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    • 专利摘要:
    The present invention relates to an acid composition, particularly an acidic food composition, comprising at least one phycocyanin resistant to acidic pH.
    公开号:FR3041505A1
    申请号:FR1653525
    申请日:2016-04-21
    公开日:2017-03-31
    发明作者:Olivier Cagnac
    申请人:Fermentalg SA；
    IPC主号:
    专利说明:

    The present invention relates to an acidic composition, particularly an acidic food composition, comprising at least one stable phycocyanin at acidic pH.
    Phycocyanins are dietary dyes that give products in which they are incorporated a blue color. Phycocyanin extracted from spirulina is today the only natural blue pigment approved by the US-FDA (FR Doc No: 2013-19550). It is sold in liquid form, or in powder form for use as a blue pigment in food.
    However, this phycocyanin extracted from spirulina has the disadvantage of being unstable at acid pH, less than 5, resulting in loss of color and precipitation which limit its use. In the best case, the loss of stability will occur around a pH of 4 (see technical specifications for phycocyanin extracted from Linablue® Spirulina; http: //www.dlt-spl.co.ip/business/en/ spirulina / linablue.html).
    However, many acidic food compositions exist, including carbonated or non-carbonated beverages, for which phycocyanin extracted from spirulina can be used, both as a food coloring agent and for its antioxidant properties.
    There is a need to identify new phycocyanines stable at acidic pH, particularly stable at acid pH below 4. Resistance or pH stability is measured here as a loss of color less than 10%, after a minimum of 10 minutes exposure to acidic pH. PH stability can be measured by other methods such as physical characterization of phycocyanins within acidic compositions as a function of time.
    Thus, the present invention relates to an acid composition, particularly an acidic food composition, which may comprise at least one phycocyanin resistant to acidic pH. Advantageously, said phycocyanin may be a phycobiliprotein whose apoprotein may comprise at least the protein of SEQ ID NO 1 or SEQ ID No. 2 or a variant thereof.
    According to the invention, the phycocyanin may be in particular a phycocyanin extracted from a Galdieriaceae, more particularly extracted from Galdieria.
    The acid composition according to the invention, particularly the acidic food composition may be either solid or pasty, or liquid, in particular a carbonated beverage or not.
    By acidic composition, particularly acidic food composition is meant according to the invention, a composition of pH less than or equal to 4, preferably pH ranging from 2 to 4, more preferably from about 2.5 to 3.5.
    Phycocyanins and allophycocyanins are phycobilliproteins comprising alpha and beta subunits composed of an apoprotein covalently bound to a chromophore. The different phycocyanins are essentially distinguished by the sequence of their apoproteins alpha and beta subunits.
    According to the invention, the acidic composition, particularly the acidic food composition, may comprise phycocyanin resistant to acidic pHs, the apoprotein of which may comprise SEQ ID NO 1 (accession number YP_009051179.1) and apoprotein of the β subunit may comprise SEQ ID 2 (accession number YP_009051180.1) or variants thereof.
    According to a preferred embodiment of the invention, the acidic composition, particularly the acidic food composition, may comprise a phycocyanin resistant to acidic pH, the apoprotein of the α-subunit may consist of SEQ ID NO. accession YP_009051179.1) and the apoprotein of the β subunit may consist of SEQ ID 2 (accession number YP_009051180.1) or variants thereof.
    According to another form of the invention, the acid composition, particularly the acidic food composition, may further comprise an allophycocyanin associated with phycocyanin.
    Advantageously, the apoprotein of the alpha subunit of said allophycocyanin may comprise SEQ ID NO 3 (accession number YP_009051103.1) and the apoprotein of the β subunit may comprise SEQ ID NO 4 ( YP_009051104.1) or variants thereof.
    According to another preferred embodiment of the invention, the apoprotein of the alpha subunit of said allophycocyanin may consist of SEQ ID NO 3 (accession number YP_009051103.1) and the apoprotein of the β subunit. may consist of SEQ ID NO 4 (YP_009051104.1) or variants thereof.
    The amino acid composition of a protein may impart to said protein different properties according to said amino acid composition. The characteristics of a protein depend inter alia on its amino acid composition and the value of its isoelectric point (p1). The isoelectric point is the pH value of the solution from which the net charge of the protein is zero or, in other words, the pH for which the molecule is electrically neutral and the proteins tend to attract, aggregate and precipitate. At a pH above their isoelectric point the proteins tend to be negatively charged and repel. The comparative study of the isoelectric points of the different proteins via the calculation method described by Patrickios and Yamasaki (Amino-Acid-Composition Polypeptide and IsoelectricPoint .11. Comparison between Experiment and Theory, Analytical Biochemistry, 231, 1, 1995: 82-91.1995 ) shows some correlation between the theoretical calculations and the experimentally observed acid pH resistance.
