METHOD FOR PRODUCING A STABLE PRECIPITATE ENRICHED IN PHYCOBILIPROTEINS.

Abstract

The invention relates to a novel method for producing a stable precipitate enriched in phycobiliproteins by means of salicylic acid precipitation. The invention also relates to the use of said precipitate enriched in phycobiliproteins for producing cosmetic or dermatological, and food or nutraceutical compositions.

Claims

1. A process for obtaining a stable precipitate enriched in phycobiliproteins, comprising at least one precipitation step consisting of the addition of salicylic acid to an aqueous extract of phycobiliproteins, said precipitate having a phycobiliprotein content at least equal to 50% of the amount initially contained in said aqueous extract, and having a clarity at least equal to 70%.

2. The process as claimed in claim 1, wherein said precipitate has a phycobiliprotein content at least equal to 50% of the amount initially contained in said aqueous extract and has a clarity at least equal to 80%.

3. The process as claimed in claim 1, wherein said precipitate has a phycobiliprotein content at least equal to 70% of the amount initially contained in said aqueous extract and is free of membrane fragments.

4. The process as claimed in claim 1, wherein said aqueous extract of phycobiliproteins contains at least 1.5 g/l of said phycobiliproteins.

5. The process as claimed in claim 1, wherein the salicylic acid is added to the aqueous extract of phycobiliproteins in a range of from 4 g/l to 20 g/l.

6. The process as claimed in claim 1, wherein the salicylic acid is added to the aqueous extract of phycobiliproteins in a range of from 4 g/l to 13 g/l.

7. The process as claimed in claim 1, wherein the salicylic acid is added to an aqueous extract of phycobiliproteins in a range of from 13 to 14 g/l.

8. The process as claimed in claim 1, wherein a step of clarification by decanting is performed prior to the step of precipitation by salicylic acid.

9. The process as claimed in claim 1, wherein a step of dissolving the precipitate is performed.

10. The process as claimed in claim 9, wherein the precipitate is dissolved in a natural polyol chosen from glycerin, glycerol, or any derivative or any form of the polyol propane-1,2,3-triol (or 1,2,3-propanetriol).

11. The process as claimed in claim 1, which comprises, beforehand, a step of enrichment in phycobiliproteins of a biomass of photosynthetic microalgae.

12. The process as claimed in claim 11, wherein the prior step of enrichment in phycobiliproteins comprises an induction of the phycobiliprotein synthesis in a biomass of which the growth is blocked.

13. The process as claimed in claim 1, which comprises: a. a step of blocking the growth of the biomass b. a step of inducing the synthesis of the phycobiliproteins c. a step of precipitating using salicylic acid d. a step of dissolving the precipitate obtained in step c.

14. The process as claimed in claim 1, wherein the aqueous extract of phycobiliproteins is obtained from Arthrospira platensis photosynthetic microalgae.

15. A stable precipitate enriched in phycobiliproteins, characterized by a phycobiliprotein content of at least equal to 50% of the amount initially contained in the starting aqueous extract, and having a clarity at least equal to 70%.

16. A stable precipitate enriched in phycobiliproteins which can be obtained by means of the process of claim 1, in powder form or in suspension.

17. The stable precipitate as claimed in claim 15, which is in powder form or in suspension and is totally free of membrane fragments.

18. The use of a precipitate as claimed in claim 15, in the preparation of cosmetic, therapeutic or nutraceutical compositions.

19. A cosmetic or dermatological composition containing a precipitate as claimed in claim 15, obtained by at least one precipitation step consisting of the addition of salicylic acid to an aqueous extract of phycobiliproteins, said precipitate having a phycobiliprotein content at least equal to 50% of the amount initially contained in said aqueous extract, and having a clarity at least equal to 70%.

20. A nutraceutical composition comprising a precipitate as claimed in claim 15, which can be obtained by at least one precipitation step consisting of the addition of salicylic acid to an aqueous extract of phycobiliproteins, said precipitate having a phycobiliprotein content at least equal to 50% of the amount initially contained in said aqueous extract, and having a clarity at least equal to 70%.

Description

DESCRIPTION OF THE FIGURES

[0130] FIG. 1 represents the curve of iso-half-life of the dissolved phycobiliproteins as a function of the variation in temperature (temp in ° C.) and in light intensity (Lum in μmol photon/m.sup.2/s).

[0131] FIG. 2 represents the variation in the degree of precipitation of the phycobiliproteins (phy) and of the fragments (frag) as a function of the salicylic acid concentration (g/l).

[0132] FIG. 3 represents the variation in the clarity of the precipitate as a function of the concentration of fragments (g/l) and of the initial concentrations of phycobiliproteins. The salicylic acid content is 13 g/l.

EXAMPLES

Example 1: Precipitation with Salicylic Acid

[0133] The process of the present disclosure is subdivided into two essential steps:

[0134] 1) Commercial grade powdered salicylic acid is added to the phycobiliprotein extract. The solution (salicylic acid+extract) is stirred for 15 minutes. This step makes it possible to homogenize the solution and to promote the bringing of the acid into contact with the phycobiliprotein molecules in solution.

