METHOD FOR ENRICHING PROTISTS WITH LIPIDS RICH IN POLYUNSATURATED FATTY ACIDS, MORE PARTICULARLY OF THE OMEGA 3 CLASS, AND IMPLEMENTATION OF SAME FOR THE PRODUCTION OF SAID LIPIDS

Abstract

The invention relates to a method for enriching protists with lipids rich in polyunsaturated fatty acids (PUFA) in particular of the omega 3 (3) class, said method comprising the culture of protists in a culture medium comprising a selenium-containing compound as well as the protists enriched with lipids rich in PUFA obtained by this method.

The invention also relates to a method for producing lipids rich in PUFA, comprising, after the enrichment method according to the invention, a method for treating the protists by extraction from the biomass of selenium-containing lipids rich in PUFA.

The invention finally relates to any food, cosmetic or pharmaceutical product comprising either selenium-containing lipids thus extracted, or selenium-containing biomass originating from the enrichment method.

Claims

1. Method for enriching protists with lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids with respect to the total lipids, said method comprising the culture of protists in a culture medium comprising at least one selenium-containing compound, the concentration of said selenium-containing compound in the culture medium being such that the concentration of selenium in the culture medium is comprised between 1 and 8 mg/L.

2. Method according to claim 1, in which the selenium-containing compound is selected from the group formed by 2 hydroxy 4 methylselenobutyric acid or one of its salts, selenite, selenic acid, selenate, selenocysteine, selenomethionine, selenocystathionine, selenohomocysteine and seleno adenosylselenomathionine.

3. Method according to claim 1, in which the selenium-containing compound is selected from 2 hydroxy 4 methylselenobutyric acid or one of its salts, selenite, selenic acid and selenomethionine.

4. Method according to claim 1, in which the microalgae are of the division Stramenopiles, preferably of the family Thraustochytrids or Labyrinthulids, even more preferably of the genus Schizochytrium, Ulkenia, Aurantiochytrium or Thraustochytrium.

5. Method according to claim 1, in which the microalgae are of the division Alveolata, preferably of the family Dinoflagellata, even more preferably of the genus Crypthecodinium.

6. Method according to claim 1, in which the microalgae are heterotrophic marine microalgae of the species Schizochytrium limacinum or Schizochytrium mangrovei, or microalgae of the species Crypthecodinium cohnii.

7. Method according to claim 5, in which the heterotrophic microalgae are microalgae of the species Schizochytrium limacinum or microalgae of the species Crypthecodinium cohnii.

8. Protists obtained by the method of enrichment with lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids, in particular of the omega 3 class, with respect to the total lipids, according to claim 1.

9. Food or cosmetic product comprising protists according to claim 8.

10. Method for producing lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids, with respect to the total lipids, said method successively comprising: a method for enriching protists according to claim 1, so as to obtain protists enriched with lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids, with respect to the total lipids; and a method for treating protists enriched by extracting from the biomass lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids, with respect to the total lipids so as to obtain lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids, with respect to the total lipids and a selenium-containing biomass depleted of lipids.

11. Method for producing lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids, with respect to the total lipids, according to claim 10, in which extraction is followed by purification of the lipids thus extracted comprising from 55% to 80% by weight of polyunsaturated fatty acids, with respect to the total lipids.

12. Lipids obtained by the method according to claim 10, said lipids comprising from 55% to 80% by weight of polyunsaturated fatty acids, with respect to the total lipids.

13. Lipids according to claim 12, such that they comprise more than 1 ppm, even more preferably more than 2 ppm, by weight, of selenium with respect to their total mass.

14. Food or cosmetic product comprising selenium-containing lipids according to claim 13.

15. Food product according to claim 14, for human or animal nutrition.

Description

[0053] The invention will be better understood in the light of the embodiment examples below, with reference to the attached drawings in which:

[0054] FIG. 1 is a graph giving the weight percent of polyunsaturated fatty acids (PUFA) in the total lipids for Schizochytrium limacinum after incubation for 187 h;

[0055] FIG. 2 is a graph giving the weight percent of docosahexanoic acid (DHA) in the total lipids for Schizochytrium limacinum after incubation for 187 h;

[0056] FIG. 3 is a graph giving the weight percent of saturated fatty acids (SFA) in the total lipids for Schizochytrium limacinum after incubation for 187 h; and

[0057] FIG. 4 is a graph giving the weight percent of polyunsaturated fatty acids (PUFA) in the total lipids, the weight percent of docosahexanoic acid (DHA) in the total lipids and the weight percent of saturated fatty acids (SFA) in the total lipids, for Crypthecodinium cohnii after incubation for 187 h.

