WO2016175670A1

WO2016175670A1 - A process of isolation and purification of diatoxanthin and diadinoxanthin

Info

Publication number: WO2016175670A1
Application number: PCT/PL2016/000047
Authority: WO
Inventors: Paulina KUCZYŃSKA; Małgorzata JEMIOŁA-RZEMIŃSKA
Original assignee: Uniwersytet Jagiellonski
Priority date: 2015-04-29
Filing date: 2016-04-28
Publication date: 2016-11-03

Abstract

The present invention discloses a process of isolation and purification of diatoxanthin and diadinoxanthin, which consists of five steps including: diatoms cultivation, pigment extraction, pigment partitioning, silica gel alkalization, open column chromatography, which performed in accordance with the invention allows to obtain diatoxanthin and diadinoxanthin of a purity of more than 99%.

Description

A process of isolation and purification of diatoxanthin and diadinoxanthin

The invention relates to a process of the isolation and purification of diatoxanthin and diadinoxanthin which consists of five steps including diatom cultivation, pigment extraction, partitioning of chlorophylls and carotenoids, alkalization of chromatography gel and open column chromatography, and finally allows to obtain highly pure diatoxanthin and diadinoxanthin provided that carried out in accordance with the invention conditions.

Open column chromatography is the most valuable preparative method and suitable to industrial use. Moreover, it does require neither the expensive equipment nor authorized staff. Alternatively, thin layer chromatography (TLC) can be used, however it is a method for analytical purposes only and inappropriate for industrial use. Furthermore, HPLC technique is extensively used to obtain pure samples, however the scale of such production is incomparably smaller and much more expensive.

To reduce production costs, the chemically synthesized substitutes of natural substances are often used. In this instance, however, the bioavailability and biological effects are substantially reduced. Manufacturers of carotenoid industry foresee a great potential in natural pigments obtaining from marine microorganisms, mainly due to the fact that of microorganisms can be cultivated in bioreactors. This applies to industrial scale and thus the costs of pigment isolation may be lower than one of their chemical synthesis. Carotenoids receive much attention in medical, cosmetic and food industries. Their antioxidant properties, ability to scavenge free radicals and prevention against DNA damages makes them good candidates to be used in pharmaceuticals, food supplements, biologically active cosmetics, and as a food additives of high nutrient value. Recently, a numerous studies have been published on modulation of the carotenoid bioaccessibility through liposomal encapsulation, which is important in terms of protection against degradation and increase their bioavailability.

Diatoxanthin and diadinoxanthin are desired carotenoids. The aim of invention is to provide a process of diatoxanthin and diadinoxanthin preparation of a purity of more than 99%, which is suitable for industrial use. The invention relates to a process of the preparation of diatoxanthin and diadinoxanthin characterized in that:

a) cultivation of diatoms is carried out, particularly Phaeodactylum tricornutum species, in white light of the intensity of 100 μΕ m^'V¹ and 16 h/8 h day/night photoperiod, favourably to obtain culture of density of 2 10⁶ cells/ml, subsequently (i) when diadinoxanthin is to be obtained, the culture is transferred to darkness for 15-20 h and then diatom cells are collected (ii) when diatoxanthin is to be obtained, the culture is transferred to white light of the intensity of 1250 μΕ m^"V for not more than 5 h, preferably 2 h, and then diatom cells are collected,

b) collected cells are subjected to total pigment extraction through the use of extraction mixture consisting of component A and component B in a volume ratio of 9:1, wherein the component A is a mixture of methanol with 0.2 M ammonium acetate in a volume ratio of 9: 1, while as component B ethyl acetate is used, the extraction mixture is added in the amount of 10 ml per 2 10⁹ cells, suspension is centrifuged and the resulting supernatant is pooled for use,