[0135] The solution is placed in a separating funnel. Virtually all of the phycobiliproteins, more than 90%, precipitate after at most one hour. The amount of salicylic acid required for the precipitation of virtually all of the phycobiliproteins after one hour depends on the amount of the latter in the initial extract. The present disclosure establishes the following equation which makes it possible to determine the minimum amount of salicylic acid to be used for any amount of soluble phycobiliproteins in the initial extract.

Salicylic acid(g)=0.007×phycobiliproteins(mg)/0.52

[0136] For uses requiring a greater purity of phycobiliproteins, the disclosure shows that the phycobiliprotein extract is clarified when the selective precipitation using salicylic acid is applied. The clarification is used after a decantation step which makes it possible to remove the decantable residues from the extract. It consequently aims to separate the phycobiliproteins from the fine membrane particles which remain in the suspension after decantation.

[0137] The precipitated phycobiliproteins, possibly clarified by decantation, are recovered by coarse filtration through gauze or filter paper or simply by pouring the supernatant from the top of the separating funnel.

[0138] 2) The precipitated phycobiliproteins, possibly clarified by decantation, are dissolved in glycerin. The disclosure determines that 1.2 l of glycerin are sufficient to dissolve 27 g of precipitated phycobiliproteins. The dissolution yield is between 95% and 99%. It should be pointed out that the fraction not dissolved in the glycerin can be recovered in absolute alcohol.

Example 2

[0139] The half-life of the phycobiliproteins precipitated with salicylic acid and redissolved in glycerol according to the process of the present disclosure is studied as a function of the temperature and of the light intensity.

[0140] The term “half-life” (Y) denotes the number of days resulting in the degradation of half the phycobiliproteins.

[0141] The temperature is studied in a range of 20 to 60° C., the light intensity is studied in a range of 50 to 150 μmol photon/m.sup.2/s.

[0142] Eleven experiments were carried out, including eight representing the combination between the three levels corresponding to the two factors. The other three represent central points and make it possible to evaluate the experimental error.

[0143] The mathematical model proposed is:

Log.sub.10Y=4−0.08Temp−0.013×Lum+0.0005×Temp.sup.2+0.0002×temp×Lum.

[0144] This model is valid at 94%, with a correlation coefficient R.sup.2=98% and is predictable (Q) at 95%.

[0145] The variation in the half-life of the phycobiliproteins (in days) according to the temperature and light conditions is given by the iso half-life curve (FIG. 1).

[0146] The half-life of the phycobiliproteins precipitated with salicylic acid and redissolved in glycerol can exceed 110 days, for a temperature below 20° C. and a light intensity below 50 μmol photon/m.sup.2/s.

[0147] By prediction outside the study range, it is possible to estimate the half-life of the stabilized phycobiliproteins at 11 years when the storage conditions are 4° C. and in total darkness.

[0148] Under the same temperature (20° C.) and luminosity (50 μmol photon/m.sup.2/s) conditions, the stability tests show that, for the untreated aqueous extracts, i.e. those which have not been subjected to the process of the disclosure based on the addition of salicylic acid and/or glycerin, all of the phycobiliproteins are degraded after 12 hours. For the powdered phycobiliproteins, precipitated and stabilized with salicylic acid without redissolving in glycerin, the degradation does not exceed 10% after more than 2 months of incubation.

[0149] For the aqueous extracts dissolved in glycerin but not precipitated with salicylic acid according to the process of the present disclosure, the degradation is 50% after one month of incubation.

[0150] The aqueous extracts precipitated with salicylic acid and dissolved in glycerin show better stability.

Example 3

[0151] It is sought to clarify the extracted phycobiliproteins by selective precipitation with salicylic acid. The extraction can be carried out from dry or fresh spirulina biomass.

[0152] 13 g of dry spirulina (or 130 g of fresh spirulina) are used, suspended in 0.2 l of sterile sea water, well homogenized.

[0153] The whole mixture is frozen at −4° C. overnight. It is thawed in 0.4 l of sterile sea water and subjected to stirring for two hours under cold conditions. A second freezing-thawing followed by thawing in 0.4 l of sterile sea water with stirring under cold conditions for two hours is carried out.

[0154] The suspension obtained is transferred into a decanting receptacle overnight under cold conditions in order to separate the aqueous phase of the extract, from the decantable biomass.

[0155] The phycobiliprotein extraction yield is estimated at 12-15% of the dry weight of the spirulina.

[0156] The phycobiliprotein content is estimated on the basis of the optical density at 620 nm and 650 nm and by applying the formulae of Bryant et al. (1976). The fragment content is evaluated by measuring the optical density at 680 nm according to an established correlation curve linking the OD680 to the dry spirulina biomass.

[0157] The phycobiliprotein concentration of the extract, thus evaluated, is 1.5 g/l of sea water.

[0158] The content of fragments and of membrane elements is evaluated at 1.9 g/l.