[0058] FIGS. 1 to 4 are explained in Examples 1 and 2 below.

EXAMPLES

[0059] The following examples illustrate the invention without, however, limiting its scope.

Example 1: Culture of Schizochytrium Protist in a Medium Containing Separately 2-Hydroxy-4-Methylselenobutyric Acid (HMSeBA); Selenite; Selenic Acid; or Selenomethionine

[0060] This culture has led to improvement in the production of polyunsaturated fatty acids and of DHA in particular.

[0061] Schizochytrium is a protist, and more precisely a heterotrophic Thraustochytride microalga.

[0062] A strain of Schizochytrium limacinum ATCC-MYA1381 was obtained from the ATCC bank (www.atcc.org).

[0063] The growth characteristics as well as the production of lipids by Schizochytrium limacinum were measured in the presence of different forms of selenium and compared in the absence of such additives (control referenced Without Se in the figures).

[0064] Pre-culture of the strain Schizochytrium limacinum ATCC-MYA1381 was carried out in standard liquid medium, the composition of which is given in Table 1. To this end, starter cultures of 50 mL were started in a 250 mL Erlen flask from a colony isolated on 633 M3 chytrid agar (www.atcc.org) and cultured at 25 C.1 C. in heterotrophy for 48 h in this standard liquid medium. The 100 mL inoculum is then started in a 500 mL Erlen flask from these starter cultures at an initial concentration of 1.010.sup.6 cells/m/L in the same culture conditions as the latter for 4 days. The cultures were continuously aerated by orbital stirring at 100 rpm (revolutions per minute).

[0065] The cultures for enrichment with selenium-containing compounds were started from the inoculum at an initial concentration of 1.010.sup.6 cells/mL for a final volume of 400 mL in a 2.0 L Erlen flask at 25 C.1 C. in heterotrophy for 187 h. The cultures were continuously aerated by orbital stirring at 100 rpm. Enrichment was carried out at TO by the addition of concentrated selenium solution (2-hydroxy-4-methylselenobutyric acid solution sold by the company Tetrahedron at 0.0125 g/L referenced HMSeBA in FIGS. 1 to 3, sodium selenite 0.0110 g/L referenced Selenite in FIGS. 1 to 3, selenic acid 0.0082 g/L referenced Selenic acid in FIGS. 1 to 3 and selenomethionine 0.0124 g/L referenced SeMet in FIGS. 1 to 3) for a final concentration of 5 mg of selenium/L in the medium, whatever the source of the selenium-containing compound.

[0066] The Schizochytrium limacinum biomass was then harvested after incubation for 72 h and 187 h.

TABLE-US-00001 TABLE 1 Summary of the concentrations of each reagent composing the liquid culture medium Reagents Final concentration (g/L) Artificial seawater 33 Yeast extract 8.7 Glucose monohydrate (G8270 Sigma) 17.0

[0067] Artificial seawater is a common commercial product; in this case it was the product Instant Ocean Salt from the company AQUARIUM SYSTEMS. Its composition is given below by way of information: Na.sup.+ (0.35 g/L), K.sup.+ (0.01 g/L), Mg.sup.2+ (0.04 g/L), Ca.sup.2+ (0.01 g/L), Sr.sup.+ (0.00054 g/L), Cl (0.641 g/L), S (SO.sub.4.sup.2) (0.07 g/L), P (PO.sub.4) (0.000157 mg/L), N (NO.sub.3) (0.002 mg/L), N (NH.sub.4) (0.006 mg/L); Si (SiO.sub.3) (0.0105 mg/L); Li (0.0124 mg/L); Si (0.0148 mg/L); Mo (0.0057 mg/L); Ba (0.00385 mg/L); V (0.0048 mg/L); Ni (0.00329 mg/L); Cr (0.0129 mg/L); Al (0.214 mg/L); Cu (0.00377 mg/L); Zn (0.00108 mg/L); Mn (0.00218 mg/L); Fe (0.000442 mg/L); Cd (0.000890 mg/L); Pb (0.0144 mg/L); Co (0.00253 mg/L); Ag (0.00819 mg/L); Ti (0.00106 mg/L).

