01% and 0.06%. The individual concentrations of phycocyanin and carrageenan applied are the same as in Table 13 already presented. The "model" or "control" beverages have no carrageenan added. The preparations were tested for color change through HTST sterilization. The model beverage and the carrageenan-containing beverage were placed in 20 mL scintillation vials and immersed in a 80°C water bath for 4 minutes. Before heat treatment, the color tone is different between the Gardieria-based beverage with carrageenan and the Spirulina-based beverage. The Gardieria has a yellower color tone than the Spirulina. The dE CMC was measured on a ColorQuest colorimeter and reported in Figure 20. Figure 20 shows a comparison of the Gardieria and Spirulina beverages with added carrageenan at pH 3.0 and 4.0. The results further demonstrate that the Spirulina-carrageenan-containing beverage behaves differently at pH 4.0 than the Gardieria-carrageenan beverage at the same pH. These results show that the beverage containing both Gardieria extract and carrageenan is more heat stable than the control beverage without carrageenan and than the beverage containing both Spirulina extract and carrageenan. Furthermore, it can be concluded that when the pH is above 3.0 and below 4.0, the acidic composition containing Galdieria phycocyanin and carrageenan is even more stable under heat treatment than that containing Spirulina phycocyanin.

[0098] Sequence information: SEQ ID NO:1: YP_009051179.1= 1 mktpiteaia aadnqgrfls ntelqavngr yqraaaslea arsltsnaqr lingaaqavy 61 skfpytsqmp gpqyassavg kakcardigy ylrmvtyclv vggtgpmdey liagleeinr 121 tfdlspswyv ealnyvksnh glsgqaanea ntyidyaina ls SEQ ID NO:2: YP_009051180.1= 1 mldafakvva qadargefls ntqldalskm vsegnkrldv vnritsnasa ivtnaaralf 61 seqpqliqpg gnaytnrrma aclrdmeiil ryvsyaiiag dssvlddrcl nglretyqal 121 gvpgasvavg vekmkdsaia iandpsgitt gdcsalmaev gtyfdraata vq SEQ ID NO:3: YP_009051103.1= 1 msivtksivn adaearylsp geldriksfv lsgqrrlria qiltdnreri vkqagqqlfq 61 qrpdivspgg naygeemtat clrdldyylr lvtygvvagd ispieeiglv gvkemynslg 121 tpisavaegi kamknvacsl lsgddsaeag fyfdytigam q SEQ ID NO:4: YP_009051104.1= 1 mqdaitavin tadvqgkyld nssieklkgy fqtgelrvra aatiaanaag iikdavaksl 61 lysditrpgg nmyttrryaa cirdldyylr yatysmlagd psildervln glketynslg 121 vpigatiqsi qamkevtssl vgseagkemg iyfdyicsgl s

[0099] All references cited herein, and the references therein, where appropriate, for teachings of additional or alternative details, features, and / or technical background, are incorporated herein by reference in their entirety. While the present disclosure has been shown and described in detail with reference to specific embodiments, it will be appreciated that the above-disclosed and other features and functions, or alternative variations thereof, may be desirably combined into many other different systems or applications, and various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art, which are also intended to be encompassed by the following claims.

Claims

1. 1. An acidic composition comprising phycocyanin, wherein the phycocyanin is present in the composition in an amount ranging from about 0.001% to about 0.1% by weight.

2. The acidic composition described in claim 1, further comprising a chelating agent.

3. The acidic composition of claim 2, wherein the chelating agent is a synthetic or natural compound, preferably selected from the group consisting of ethylenediaminetetraacetic acid and / or its Na, K, Ca salts (EDTA), L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA), sodium hexametaphosphate, glutathione, metallothionein, 2,3-dimercapto-1-propanesulfonic acid, chlorella, garlic, coriander, selenium, milk thistle, vitamin C, vitamin E, citrate, grape seed extract, quercetin, lipoic acid, and mixtures thereof, preferably the chelating agent is vitamin C and / or EDTA.

4. 4. The acidic composition of claim 1, wherein phycocyanin is present in the composition in an amount selected from the group consisting of: an amount ranging from about 0.001% to about 0.05% by weight; an amount ranging from about 0.001% to about 0.025% by weight; an amount ranging from about 0.0025% to about 0.010% by weight; and an amount ranging from about 0.0025% to about 0.005% by weight.

5. 2. The acidic composition of claim 1, comprising an amount of at least one sulfated polysaccharide, preferably carrageenan, more preferably the polysulfated carrageenan λ-carrageenan.

6. The acidic composition of claim 5, wherein the phycocyanin and the at least one sulfated polysaccharide are present in the composition in a ratio of about 0.20:1 to about 20:1, preferably about 0.25:1 to about 10:1 or about 1:1 to about 10:1, and more preferably about 0.3:1 to about 1.5:

1.