c) pre-purification is conducted through partitioning of pigments present in supernatant obtained in step b), wherein the partitioning is carried out in a mixture consisting of component A, component B and component C in a volume ratio of 4; 1 :2, wherein as a component A hexane-diethyl ether in a volume ratio of 1:1 is used, as a component B petroleum is used and as a component C water is used, wherein the fraction of lower density is collected as the fraction containing carotenoids,

d) chromatography column is prepared, wherein as a stationary phase a silica gel of pH 9 is used, particularly alkalised with sodium hydrogen carbonate, then balanced with a mixture of hexane and acetone in a volume ratio of 90:10, wherein hexane is a mixture of isomers,

e) separation of pigments obtained in step c) on the column obtained in step d) is carried out, wherein pigment mixture obtained from 2 10⁹ cells is applied to 20 g of silica gel, wherein pigments are dissolved in a mixture of hexane and acetone in a volume ratio of 90:10, and then elution is carried out with a mixture of hexane and acetone in a volume ratio from 90: 10 to 70:30, wherein hexane is a mixture of isomers, until the fraction containing diatoxanthin which is the third fraction eluted from the column is collected, preferably eluted using mixture of hexane and acetone in a volume ratio of 80:20, and/or the fraction containing diadinoxanthin which is the fourth fraction eluted from the column is collected, preferably eluted using mixture of hexane and acetone in a volume ratio of 70:30.

Preferably, step a) is performed at temperature from 15 to 20° C, preferably at 15 °C.

Preferably, in step a) diatom cells are collected through centrifugation at 5500xg at 4°C for 5 Preferably, supernatant obtained in step b) is concentrated by evaporation in oxygen-free atmosphere, particularly nitrogen. Other methods of evaporation can be used provided that they ensure the avoidance of oxygen.

Preferably, carotenoid fraction obtained in step c) is additionally washed by deionised water. Preferably, partitioning in step c) is repeated several times, and obtained carotenoid fraction is concentrated by evaporation in oxygen-free atmosphere, preferably nitrogen.

Preferably, in step d) silica gel of 60 g mol, particle size 0.040-0.063 mm, mesh 230-400 is used.

Preferably, in step e) elution with a mixture of hexane and acetone in a volume ratio of 90:10 is carried out until the first fraction is removed.

Preferably, in step e) elution with a mixture of hexane and acetone in a volume ratio of 80:20 is carried out following the first fraction, until the second and subsequently third fractions are collected.

Preferably, in step e) elution with a mixture of hexane and acetone in a volume ratio of 70:30 is carried out following the third fraction, until the fourth fraction is removed.

Preferably, in step e) elution with acetone is carried out following the fourth fraction, until the fifth fraction is removed and silica gel is cleansed.

Preferably, obtained in step e) fraction containing purified diatoxanthin is evaporated under nitrogen and stored at -80°C, while protected from light.

Preferably, obtained in step e) fraction containing purified diadinoxanthin is evaporated under nitrogen and stored at -80°C, while protected from light.

A process in accordance to the invention allows to obtain about 30 μg of diatoxanthin or 50 μg of diadinoxanthin of a purity of more than 99% from 2x10⁹ diatom cells in about 22 hours.

Described procedure of isolation and purification of diatoxanthin and diadinoxanthin is so far the only one which allows to obtain highly pure pigments (>99%), suitable to perform on an industrial scale. Its primary advantages are low cost and short time. Starting from the centrifugation of diatom culture it takes approximately 22 hours to obtain pure pigment.

The invention will be more completely understood by reference to the following figures: Fig. 1. Exemplary HPLC chromatogram of pigment extract from diatoms cultivated under continuous white light of the intensity of 1250 μΕ m^'V. Samples were collected for seven days. The content of diatoxanthin (peak with retention time 12.044 min.) in these conditions was about 15%, but in each sample the presence of both isomer diatoxanthin (peak with retention time 12.378 min.) and pigments of the violaxanthin cycle (peaks with retention times of 11.152 and 12.777 min.) was detected.