    The Applicant's studies show that the acid pH resistance of phycocyanin could be related to the amino acid sequence of the α-subunit of said phycocyanin. It should be noted that, within the amino acid sequence of phycocyanin subunit a, the identity of the first 26 amino acids seems particularly important. This is particularly the case of phycocyanin obtained by cultivation of microalgae strains of the genera Cyanidioschyzon, Cyanidium or Galdieria, particularly Galdieria sulphuraria, Cyanidium caldarium and Cyanidioschyzon merolae strains.
    Thus, advantageously, the composition according to the invention may comprise at least one phycocyanin of which at least one apoprotein, particularly that the α-subunit, may have a low isoelectric point allowing a better stability at acidic pH.
    By low isoelectric point is meant an isoelectric point of a value less than or equal to 3, preferably less than or equal to 2.5, more preferably less than or equal to 2.2.
    Thus, even more advantageously, the composition according to the invention may comprise at least one phycocyanin of which at least one apoprotein, particularly that of the α-subunit, may have an isoelectric point less than or equal to 3, preferably less than or equal to 2.5, plus preferably less than or equal to 2.2.
    Thus, according to one particular embodiment of the invention, the acid composition, particularly the acidic food composition, may comprise at least one phycocyanin, the apoprotein of which may have a low isoelectric point, in particular at least one phycocyanin of which the apoprotein of the -subunit may comprise SEQ ID NO: 1, or a variant.
    Thus, according to another particular embodiment of the invention, the acid composition, particularly the acidic food composition, may comprise at least one phycocyanin, the apoprotein of which may have a low isoelectric point, particularly at least one phycocyanin of which the apoprotein of the -subunit may consist of SEQ ID NO: 1, or a variant.
    By variant is meant according to the invention a protein sequence corresponding to a reference sequence, in this case the protein represented by SEQ ID NO 1 or SEQ ID NO 2, or SEQ N03, or SEQ N04, modified by one or more substitutions, insertions or deletions of one or more amino acids of the reference sequence and which has the same functional properties as said reference sequence.
    The variants according to the invention may have a sequence identity of at least 83% for phycocyanin α subunits, and at least 82% for phycocyanin beta subunits.
    Likewise according to the invention for allophycocyanins, the variants may have a sequence identity of at least 89% for the α subunits of allophycocyanin, and at least 90% for the β subunits of the allophycocyanin. 'allophycocyanin. Those skilled in the art know how to measure an identity of protein sequences according to the usual methods at their disposal, in particular the BLASTP program (http://blast.ncbi.nlm.nih.gov/Blast.cqn.
    Similarly, those skilled in the art know how to identify variants of said sequences and verify that they retain the same structural properties by simple acid pH stability test, for example by performing a test such as the test presented in Example 3 .
    It is known to those skilled in the art that a polypeptide may be modified by substitution, insertion and / or deletion of at least one amino acid without substantially modifying its function.
    For example, the substitution of an amino acid at a given position by another chemically equivalent amino acid is a known example of sequence variation that does not substantially affect the properties of the protein.
    These "conservative" substitutions can be defined as exchanges within the following amino acid groups - Ala, Ser, Thr, Pro, Gly-Asp, Asn, Glu, Gin-His, Arg, Lys
    Met, Leu, Ile, Val, Cys and Phe, Tyr, Trp
    Thus according to the invention, for the apoprotein variants of the phycocyanins and / or allophycocyanines that may be used in the acid compositions, resulting from substitutions, insertions or deletions, they may comprise from 1 to 30 amino acids of difference in number relative to to the corresponding reference sequence, particularly with respect to the α and / or β subunits of phycocyanin, insofar as the variant obtained retains the properties of the reference protein and the homology / identity percentages stated above. -above.
    More precisely according to the invention, for the apoprotein variants of the α-subunit phycocyanins that may be used in the acid compositions according to the invention, resulting from substitutions, insertions and / or deletions, they may comprise from 1 to 27 amino acids of difference relative to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above; for variants of the apoproteins of the β subunit of the phycocyanins that can be used in the acid compositions according to the invention, derived from substitutions, insertions and / or deletions, they may comprise from 1 to 30 amino acids of difference relative to to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above; for the apoprotein variants of the α-subunit of allophycocyanines that may be used in the acid compositions according to the invention, resulting from substitutions, insertions or deletions, they may comprise from 1 to 24 amino acids of difference relative to the so-called corresponding reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above; for the apoprotein variants of the β subunit of the allophycocyanines that can be used in the acid compositions according to the invention, resulting from substitutions, insertions and / or deletions, they may comprise from 1 to 20 amino acids of difference relative to to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above.