[0068] Yeast extract is a standard commercial product provided by the company Merck.

[0069] Culture Monitoring

[0070] The total concentration of biomass was monitored by measuring the dry mass (filtration on GFC filter, Whatman, then drying in an oven under vacuum, 70 C. and 0.8 bar (80 kPa), for 24 h minimum before weighing). 10.sup.7 cells/mL were extracted in order to quantify the total lipids. The method for extracting lipids is known to a person skilled in the art and is as described by the publication Bligh E. G. & Dyer W. J., A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol 37 1959 911-917, this method having been slightly adapted for microalga cells. Thus, for each measurement, approximately 10 mL of culture (biomass and medium), freshly sampled, was washed with demineralized water so as to remove the extracellular salts before lyophilization, then placed in a glass tube and centrifuged for 10 mins at 3600 g and at 4 C. After centrifugation, the supernatant was removed and the pellet containing the cells was lyophilized. Extraction was carried out with 6 mL of a monophasic CHCl.sub.3/MeOH (chloroform/methanol) mixture in proportions of 2/1 (V/V). In order to ensure complete extraction of the lipids, the tube was kept under stirring on a rocker for 6 hours in darkness, then stored at 20 C. before being analyzed by GC-FID (gas chromatography with flame ionization detection) in order to determine the quantity and the profiles of the total fatty acids extracted.

[0071] The results obtained are shown in FIGS. 1, 2 and 3 which give respectively, for Schizochytrium limacinum after incubation for 187 h, the percentage of PUFA of total lipids, the percentage of DHA in the total lipids and the percentage of SFA in the total lipids on the y-axis, for each of the selenium-containing sources shown on the x-axis.

[0072] It has been noted that the presence of a selenium-containing compound at 5 mg/L in the culture medium made it possible to obtain a percentage of PUFA markedly higher than that of the control (at least 67% against 53%), whatever the selenium-containing species used, the most interesting result being given by selenite (73%).

[0073] It has also been noted that the presence of a selenium-containing compound at 5 mg/L in the culture medium made it possible to obtain a percentage of DHA markedly higher than that of the control (at least 50% against 41%), whatever the selenium-containing species used, the most interesting result being given by selenite (54%).

[0074] Finally, it has been noted that the presence of a selenium-containing compound at 5 mg/L in the culture medium made it possible to obtain a percentage of SFA markedly lower than that of the control (at least 31% against 44%), whatever the selenium-containing species used, the most interesting result being given by selenite (26%).

Example 2. Culture of Crypthecodinium cohnii Protist in a Medium Containing Selenomethionine

[0075] This culture has led to improvement in the production of polyunsaturated fatty acids and of DHA in particular.

[0076] It was produced in the same way as in Example 1, except that the protist is Crypthecodinium cohnii instead of Schizochytrium limacinum, and that a single source of selenium was used: selenomethionine (SeMet).

[0077] Crypthecodinium cohnii is a protist, and more precisely a heterotrophic microalga. The strain Crypthecodinium cohnii COMP 316 was obtained from the NCMA bank (https://ncma.bigelow.org).

[0078] The results obtained are shown in FIG. 4 which gives respectively, for Crypthecodinium cohnii after incubation for 187 h, the percentage of compound in the total lipids on the y-axis, for each of the compounds (PUFA, DHA and SFA) shown on the x-axis, in the presence of selenomethionine (reference With SeMet), or in the absence of a selenium-containing compound (reference Without Se).

[0079] It has been noted that the presence of selenomethionine at 5 mg/L of selenium in the culture medium has made it possible to obtain a percentage of PUFA markedly higher than that of the control (35% against 25%).

[0080] It has also been noted that the presence of selenomethionine at 5 mg/L of selenium in the culture medium has made it possible to obtain a percentage of DHA markedly higher than that of the control (34% against 24%).

[0081] Finally, it has been noted that the presence of selenomethionine at 5 mg/L of selenium in the culture medium has made it possible to obtain a percentage of SFA markedly lower than that of the control (53.5% against 64%).

[0082] Moreover, an increase in the total lipid content in the dry biomass has been noted, due to the use of selenomethionine. In fact, a quantity of 34% more total lipids is obtained using the selenium-containing culture medium relative to the control medium.