Fig. 2. Exemplary HPLC chi matogram of pigment extract from diatoms cultivated under continuous white light of the intensity of 1250 μΕ m"V^!. Samples were collected for seven days. The content of diadinoxanthin (peak with retention time 10.420 min.) in these conditions was about 15%, but in each sample the presence of both isomer diadinoxanthin (right-sided asymmetry of peak of retention time of 10.420 min.) and pigments of the violaxanthin cycle (peaks with retention times of 9.366, 11.155 and 12.782 min.) was detected.

Fig. 3. Exemplary HPLC chromatogram of pigment extract from diatoms cultivated under white light of the intensity of 700 μΕ m^"2s^"' in 6/18 h day/night photoperiod. Samples were collected for seven days. The content of diatoxanthin (peak with retention time 11.837 min.) in these conditions was about 9%, but in each sample the presence of both isomer diatoxanthin (peak with retention time 12.170 min.) and pigments of the violaxanthin cycle (peaks with retention times of 10.967 and 12.558 min.) was detected.

Fig. 4. Exemplary HPLC chromatogram of pigment extract from diatoms cultivated under white light of the intensity of 700 μΕ m^"V in 6/18 h day/night photoperiod. Samples were collected for seven days. The content of diadinoxanthin (peak with retention time 10.224 min.) in these conditions was about 18%, but in each sample the presence of both isomer diadinoxanthin (right-sided asymmetry of peak of retention time of 10.224 min.) was detected.

Fig. 5. Exemplary HPLC chromatogram of pigment extract from diatoms cultivated under white light of the intensity of 100 μΕ m^"V in 16/8 h day/night photoperiod and illuminated with white light with the intensity of 1250 μΕ m^~2s^~' for 2 h. The content of diatoxanthin (peak with retention time 11.412 min.) in these conditions was about 8%, wherein neither isomer diatoxanthin nor violaxanthin cycle pigments were detected. Fig. 6. Exemplary HPLC chromatogram of pigment extract from diatoms cultivated under white light with the intensity of 100 μΕ m'V in 16/8 h day/night photoperiod and incubated in darkness for 15 h. The content of diadinoxanthin (peak with retention time 10.095 min.) in these conditions was about 8%, wherein neither isomer diadinoxanthin nor violaxanthin cycle pigments were detected.

Fig. 7. Pigment partitioning steps: (A) a saponified pigment extract upon the addition of hexane: diethyl ether (1 : 1 v/v) - no fraction separation was observed; (B) a saponified pigment extract upon the addition of hexane :diethyl ether (1 :1 v/v) and petroleum— deficient fraction separation was observed; (C) a saponified pigment extract upon the addition of hexane: diethyl ether (1 :1 v/v), petroleum and water - clear fraction separation was observed; (D) carotenoid fractions collected through partitioning - visible turbidity of the solution; (E) washing the carotenoid fraction with water to remove polar, non-pigmented compounds; (F) carotenoid fraction upon the second washing with water - a transparent solution was obtained; white colour and dark grey colour in pictures A, B and C correspond to the yellow carotenoid fraction and the green chlorophyll fraction, respectively.

Fig. 8. Exemplary HPLC-DAD chromatogram (left) and the absorption spectrum (right), which confirm the purity of the obtained sample - diatoxanthin.

Fig. 9. Exemplary HPLC-DAD chromatogram (left) and the absorption spectrum (right) which confirm the purity of the obtained sample - diadinoxanthin.

Example 1. Diatoms cultivation.

The culture of P. tricornutum was grown under sterile conditions in f/2-Si medium (Guillard, 1975) made with artificial seawater supplemented with inorganic nutrients and vitamin mixture [Tab. 1], The culture was grown in a chamber at 15°C, under white light of the intensity of 100 μΕ m'V in 16/8 h day/night photoperiod. During the exponential phase of growth 1 liter of the culture was adjusted to density of 2 10⁶ cells/ml. Then, (i) when diadinoxanthin is to be obtained, the culture was transferred to darkness for 15-20 h, (ii) when diatoxanthin is to be obtained, the culture was illuminated with white light of the intensity of 1250 μΕ in V¹ at 15°C for 2 h. Tab. 1. Components of the f/2-Si medium to Phaeodactylum tricornutum cultivation (GuiHard, 1975).