    Very particularly according to the invention, and whatever the reference sequence considered (subunit a and / or β of phycocyanin and / or subunit a and / or β of allophycocyanin) the variants of said subunits may advantageously comprise from 1 to 15 amino acids of difference, preferably from 1 to 10 amino acids of difference, in particular 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 amino acids of difference relative to to the so-called reference sequence, insofar as the variant obtained retains the properties of the reference protein and the percentages of identity stated above.
    According to the invention, the phycocyanin or its variants, which can be used, alone or as a mixture with an allophycocyanin or its variants, in acidic compositions, particularly in acidic food compositions, can be obtained by culturing a natural organism naturally expressing phycocyanin. or its desired variant or by culturing a genetically transformed organism to express phycocyanin or its selected desired variant on its ability to produce said phycocyanin or its variants. As natural organisms naturally expressing a phycocyanin that can be used in the compositions according to the invention or its desired variant, mention will be made of algae (or microalgae) of the order of Cyanidiales. The order Cyanidiales, includes the families Cyanidiaceae or Galdieriaceae, themselves subdivided into the genera Cyanidioschyzon, Cyanidium or Galdieria, to which among others belong the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum , Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria. In particular, the strain Galdieria sulphuraria (also called Cyanidium caldarium) UTEX # 2919 is mentioned.
    Thus according to the invention, the acidic composition, particularly the acidic food composition, may comprise a phycocyanin resistant to acidic pH that may come from natural organisms such as algae or microalgae of the order of Cyanidiales,
    Particularly according to the invention, the acid composition, particularly the acidic food composition, may comprise a phycocyanin resistant to acidic pH that may come from natural organisms such as algae or microalgae that may belong to the families of Cyanidiaceae or Galdieriaceae,
    Very particularly according to the invention, the acidic composition, particularly the acidic food composition, may comprise a phycocyanin resistant to acidic pH that may come from natural organisms such as algae or microalgae that may belong to the genera Cyanidioschyzon, Cyanidium, Galdieria,
    Even more particularly according to the invention, the acid composition, particularly the acidic food composition, may comprise a phycocyanin resistant to acidic pH that may come from natural organisms such as algae or microalgae that may belong to the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium Caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria.
    The preferred form of the acidic composition, particularly the acidic food composition, according to the invention may comprise phycocyanin resistant to acidic pH that may come from a natural microalga such as Galdieria sulphuraria, Cyanidium caldarium or Cyanidioschyzon merolae. As a transformed organism for expressing phycocyanin or its selected desired variant on its ability to produce said phycocyanin or its variants can be mentioned a microorganism transformed so as to express the apoprotein of SEQ ID NO 1 and / or SEQ ID NO 2 and / or SEQ ID NO 3 and / or SEQ ID NO 4, said microorganism also comprising the biosynthetic pathways necessary for the production of the chromophore and its binding to the apoprotein. In particular yeasts as microorganisms may be modified to produce phycocyanin and / or allophycocyanin used in food compositions according to the invention. Those skilled in the art will have no difficulty in finding in the prior art the description of the culture processes of natural and / or modified organisms which can produce a phycocyanin that can be used in the compositions according to the invention.
    For example, the culture of Cyanidiaceae or Galdieriaceae, belonging to the order of Cyanidiales, well known to those skilled in the art can advantageously be performed in mixotrophic mode, the light being generally necessary for the biosynthesis of pigments.
    Such an industrial culture can advantageously be carried out in large volume fermenters of the 1000 liter, 10,000 liter, 20,000 liter, 100,000 liter type. The culture can be carried out according to the conditions known to those skilled in the art. It can be performed in batch mode called "batch", in semi-continuous mode said "fed batch" or in continuous mode.
    The phycocyanin that can be used in the compositions according to the invention can more particularly be extracted from a biomass obtained by cultivating an alga of the order of cyanidiales as defined previously cultivated in a mixotrophy with a light of wavelength between 400 nm and 550 nm, advantageously between 420 nm and 500 nm, preferably between 430 and 480 nm, more preferably about 455 nm. It may be a so-called "white" light, broad spectrum comprising the light of said wavelength. It can also be advantageously a so-called narrow spectrum light consisting of said wavelength.