7. 1. An acidic composition comprising phycocyanin and at least one sulfated polysaccharide, wherein the phycocyanin and the at least one sulfated polysaccharide are present in the composition in a ratio of about 0.20:1 to about 20:1, preferably about 0.25:1 to about 10:1, or about 1:1 to about 10:1, more preferably about 0.3:1 to about 1.5:1, such as about 1:1 to about 4:1, e.g., about 1:1 to about 3:1, or about 1:1 to 2:1, and wherein the sulfated polysaccharide is carrageenan, more preferably a polysulfate. and a chelating agent selected from the group consisting of ethylenediaminetetraacetic acid and / or its Na, K, Ca salts (EDTA), L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA), sodium hexametaphosphate, glutathione, metallothionein, 2,3-dimercapto-1-propanesulfonic acid, chlorella, garlic, coriander, selenium, milk thistle, vitamin C, vitamin E, citrate, grape seed extract, quercetin, lipoic acid, and mixtures thereof, more preferably the chelating agent is vitamin C and / or EDTA.

8. An acidic composition described in claim 1 or 7, having a pH of about 4.0 or less, preferably a pH of greater than about 3 and less than about 4.

9. A concentrated liquid composition comprising phycocyanin, wherein the phycocyanin is present in the concentrated composition in an amount ranging from about 50 mg / L to about 2500 mg / L, preferably from about 500 mg / L to about 1000 mg / L, and may further comprise at least one sulfated polysaccharide and / or chelating agent, preferably the at least one sulfated polysaccharide is carrageenan, more preferably the carrageenan is λ-carrageenan, and preferably the chelating agent is ethylenediaminetetraacetic acid and / or its Na, K, Ca salts (EDTA), L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA), sodium hexametaphosphate, glutathione, metallothionein, 2,3-dimercapto-1-propanesulfonic acid (2,3-dimercapto-1-propanesulfonic acid acid), chlorella, garlic, coriander, selenium, milk thistle, vitamin C, vitamin E, citrate, grape seed extract, quercetin, lipoic acid and mixtures thereof, more preferably the chelating agent is vitamin C and / or EDTA.

10. The acidic composition according to claim 1, 7 or 9, wherein the phycocyanin comprises an acidic pH-resistant phycocyanin, the α-subunit apoprotein of which has the amino acid sequence shown in SEQ ID NO: 1 (Registration No. YP_009051179.1) or a variant thereof, and the β-subunit apoprotein of which has the amino acid sequence shown in SEQ ID NO: 2 (Registration No. YP_009051180.1) or a variant thereof.

11. Phycocyanin is extracted from algae or microalgae of the family Cyanidiaceae and / or Galdieriaceae, preferably from the genus Cyanidioschyzon, Cyanidium, or Galdieria, preferably Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, or Cyanidium lumpens.

10. The acidic composition of claim 1, 7 or 9, wherein the algae or microalgae is extracted from a species selected from the group consisting of Galdieria sulphuraria, ...

12. A composition comprising an extract of the genus Galdieria, wherein the extract of the genus Galdieria is present in the composition in an amount ranging from about 0.004 to about 0.4% by weight, and optionally further comprising at least one sulfated polysaccharide and / or chelating agent, preferably wherein the at least one sulfated polysaccharide is carrageenan, more preferably wherein the carrageenan is λ-carrageenan, and preferably wherein the chelating agent is ethylenediaminetetraacetic acid and / or its Na, K, Ca salts (EDTA), L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA), sodium hexametaphosphate, glutathione, metallothionein, 2,3-dimercapto-1-propanesulfonic acid (2,3-dimerapto-1-propanesulfonic acid acid), chlorella, garlic, coriander, selenium, milk thistle, vitamin C, vitamin E, citrate, grape seed extract, quercetin, lipoic acid and mixtures thereof, more preferably the chelating agent is vitamin C and / or EDTA.

13. 10. A food product comprising a liquid composition as defined in any one of claims 1, 7 or 9, having a pH of less than 4, and which may be characterized by having been heated and / or exposed to light, and / or wherein the product has been subjected to sterilization, such as flash sterilization.

14. a) adding and dissolving at least one sulfated polysaccharide in water; and b) adding phycocyanin and mixing until dissolved at a pH of at least 5, preferably between 5 and 10; A method for improving the thermal stability and / or photostability of phycocyanin, comprising:

15. 1. A method for producing a green color comprising heating phycocyanin in an aqueous solution at a temperature of about 30°C to about 90°C and at a pH of about 3.6 or less, wherein the phycocyanin is selected from the group consisting of phycocyanins of the genus Cyanidioschyzon, Cyanidium, and Galdieria, preferably Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, and Cyanidium lumpens. rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria phlegrea, Galdieria sulphuraria, and preferably from algae or microalgae selected from the species Galdieria sulphuraria, Cyanidium caldarium, and Cyanidioschizon melorae.

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