* Tropic Marin BIO-ACTIF SEA SALT as an artificial seawater was used

The f/2 medium is a standard medium to marine algae cultivation. One of its components, essential to diatoms growth is sodium silicate. However, Phaeodactylum tricornutum is a species that does not require the presence of silicon, which also hinders pigment extraction, thus cells must be additionally homogenized, in the exemplary implementation, Phaeodactylum tricornutum was grown without sodium silicate. Due to reduced growth rate in deteriorated oxygen conditions, the volume of the culture should not exceed half of the flask volume. To obtain for isolation the same amount of material with expected pigment composition in a repetitive way, the culture should be of density of no more than 2 10⁶ cells/ml and in the exponential phase of growth. Inoculation of diatoms to fresh medium every 3-4 days ensures their optimal growth. As the most reliable method of assessing the quality of cultivation, a microscopic cell counts should be considered. In a preferred implementation, the range of temperature conditions is 15-20°C. Light conditions are crucial to the process, which is aimed at obtaining the cells with the highest diatoxanthin or diadinoxanthin content while maintaining reduced amount of pigment isomers and irremovable violaxanthin-cycle pigments. Light stress induces an mcrease in diadinoxanthin cycle pigments content. Therefore, cell illumination with light intensities of 1250 μΕ rcfV¹ and 700 μΕ m'V was tested. Notwithstanding the fact, that the light stress is required to produce diatoxanthin, high intensity of the light induces the violaxanthin cycle, which results in the presence of undesired pigments. The exposure to light of 1250 μΕ m'V¹ and 700 μΕ m^" V¹ for several hours resulted in the synthesis of additional pigments and formation of cis- isomers [Fig. 1, 3]. Therefore, as a particularly preferable conditions to diatoxanthin production, the illumination with light intensity of 1250 μΕ m^'V¹ for 2 h at 1 °C have been chosen, which ensured a desired pigment composition [Fig. 5]. To obtain a high amount of diadinoxanthin, cells cultivated under strong light (1250 μΕ m^"2s^"' or 700 μΕ m^'V¹) were transferred into darkness to convert diatoxanthin to diadinoxanthin. This resulted in a significant increase of the diadinoxanthin level, however concurrently additional pigments and isomers were detected [Fig. 2, 4]. Therefore, selected conditions, i.e. white light of the intensity of 100 μΕ m^'V¹ in 16/8 h day/night photoperiod both ensure a slight increase in the content of diadinoxanthin in comparison with a typical conditions (approx. 40 μΕ m'V¹) and do not cause the formation of undesired pigments and isomers [Fig. 6],

Example 2. Pigment extraction.

1 litre of diatom culture was centrifuged at 5500xg at 4°C for 5 min. The pellet was suspended in 20 ml of PBS buffer of pH 7 and centrifuged again using the same parameters. The obtained pellet was frozen in liquid nitrogen. The extraction was performed by the addition of the extraction mixture A:B (9: 1 v/v); A: methanol and 0.2 M ammonium acetate (9: 1 v/v), B: ethyl acetate; to pellet in a proportion of 10 ml of extraction mixture to 2 10⁹ cells. Suspension was vortex vigorously 2 x 10 s and then centrifuged at 14000xg at 4°C for 4 min. Supernatant was collected and evaporated under nitrogen.