    Such a process for the industrial preparation of biomass of Cyanidiales in mixotrophy, and the biomass thus obtained, are described in particular in the patent application FR 15 59072 filed on September 25, 2015.
    Advantageously according to the invention acid composition according to the invention means any imaginable composition which has an acidic pH (less than 4) and requires that a phycocyanin resistant to acidic pH is incorporated therein.
    By acidic food composition according to the invention is meant any composition intended to be ingested by humans or animals which falls within the above definition. Nutraceutical acid compositions must be considered as falling within the definition of acidic food compositions within the meaning of the invention.
    The acidic food compositions according to the invention are well known to those skilled in the art. They may comprise a vehicle that may comprise structural constituents associated with active compounds identified with regard to their nutritive contributions or for their properties beneficial to the health of humans or animals. The acidic food composition according to the invention may also comprise food additives such as texturizing agents, flavoring agents, preserving agents, all components well known to those skilled in the art. The vehicle may comprise water and / or proteins and / or fats and / or fibers and / or sugars. The constituents of the vehicle may have only structural properties but they are generally known for their nutrient inputs.
    As regards the non-food acidic compositions according to the invention, they may be, for example, pharmaceutical, veterinary or cosmetic, and furthermore include any additives and / or active agents known and used in this type of composition.
    The acid composition, particularly the acidic food composition, according to the invention may be a solid or pasty composition, or a liquid composition. It can be ready for use or as a food additive that is added to a solid, pasty or liquid preparation to prepare the food that can be ingested.
    In a solid, liquid or pasty acid composition according to the invention, the phycocyanin may be incorporated, for example, in the form of a powder. Said acid composition, particularly said acidic food composition may then be in any known known form such as creams, gels, foams, pastes, etc. Especially for a solid food composition include cakes or biscuits, dry foods to cook, the powders to be diluted, the solid gelatinous compositions or "jelly", foams etc.
    According to the invention, said liquid acid composition may be an aqueous composition in which the phycocyanin is dissolved. It can be in the form of a ready-to-use composition or as a liquid concentrate to be diluted for ingestion or to be added to a solid food either for its preparation or for its ingestion, for example a concentrated liquid composition of coating or "topping" which will be deposited on a cake to bring its color. Among these concentrated compositions include syrups, alcoholic or not.
    The liquid acid composition according to the invention may be of variable viscosity and may or may not comprise additives such as viscosity agents, gelling agents, and other structuring additives known to those skilled in the art and customary for the preparation of liquid food compositions.
    According to a particular embodiment of the invention, the liquid food composition may be an acidic beverage, gaseous or not. Examples include sodas, juices, sports drinks, exercise drinks, salvage drinks, etc.
    According to the invention, the phycocyanin content in the compositions according to the invention may be in accordance with the practice of those skilled in the art.
    For example, when the phycocyanin will be used to color the acidic composition, then the phycocyanin content in said composition may be in accordance with the practices of a person skilled in the art of coloring.
    In a liquid acid composition within the meaning of the invention the phycocyanin content may be between 2.5 mg / L and 2500 mg / L, preferably between 25 mg / L and 300 mg / L.
    In a ready-to-use beverage liquid composition, the phycocyanin content may generally be between 25 mg / l and 300 mg / l, preferably between 50 mg / l and 100 mg / l.
    In a concentrated liquid composition to be diluted for its use, such as a syrup, the phycocyanin content may generally be between 250 mg / l and 2500 mg / l, preferably between 500 mg / l and 1000 mg / l.
    In a solid composition, the phycocyanin content may generally be between 0.01 mg / g and 10 mg / g, preferentially between 0.1 mg / g and 5.0 mg / g, very preferably between 0.25 mg / g g and 2.5 mg / g.
    One of the advantages of the invention resides as can be seen in the following examples in that the coloration provided by the phycocyanines resistant to acidic pHs have a better behavior over time. Other aspects and characteristics of the invention may appear on reading the examples and figures.
    Figure 1 depicts the amino acid sequences of the apoproteins of phycocyanin and allophycocyanin of Galdieria sulphuraria with: SEQ ID NO: 1: YP_009051179.1: α-subunit Phycocyanin; SEQ ID NO: 2 YP_009051180.1: β Subunit of Phycocyanin; SEQ ID NO: 3 YP_009051103.1: A subunit of Allophycocyanin; SEQ ID NO: 4 YP_009051104.1: β Subunit of Allophycocyanin;
    Figure 2 shows the pH stability curve of phycocyanins extracted from Galderia sulphuraria (UTEX2919) and Cyanidioschyzon merolae (ACUF 199) whose apoprotein sequence consists of SEQ ID NO 1 or a variant, on which is reported the stability curve of phycocyanin extracted from Linablue® spirulina.