[0159] 1 to 2 g of powdered salicylic acid are intermittently added with continuous stirring.

[0160] At each addition, the solution is left to stand for two hours, after which time the phycobiliproteins precipitate. The amount of phycobiliproteins and of fragments in solution is then evaluated in order to determine the degrees of precipitation.

[0161] The results of the degrees of precipitation are presented in FIG. 2.

[0162] It is shown that it is possible to obtain a 100% clarified precipitate for a concentration of less than 12-13 g/l of salicylic add.

[0163] The maximum yield of precipitated and totally clarified phycobiliproteins thus obtained is 70% for an addition of 13 g/l of salicylic acid.

[0164] All of the membrane fragments and complexes remain in the supernatant and are only precipitated at high concentrations of salicylic acid.

Example 4: Variation in the Level of Clarity of the Precipitated Phycobiliproteins

[0165] The variation in the level of clarity of the precipitated phycobiliproteins is studied for various initial phycobiliprotein and fragment concentrations in the extract (obtained by various dilution and centrifugation) and for various salicylic acid concentrations.

[0166] The study is carried out according to the experimental design methodology, by means of a complete factorial design having three levels with interaction. The factors are: the initial phycobiliprotein concentration of the extract, ranging from 0.3 to 1.52 g/l, the fragment concentration ranging from 0.7 to 1.9 g/l and the salicylic acid concentration in a range of 10 to 20 g/l.

[0167] The experimental matrix given by the model is composed of fifteen experiments representing the combination between the three levels (minimum, maximum and center) corresponding to the three factors. Three repetitions of the same combination make it possible to evaluate the experimental error.

[0168] After addition of the salicylic acid, the amount of phycobiliproteins and of fragments not precipitated is determined separately for each experiment by spectrophotometry. The contents of precipitated phycobiliproteins and fragments are deduced therefrom. The clarity is calculated.

[0169] “Clarity” is denoted by clarity=100×amount of precipitated phycobiliproteins/amount of precipitated phycobiliproteins+fragments.

[0170] The model makes it possible to plot the iso-clarity curves that are presented in FIG. 3.

[0171] It is shown that it is possible to obtain a level of total clarification of 100% only for a phycobiliprotein concentration greater than 1.5 g/l and a fragment content less than 0.9 g/l. The salicylic acid content required for total clarification under these conditions is less than 13 g/l.

Example 5: Anti-Wrinkle Cream

[0172] An anti-wrinkle cream is obtained with the following composition:

[0173] Water: 59%,

[0174] Sweet almond oil: 18%,

[0175] Stable precipitate enriched in phycobiliproteins dissolved in glycerin: 7%,

[0176] Palm oil: 5%, Castor oil: 3%,

[0177] Extract of algae: 3% (beta-carotene),

[0178] Beeswax: 2%,

[0179] Xanthan gum: 1.7%,

[0180] Fragrance: 1%,

[0181] Ethyl paraben: 0.1%, methyl paraben: 0.1%, benzyl paraben: 0.1%.

Example 6: Anti-Mark Cream

[0182] An anti-mark cream is obtained with the following composition:

[0183] Anti-mark cream:

[0184] Water: 59%,

[0185] Sweet almond oil: 19.5%,

[0186] Stable precipitate enriched in phycobiliproteins dissolved in glycerin: 7%,

[0187] Palm oil: 5%, Castor oil: 3%,

[0188] Extract of algae: 1.5% (beta-carotene),

[0189] Beeswax: 2%,

[0190] Xanthan gum: 1.7%,

[0191] Fragrance: 1%,

[0192] Ethyl paraben: 0.1%, methyl paraben: 0.1%, benzyl paraben: 0.1%.

REFERENCES

[0193] Arad and Ai., 1997. Coloring material. U.S. Pat. No. 5,643,585 [0194] Bryant D. A., Glazer, A. N. and Eiserling F. A., 1976. Characterisation and Structural properties of the major biliproteins of Anabaena sp. Arch. Microbiol, 110, 61-75. [0195] Costa J A V, Colla L M, Duarte Filho P F, Kabke K, Weber A., 2002. Modelling of Spirulina platensis growth in freshwater using response surface methodology. World J Microbiol Biotechnol 18:603-607 [0196] Minard. F., 2005. Method for the photostabilisation of phycobiliproteins in an aqueous extract, compositions containing stabilised phycobiliproteins and use of stabilised phycobiliproteins. Patent No. WO2005065697 A1. [0197] Rebeller M. Yout P and Lonchamp D., 1979. Procédé d'extraction selective des colorants contenus dans les algues cyanophycdes [Process for selective extraction of dyes contained in cyanophyceae algae]. (Patent filed by the Institut français du pétrole entered under classification C 09 B 61/00). [0198] Frédéric Pottecher, 2014. Procédé d'extraction et de stabilisation dephycocyanine et ses application [Process for extraction and stabilization of phycocyanin and applications thereof]. WO 2014045177 A1. Patent filed by Ecosystem.