A proportion between extraction mixture volume and the number of cells is essential for the extraction efficiency. The reduction in volume of the mixture with respect to the number of cells results in a decrease in extracted pigment amount, including diatoxanthin and diadinoxanthin. An attempt to increase a volume of mixture has no impact on extraction efficiency, and it only elevates the cost of the process and prolongs time of evaporation. The composition of extraction mixture has a primary impact on the process efficiency. Abele et al. (2012) have shown that the mixture used in the invention, unlike acetone and methanol, ensured the efficient extraction of xanthophylls with concurrent low-yield extraction of chlorophylls and β-carotene. Additionally, the presence of ammonium ions prevents isomerization. Pilot studies have shown that the additional cells homogenization with glass beads did not increase the extraction yield. Re-extraction of pigments using a pellet obtained after the first extraction does not increase the efficiency of this step.

Example 3. Pigments partitioning.

A saponification is carried out by dissolving the extracted pigments described in Example 2 in 35 ml of 95% ethanol, and addition of 3.5 ml of 60% KOH followed by gentle agitation overnight at 4°C. Then, chlorophylls are separated from carotenoids through:

1) adding to a pigment mixture consecutively 40 ml of hexane: diethyl ether (1 :1 v/v), 10 ml of petroleum and 20 ml of distilled water; gentle mixing and allowing to separate into two phases; then collecting the upper carotenoid fraction;

2) adding to remained chlorophyll fraction consecutively 10 ml of hexane: diethyl ether (1 :1 v/v), 2,5 ml of petroleum and 5 ml of distilled water; gentle mixing and allowing to separate into two phases; then collecting the upper carotenoid fraction;

3) repeating the step 2) to increase the efficiency of the procedure.

In a preferred embodiment, a washing with water prior to collection of carotenoid fractions are performed. For this purpose, distilled water in a ratio 1 :1 v/v is added to the combined carotenoid fractions; mixed vigorously and left to phase separation. Then, the upper carotenoid fraction is collected. Washing is repeated twice. Finally, the pigment mixture is evaporated in a nitrogen atmosphere.

A saponification allows for removal of lipophilic compounds and is commonly used to isolate photosynthetic pigments. Partitioning of saponified pigment extract allows to remove the chlorophylls which represent more than 30% of diatom pigments. For a successful pigment partitioning, mixture composition as well as the solvent proportions are essential. They ensure to obtain the upper fraction containing the highest amount of diatoxanthin or diadinoxanthin and to remove soap formed as a result of the use of KOH. The mixture of hexane: diethyl ether (1 : 1 v/v) which is commonly used for partitioning of the photosynthetic pigments of higher plants and algae does not lead to the phase separation in the case of diatoxanthin and diadinoxanthin isolation [Fig. 7A]. The addition of petroleum results merely in partial phase separation, and the phase boundary is diffused [Fig. 7B]. The addition of water as a fourth component exclusively allows to excellent sepatarion [Fig. 7C], wherein diatoxanthin and diadinoxanthin are preferentially dissolved in the upper phase. Additional two replicate partitioning step using 10 ml of hexane:diethyl ether (1 :1 v/v), 2.5 ml of petroleum and 5 ml of distilled water per each one, can recover all available diatoxanthin and diadinoxanthin pool. An increase in the number of repetitions results in the adverse effect as it contributes to the enhanced level of fucoxanthin. The partitioning step is crucial for diatoxanthin and diadinoxanthin purification and ensures to remove chlorophylls and significant amount of fucoxanthin. Skipping of this step precludes obtaining diatoxanthin and diadinoxanthin of high purity.

Example 4. Preparation of chromatography column.

A silica gel (Merck Silica gel 60 (0.040-0.063 mm)) is alkalised in accordance with a procedure described by Nagy et al., 2009. For this purpose 200 g of silica gel is added to 2 L of saturated sodium hydrogen carbonate solution and stirred for 2 h at room temperature. The gel filtration is carried out on filter paper in a Buchner funnel using a vacuum pump. Upon the removal of sodium hydrogen carbonate solution, the gel is washed with 2 L of distilled and then with 500 ml of acetone. Silica gel is dried under the hood for 24 h. An amount of 20 g of obtained silica gel suspended in a mixture of hexane: acetone (90: 10 v/v) is applied to the glass column with a diameter of 2 cm and a length of at least 30 cm.