    Phycocyanin of Galdieria sulphuraria (UTEX # 2919)
    Phycocyanin from Cyanidioschyzon merolae (ACUF 199)
    Lina Blue®: Phycocyanin from Spirulina platensis (Arthrospira platensis) (data obtained from http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html). with indication of the pH at which precipitation of Phycocyanin appears.
    Figure 3 shows the evolution of the color of an acidic beverage comprising phycocyanin over time. S0, S2, S4 and S6: week 0, 2, 4, and 6
    EXAMPLES
    Example 1: Production and extraction of phycocyanin from Galdieria sulphuraria UTEX # 2919 Material and methods
    Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX # 2919 Culture medium
    Mixotrophy: 30 g / L glycerol, 8 g / L (NH4) 2SO4, 1 g / L KH2PO4, 716 mg / L MgSO4, 44 mg / L CaCl2, 3 mL / L of Fe-EDTA stock solution (FeS04 at 6.9 g / L and EDTA-Na2 at 9.3 g / L) and 4 ml / L of metal trace solution (3.09 g / L EDTA-Na 2, 0.080 g / L CuSO 4, 5H 2 O, 2.860 g / L H 3 BO 3, 0.040 g / L NaV03, 4H2O, 1.820g / L MnCl2, 0.040g / L CoCl2.6H2O, 0.220g / L ZnSO4.7H2O, 0.017g / L Na2SeO3, 0.030g / L (NH4) 6Mo7024, 4H2O).
    Culture conditions:
    The cultures are carried out in reactors of 1 to 2 L of useful volume with dedicated automata and supervision by computer station. The pH of the culture is regulated via the addition of base (14% ammonia solution (wNH3 / w) and / or acid (4N sulfuric acid solution) .The culture temperature is set at 37 ° C. The culture is illuminated by pale counters equipped with a white LED system or blue LED (455 nm) in a manner similar to that described in patent PCT / FR2014 / 050891. The monitoring of cell growth is performed at different
    time per absorbance measurement at 800 nm. And an estimate of the dry mass is carried out by filtration. - Results
    The performances of the culture at the end of growth are summarized in Table 1 below
    The estimation of the intracellular phycocyanin content per gram of dry matter was carried out using the extraction and assay method described by Moon et al. [Moon et al., Korean J. Chem. Eng., 2014, 1-6] by replacing the phosphate buffer with water.
    Example 2 Extraction of Phycocyanines
    The Galdieria sulphuraria (UTEX # 2919) and / or Cyanidioschizon merolae (ACUF199) strains were cultured under the conditions of Example 1.
    The phycocyanin is then extracted according to a modification of the protocol described by Moon et al., 2014 (op.citus). This modification consists of replacing the phosphate buffer used to solubilize phycocyanin with demineralized water.
    An extract (also called "phycocyanin extract" or "crude extract") is then obtained which comprises, in addition to the desired phycocyanin, other water-soluble proteins. There are several qualities of phycocyanin extract possible depending on the extraction and / or purification methods used. For example, a crude extract will contain a water-soluble amount of protein, other than phycocyanin, greater than that found in a purified extract. Purified extract comprises a crude extract of which a portion of the water-soluble proteins have been removed by ultrafiltration, hollow-fiber filtration or ion-exchange chromatography methods known to those skilled in the art, while retaining phycocyanin. The purity index is conventionally expressed by making the absorbance ratio of the solution at 618 nm (specific absorbance of phycocyanin) to that of the specific 280 nm absorbance value of the aromatic amino acids giving an idea of the rate of global proteins. The lower the ratio, the more protein other than phycocyanin in solution. The crude extract was purified using the KrosFIo® tangential flow filtration system from Spectrum-Labs.
    Table 2. Measurement of a purity index of a phycocyanin extract before and after purification.
    Example 3 Study of the Stability of Phycocyanin Extracted from Galdieria sulphuraria (UTEX # 2919) and Cyanidioschizon merolae (ACUF199) and Spirulina (Arthrospira) platensis Strains as a Function of pH
    The Galdieria sulphuraria (UTEX # 2919) and Cyanidioschizon merolae (ACUF199) strains were cultured under the conditions of Example 1.
    The tests are performed using the data of the commercial product LinaBlue® (http: //www.dlt-spl.co.ip/business/en/spirulina/linablue.html) from DIC Lifetec Co, Ltd. as a reference. (Tokyo, Japan) which is a phycocyanin extracted from Spirulina (Arthrospira) platensis.
