PROCESS FOR EXTRACTING PHYCOCYANINS

Abstract

The invention relates to a new process for extracting and purifying phycocyanins produced by fermenting microalgae, in particular produced by Galdieria sulphuraria, by means of selective precipitation.

Claims

1. A process for extracting phycocyanins from an initial phycocyanin solution, wherein the process comprises the following steps: a) selective precipitation which consists in: (i) adjusting the pH of the initial solution to a selected value within a range of pH values in which the phycocyanins are less soluble, (ii) concentrating the phycocyanins in the solution to promote their precipitation, and b) recovery of the precipitated phycocyanin.

2. The process according to claim 1, wherein the steps (i) of pH adjustment and (ii) of concentration are carried out simultaneously or sequentially.

3. The process according to claim 1, wherein the initial phycocyanin solution is a crude solution obtained from a lysis of a microorganisms biomass cultivated to produce phycocyanin.

4. The process according to claim 1, wherein the phycocyanin is a phycocyanin stable at acidic pH.

5. The process according to claim 1, wherein the phycocyanin is a phycocyanin of microbial origin, produced by a microorganism selected from species of the genus Cyanidioschyzon, Cyanidium or Galdieria.

6. The process according to claim 1, wherein the range of pH values in which the phycocyanins are less soluble is from 4.5 to 5.5.

7. The process according to claim 1, wherein the concentration consists in removing water so as to obtain a phycocyanin content of at least 15 g/L.

8. The process according to claim 1, wherein the concentration step is carried out by tangential filtration with a cut-off threshold allowing the phycocyanin to be retained.

9. The process according to claim 1, wherein the recovered phycocyanin is dried and optionally ground.

10. The process according to claim 1, wherein the recovered phycocyanin has a purity index of at least 2.

11. The process according to claim 10, wherein the recovered phycocyanin has a purity index higher than 4.

12. A purified phycocyanin obtained by the process according to claim 1.

13.-14. (canceled)

15. The process according to claim 2, wherein the step (i) of pH adjustment is carried out before the step (ii) of concentration.

16. The process according to claim 3, wherein the microorganism also produces glycogen.

17. The process according to claim 16, wherein the obtained phycocyanin is a phycocyanin having a glycogen/phycocyanin weight ratio lower than 6.

18. The process according to claim 1, wherein the recovered phycocyanin has a purity index of at least 3.

Description

DESCRIPTION OF THE FIGURES

[0063] FIG. 1 shows the mass of the precipitate obtained at different phycocyanin concentrations and different pHs in the initial solution.

[0064] FIG. 2 shows the phycocyanin concentration in the supernatant after precipitate recovery as a function of pH for different phycocyanin concentrations.

EXAMPLES

[0065] Material and Methods

[0066] Strain: Galdieria sulphuraria (also called Cyanidium caldarium) UTEX #2919.

[0067] Cultivation Conditions:

[0068] The biomass is obtained by fed-batch fermentation using the conditions described in patent WO2017050918A1.

[0069] Extraction Conditions:

[0070] The cells are mechanically ground using a DYNO®-MILL KD ball mill (Willy A. Bachofen AG Maschinenfabrik). Since phycocyanin (PC) is a hydrophilic molecule, it is extracted with water by adjusting the pH to the desired value with a base (NaOH, KOH, NH.sub.4OH, etc.) or an acid (H.sub.2SO.sub.4, citric acid, etc.). The crude PC extract is recovered after separation of cell debris by centrifugation at 10000 g for 10 min at room temperature. The crude extract is concentrated by tangential filtration with a ceramic or organic membrane with a cut-off threshold allowing phycocyanin to be retained. The samples are then centrifuged to separate the precipitate from the supernatant. The mass of the pellet is measured with a precision balance. The pellet is resuspended in an aqueous solution of pH 7 allowing its resolubilization, in order to quantify the precipitated phycocyanin.

[0071] PC Determination:

[0072] The estimation of phycocyanin content and of purity index was performed by absorbance measurement using the method described by Moon et al. (Moon et al., Korean J. Chem. Eng., 2014, 1-6).