The use of alkalised silica gel prevents the formation of ci -isomers during chromatography, but it does not guarantee an efficient separation. To obtain diatoxanthin and diadinoxanhin of a purity of more than 99%, the conditions described below must be kept. The ratio between the amount of pigments and gel are important to perform chromatography efficiently, i.e. the carotenoid fraction obtained from pigment mixture extracted from 2 10⁹ cells should be applied on 20 g of silica gel. A decrease in gel amount results in overloading the column and therefore reduces the separation efficiency, whereas and increase in gel amount increases the costs and procedure time without improving the efficiency. The diameter of the column is also important, a larger one reduces the distance between bands and limits their separation. Therefore, the ratio between the length and diameter of the column should be maintained.

Example 5. Open column chromatography.

Open column chromatography is carried out on the column prepared in Example 4. according to the following steps: 1) dissolving the pigment residue in 3 ml of hexane: acetone (90: 10 v/v) and applying onto the column;

2) elution with a mixture of hexane: acetone (90: 10 v/v) until the first fraction is removed,

3) elution with a mixture of hexane: acetone (80:20 v/v) until the second fraction is removed,

4) elution with a mixture of hexane:acetone (80:20 v/v) and collecting the third fraction which corresponds to diatoxanthin,

5) elution with a mixture of hexane :acetone (70:30 v/v) and collecting the fourth fraction which corresponds to diadinoxanthin

6) elution with an acetone until the fifth fraction is removed to clean up the column for its reuse.

Steps 5 and 6 may be omitted if new portion of silica gel is used each time.

A described process ensures obtaining about 30 μ of diatoxanthin or 50 μg of diadinoxanthin of a purity of more than 99% from 2 l0⁹ diatom cells in about 22 hours.

Performing the chromatography in accordance with principals described above is crucial to purify diatoxanthin and diadinoxanthin. Dissolving of pigment residue in 3 ml of hexane: acetone (90:10 v/v) is optimal to enable all pigments to get adsorbed onto the silica gel at the similar time. Preliminary studies showed a significant impact of different solvents on the separation process. The use of two eluents merely, i.e. hexane :acetone (90: 10 v/v and 60:40 v/v) results in separation of two fractions: β-carotene and a mixture of diadinoxanthin and diatoxanthin. Another two eluents used to separate diatoxanthin from diadinoxanthin, i.e. B:C (35:65 v/v and 29:71 v/v); B: 90% acetonitrile, 10% water, C: ethyl acetate; did not provide a positive result. Therefore, the use of eluents in accordance with the invention is crucial for the purification procedure. The use of mixture of hexane: acetone (90: 10 v/v) causes fast β-carotene and very slow β-cryptoxanthin epoxide migration, while other carotenoids remain on the top of the column. The change of solvent proportions to 80:20 v/v results in β-cryptoxanthin epoxide elution and starts the migration of the third fraction, which is diatoxanthin. At the same time, the fourth faction corresponding to diadinoxanthin starts migration slowly. Upon completion of diatoxanthin collection, the eluent should be changed to hexane: acetone (70:30 v/v) to run the elution of diadinoxanthin. It is of utmost importance to change the eluent just upon completion of diatoxanthin removal to guarantee the purity of collected pigments. Following diadinoxanthin removal, further elution with acetone may be continued to clean up the column and reused it. This opportunity significantly contributes to the economics of the process. Example 6. Storage of diatoxanthin and diadinoxanthin.

Obtained in Example 5 fraction of purified diatoxanthin [Fig. 8] or diadinoxanthin [Fig. 9] should be evaporated under nitrogen and stored at -80° C in a sealed glass vial, when protected from the light. Increased temperature, oxygen and light cause pigment decomposition due to its physicochemical properties typical to carotenoids. Several thawing and freezing cycles as well as two years of storage do not lead to pigment decomposition. So far the first formulation has two years and no degradation was found.