    These tests were carried out with the phycocyanines prepared in Example 2 and having a purity value of 2.12. The blue color measurement is made by absorbance measurement at 618 nm using an ultraspec 2100 pro spectrophotometer (Amersham). Percentage loss of color is measured relative to the measurement of absorbance of the sample under reference conditions (pH 6).
    For the acid resistance test, the pH is lowered gradually by adding a 5% citric acid solution (Sigma 251275) to the phycocyanin preparation. For each pH value a sample of the phycocyanin solution is taken and its absorbance measured at 618 nm, 10 minutes after the pH drop to the desired value.
    The results of these tests are shown in Figure 2:
    Phycocyanin extracted from Galdieria sulphuraria (UTEX # 2919) [(-)] has a very good pH resistance compared to that of Spirulina, with less than 10% loss of pigmentation up to pH 2.75 (98.25% at pH 3.92.4% at pH 2.75), the loss becoming greater from pH 2.5 (79% at pH 2.5, 75% at pH 2.25, 46% at pH 2 ).
    The phycocyanin extracted from the strain Cyanidioschizon merolae (ACUF 199) [(* ·· χ ···)] has good pH resistance compared to that of Spirulina, with still more than 90% of its color at pH 3, 70 % at pH 2.75; 40% at pH 2.5; 23% at pH 2.25; 20% at pH 2.
    The phycocyanin extracted from the strain Spirulina platensis [(~ o * ~)] has only 90% of its color at pH 4.80% at pH 3.8; 60% at pH 3.6; 38% at pH 3.4. Phycocyanin extracted from Spirulina platensis begins to precipitate from pH 3.8.
    In conclusion :
    The phycocyanins of Galdieria sulphuraria or Cyanidioschyzon merolae are more resistant to acidic pH than those extracted from Spirulina.
    EXAMPLE 4 Comparative Analysis of the Apoprotein Sequences of Phycocyanin and Allophycocyanin from Different Microalgae and Measurement of Their Isoelectric Points
    In order to identify a possible cause for the increase in resistance to acidic pH, the comparison of the α and β subunit sequences of phycocyanins and allophycocyanins produced by different microorganisms, particularly microalgae, was carried out using the program. BLASTP whose use is well known to those skilled in the art, on the basis of accessible published sequences (see Accession No.) in the databases. The comparison is made with respect to the apoprotein sequence of the corresponding subunit of the strain Galdieria sulphuraria.
    At the same time, the value of the isoelectric point of the apoproteins of the α and β subunits of the phycocyanins and allophycocyanins whose sequence comparison was performed was determined by the calculation method described by Patrickios and Yamasaki (1995).
    The phycocyanin and allophycocyanin produced by the strain Galdieria sulphuraria are used as reference in these studies. Results
    The results of sequence comparisons and udder calculations are presented in Tables 3 and 4 below:
    Table 3. Comparisons of alpha and beta apoprotein sequences of phycocyanin from different organisms. Blast result indicating the percentage identity between the protein sequence of Galdieria sulphuraria and the sequences of the other strains for the alpha (A) and beta (B) chains. The isoelectric point of each protein is indicated (Patrickios and Yamasaki, 1995).
    Table 4. Comparisons of alpha and beta apoprotein sequences of allophycocyanin from different organisms. (A) Blast result indicating the percentage identity between the Galdieria sulphuraria protein sequences and the sequences of the other strains for the alpha chain (A) and the beta chain (B). The isoelectric point of each protein is indicated (Patrickios and Yamasaki, 1995). Analysis of the identity percentages between the apoprotein sequence of the Galdieria sulphuraria reference alpha subunit and the apoprotein sequences of the other organisms analyzed makes it possible to observe that said apoproteins of the alpha subunit of Cyanidium caldarium (P00306.3) and Cyanidioschyzon meroiae (1MP_848986.1), in addition to that of Galdieria sulphuraria (YP_009051103.1) fall within the criteria used by the invention for use in the acidic compositions according to the invention.
    In conclusion :
    It is clear that phycocyanins resistant to acidic pHs (isoelectric point of less than 3) have a higher percentage identity of their α-subunits compared to the Galdieria sulphuraria sequence.
    Example 5: Stability in time in a drink
    The phycocyanin stability test, extracted from the UTEX strain # 2919, in an acidic medium over time was carried out by adding the phycocyanin in a lemonade-type beverage (as described in Example 6: drink 1) of pH 2.95. After addition of 0.025% o phycocyanin whose apoprotein sequence consists of a variant of SEQ ID NO 1, the beverage was exposed to a day / night cycle (16/8 hour) with artificial light for 6 hours. weeks, at room temperature.