EXAMPLE 1

Effect of Concentration and pH on the Precipitation and Purification of Phycocyanin (PC)

[0073] A crude phycocyanin solution with an initial concentration of 1 g/L of PC and an initial purity of 1.6 is concentrated by tangential filtration at pH 4 to obtain a retentate with a concentration of 20 g/L, then 30 g/L and then 45 g/L. An increase in the purity of the product can be observed during the filtration, however this purity does not exceed the value of 2 despite the degree of concentration of the product. In FIG. 1 it can be seen that for a concentration of 20 g/L, the precipitation of phycocyanin is low at pH 4 and increases slightly as the pH increases towards higher values (FIG. 1). At the same time, the measurement of the soluble phycocyanin concentration in the supernatant during this pH rise shows a relatively small decrease. For a PC concentration of 30 g/L, this phenomenon of precipitation by change of pH is much more marked and appears to be maximal for values of 4.5 and 5.5 (FIG. 1 and FIG. 2). Upon further filtration and concentration of phycocyanin up to the value of 40 g/L soluble, the formation of a significant precipitate is observed during filtration even before pH modification (FIG. 1). As before, the change in pH increases the phenomenon of phycocyanin precipitation.

[0074] By tangential filtration, the purity of phycocyanin decreases and conversely the purity of the precipitate collected and resolubilized at pH 7. This indicates a preferential precipitation of phycocyanin, which can be resolubilized under more favorable pH conditions.

[0075] Table 1 reports the measurement of phycocyanin purity after resolubilization of the phycocyanin precipitate for precipitation at pH 7.5.

TABLE-US-00001 TABLE 1 PC concentration PC purity of PC purity of pellet in the sample the supernatant resuspended at pH 7.5 20 g/L 1.96 2.18 30 g/L 1 2.27 45 g/L 0.31 2.88

EXAMPLE 2

Effect of Concentration and pH on the Precipitation and Purification of PC from an Enzymatically Digested Sample

[0076] In this example, the crude solution is subjected to enzymatic digestion to degrade the glycogen present. The enzymatic degradation is done at room temperature and pH=4 with the enzymes alpha 1-4 glucuronidase (“Pectinex Ultra SPL”) and alpha 1-6 glucosidase (“Novozyme 26062”).

[0077] Enrichment by tangential filtration is carried out to reach a phycocyanin concentration of several tens of g/L and then a pH adjustment is performed, causing precipitation. pH samples at 4.5, 5 and 5.5 are taken with a measurement of residual soluble phycocyanin and a measurement of precipitated phycocyanin after collection and resolubilization of the pellet with a buffer solution at pH 7.5.

[0078] Similar to the previous example, precipitation is significant for a pH range comprised between 4.5 and 5.5, and the purity level in this case reaches values above 3.8 upon resolubilization of the precipitated phycocyanin. Enzymatic digestion of glycogen does not affect the purification by precipitation. Table 2 below gives the purity index values of phycocyanin after precipitation by adjustment to instability pH and also after resolubilization of the phycocyanin precipitate.

TABLE-US-00002 TABLE 2 Sample [PC] (g .Math. L.sup.−1) Purity Supernatant pH 4.5 5.90 1.08 Supernatant pH 5 7.19 1.25 Supernatant pH 5.5 10.79 1.51 Pellet pH 4.5 22.26 3.82 Pellet pH 5 19.82 3.90 Pellet pH 5.5 16.55 3.87

REFERENCES

[0079] Cruz de Jesùs et al., Int J Food Nutr Sci (2016) 3(3): 1-0 [0080] Martinez-Garcia et al., Int J Biol Macromol. (2016) 89:12-8 [0081] Martinez-Garcia et al., Carbohydrate Polymers (2017) 169: 75-82 [0082] Moon et al., 2014 Korean J. Chem. Eng., 2014, 1-6 [0083] TN 2009000406, U.S. Pat. Nos. 6,074,854, 5,817,498, US 2017/159090, WO 2009/075682, WO 2016/030643, WO 2017/050917, WO 2017/050918, WO 2017/093345, WO 2018/178334