    Regular samples are taken over time to measure the absorbance at 618 nm. An aliquot is taken at weeks 0 (S0), 2 (S2), 4 (S4), and 6 (S6) and the absorbance at 618 nm measured at ultraspec 2100 pro spectrophotometer (Amersham) to define the 100% point of the colour. The residual color is expressed as a percentage of the initial reference value.
    Figure 3 shows the results of this experiment. There is a steady loss of color over time with lasting exposure to light. However after 6 weeks of exposure there is still more than 60% of the color.
    Example 6 Examples of Acidic Drinks in Liquid Form Comprising Phycocyanin
    Drinks containing phycocyanin may have the following composition: Drink 1 - Soda drink:
    - Sparkling water qs 1L - Sugars 70 g
    Citric acid 1.5 g Organic natural flavoring 4 to 8 g Phycocyanine extract obtained in Example 2 qs 100 mg
    phycocyanin
    The pH of this drink is 2.95 Drink 2 - Dietary Drink for Sportsmen:
    - Spring water qs 1L - Glucose 20 g - Fructose 10 g - Citric acid 2.7 g - Sodium citrate 1.87 g - Potassium citrate 0.327 g - Magnesium chloride 2.5 mg - Calcium chloride 3 mg - Organic natural flavoring 4 g - Phycocyanine extract obtained in Example 2 qs 150 mg of
    phycocyanin
    The pH of this drink is pH 3.5
    EXAMPLE 6 Acid Drink in the Form of a Powder to Dissolve Comprising Phycocyanin - Sugars 70 to 100 g - Citric Acid 1 to 1.5 g - Organic natural flavoring 4 to 8 g - Phycocyanine extract obtained in Example 2 qs 250 mg of
    phycocyanin
    The powder (75 to 110 g) thus prepared can be dissolved in 1L of water to obtain an acidic beverage colored blue.
    Example 7: Solid acid composition comprising phycocyanin: acidic confectionery: - Water: qs 1 kg - Sugars: 800 g - Citric acid: 1 to 2 g - Phycocyanine extract obtained in Example 2 qs 2500 mg of
    phycocyanin
    权利要求:
    Claims (15)
    [1" id="c-fr-0001]
    An acidic composition, particularly an acidic food composition, comprising at least one phycocyanin resistant to acidic pH, said phycocyanin being a phycocyanin at least one apoprotein, particularly that the α-subunit has an isoelectric point less than 3.
    [2" id="c-fr-0002]
    2. Composition according to claim 1, characterized in that said phycocyanin is a phycobiliprotein whose apoprotein comprises at least the protein of SEQ ID NO 1 or SEQ ID 2 or a variant thereof.
    [3" id="c-fr-0003]
    3. Composition according to claim 2, characterized in that the apoprotein of the α subunit of said phycocyanin comprises SEQ ID NO 1 (accession number YP_009051179.1) and the apoprotein of the β subunit of said phycocyanin comprises SEQ ID 2 (accession number YP_009051180.1) or variants thereof.
    [4" id="c-fr-0004]
    4. Composition according to claim 3, characterized in that said apoprotein of the α-subunit consists of SEQ ID NO 1 (accession number YP_009051179.1) and the apoprotein of the β subunit consists of SEQ ID 2 (accession number YP_009051180.1) or variants thereof.
    [5" id="c-fr-0005]
    5. Composition according to any one of claims 1 to 4, characterized in that it further comprises an allophycocyanin associated with phycocyanin.
    [6" id="c-fr-0006]
    6. Composition according to claim 5, characterized in that the apoprotein of the α-subunit of said allophycocyanin comprises SEQ ID NO 3 (accession number YP_009051103.1) or variants thereof and the apoprotein of the β subunit of said allophycocyanin comprises SEQ ID NO 4 (YP_009051104.1) or variants thereof.
    [7" id="c-fr-0007]
    7. Composition according to claim 6, characterized in that the apoprotein of the α subunit of allophycocyanin consists of SEQ ID NO 3 (accession number YP_009051103.1) or variants thereof and the apoprotein of the β subunit consists of SEQ ID NO 4 (YP_009051104.1) or variants thereof.
    [8" id="c-fr-0008]
    8. Composition according to any one of claims 1 to 7, characterized in that the phycocyanin is a phycocyanin extracted from an alga (or microalgae) of the order of Cyanidiales.
    [9" id="c-fr-0009]
    9. Composition according to any one of claims 1 to 8, characterized in that phycocyanin is a phycocyanin extracted from an alga (or microalgae) belonging to the family of Cyanidiaceae or family Galdieriaceae.
    [10" id="c-fr-0010]
    10. Composition according to any one of claims T to 9, characterized in that the phycocyanin is a phycocyanin extracted from an alga (or microalgae) belonging to the genera Cyanidioschyzon, Cyanidium or Galdieria.
    [11" id="c-fr-0011]
    11. Composition according to any one of claims 1 to 10, characterized in that the phycocyanin is a phycocyanin extracted from an alga (or microalgae) belonging to the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, preferentially species Galdieria sulphuraria, Cyanidium caldarium, and Cyanidioschyzon merolae.
    [12" id="c-fr-0012]
    12. Composition according to any one of claims 1 to 11, characterized in that it is solid, pasty or liquid.
    [13" id="c-fr-0013]
    13. Composition according to any one of claims 1 to 12, characterized in that it has a pH of less than or equal to 4, advantageously of pH ranging from 2 to 4, more preferably from about 2.5 to 3, 5.
    [14" id="c-fr-0014]
    14. Composition according to any one of claims 1 to 13, characterized in that the phycocyanin content is between 2.5 mg / L and 2500 mg / L in the case of a liquid composition, or between 0, 25 mg / g and 2.5 mg / g in the case of a solid composition.
    [15" id="c-fr-0015]
    15. Composition according to any one of claims 1 to 14, characterized in that said phycocyanin is a phycocyanin at least one apoprotein, particularly that the subunit has an isoelectric point less than 2.5, more preferably less than 2.2 .
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    同族专利:
    公开号 | 公开日
    US11162126B2|2021-11-02|
    FR3041653B1|2017-12-29|
    CN108431202A|2018-08-21|
    CA2998973A1|2017-03-30|
    US20180274002A1|2018-09-27|
    BR112018005928A2|2018-12-11|
    FR3041505B1|2021-06-11|
    JP2018529343A|2018-10-11|
    RU2018112989A|2019-10-25|
    FR3041653A1|2017-03-31|
    RU2018112989A3|2020-01-31|
    JP2021176341A|2021-11-11|
    EP3353282A1|2018-08-01|
    US20180271119A1|2018-09-27|
    WO2017050917A1|2017-03-30|
    RU2739847C2|2020-12-29|
    EP3353283A1|2018-08-01|
    WO2017050918A1|2017-03-30|
    MX2018003696A|2018-04-30|
    JP6976933B2|2021-12-08|
    JP2018527940A|2018-09-27|
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    法律状态:
    2017-03-31| PLSC| Publication of the preliminary search report|Effective date: 20170331 |
    2017-03-31| EXTE| Extension to a french territory|Extension state: PF |
    2017-04-28| PLFP| Fee payment|Year of fee payment: 2 |
    2018-04-26| PLFP| Fee payment|Year of fee payment: 3 |
    2019-04-29| PLFP| Fee payment|Year of fee payment: 4 |
    2020-04-30| PLFP| Fee payment|Year of fee payment: 5 |
    2021-04-29| PLFP| Fee payment|Year of fee payment: 6 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1559072A|FR3041653B1|2015-09-25|2015-09-25|PROCESS FOR CULTIVATION OF ALGAE, PARTICULARLY UNICELLULAR RED ALGAE |JP2018515313A| JP6976933B2|2015-09-25|2016-09-22|Acid composition containing phycocyanin|
    US15/762,659| US20180271119A1|2015-09-25|2016-09-22|Acid composition comprising a phycocyanin|
    MX2018003696A| MX2018003696A|2015-09-25|2016-09-22|Acid composition comprising a phycocyanin.|
    CN201680055641.4A| CN108431202A|2015-09-25|2016-09-22|Include the acidic composition of phycocyanin|
    BR112018005928-5A| BR112018005928A2|2015-09-25|2016-09-22|acid composition comprising a phycocyanin|
    RU2018112989A| RU2739847C2|2015-09-25|2016-09-22|Acidic composition containing phycocyanine|
    EP16775587.5A| EP3353282A1|2015-09-25|2016-09-22|Acid composition comprising a phycocyanin|
    PCT/EP2016/072583| WO2017050918A1|2015-09-25|2016-09-22|Acid composition comprising a phycocyanin|
    JP2021129656A| JP2021176341A|2015-09-25|2021-08-06|Phycocyanin-containing acid composition|
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