Claims

Patent claims

1. A process of diatoxanthin and diadinoxanthin purification comprising the steps of:

a) cultivation of diatoms, particularly Phaeodactylum tricornutum species, in white light of the intensity of 100 μΕ m'V¹ and 16 h/8 h day/night photoperiod, favourably to obtain culture of density of 2 10⁶ cells/ml, subsequently (i) when diadinoxanthin is to be obtained, the culture is transferred to darkness for 15-20 h and then diatom cells are collected (ii) when diatoxanthin is to be obtained the culture is transferred to white light of the intensity of 1250 μΕ m"V for no more than 5 h, preferably 2 h and then diatom cells are collected,

b) collected cells are subjected to total pigment extraction through the use of extraction mixture consisting of component A and component B in a volume ratio of 9:1, wherein the component A is a mixture of methanol with 0.2 M ammonium acetate in a volume ratio of 9:1, while as component B ethyl acetate is used, the extraction mixture is added in the amount of 10 ml per 2 10⁹ cells, suspension is centrifuged and the resulting supernatant is pooled for use,

c) pre-purification is conducted through partitioning of pigments present in supernatant obtained in step b), wherein the partitioning is carried out in a mixture consisting of component A, component B and component C in a volume ratio of 4:1 :2, wherein as a component A is hexane-diethyl ether in a volume ratio of 1 : 1, as a component B is petroleum and as a component C is water, wherein the fraction of lower density is collected as the fraction containing carotenoids,

e) separation of pigments obtained in step c) on column obtained in step d) is carried, wherein pigments mixture obtained from 2 10⁹ cells is applied to 20 g of silica gel, wherein pigments are dissolved in a mixture of hexane and acetone in a volume ratio of 90:10, and then elution is carried out with a mixture of hexane and acetone in a volume ratio from 90: 10 to 70:30, wherein hexane is a mixture of isomers, until the fraction containing diatoxanthin which is the third fraction eluted from column is collected, preferably eluted using mixture of hexane and acetone in a volume ratio of 80:20, and/or the fraction containing diadinoxanthin which is the fourth fraction eluted from column is collected, preferably eluted using mixture of hexane and acetone in a volume ratio of 70:30.

2. The process according to claim 1, characterised in that in step a) is provided at the temperature from 15 to 20° C, preferably 15 °C.

3. The process according to claim 1, characterised in that in step a) cells are separated through the centrifugation at 5500xg at 4° C for 5 min.

4. The process according to claim 1, characterised in that in step b) is concentrated through evaporation in oxygen-free atmosphere, particularly nitrogen.

5. The process according to 1, characterised in that the fraction containing carotenoids obtained in step c) is additionally washed with distilled water.

6. The process according to claim 1, characterised in that partitioning in step c) is repeated several times and obtained carotenoid fraction is- concentrated through evaporation in oxygen-free atmosphere, preferably nitrogen.

7. The process according to claim 1, characterised in that in step d) is used a silica gel of 60 g/mol, particle size 0.040-0.063 mm, mesh 230-400.

8. The process according to claim 1, characterised in that in step e) the elution with mixture of hexane and acetone in a volume ratio of 90:10 is carried out until the first fraction is removed from the column.

9. The process according to claim 1 , characterised in that in step e) the elution with mixture of hexane and acetone in a volume ratio of 80:20 is carried out following the first fraction, until the second and subsequently third fractions are collected.

10. The process according to claim 1, characterised in that in step e) the elution with mixture of hexane and acetone in a volume ratio of 70:30 is carried out following the third fraction, until the fourth fraction is removed.

11. The process according to claim 1, characterised in that in step e) the elution with acetone is performed following the fourth fraction, until the fifth fraction is removed and silica gel is cleansed.

12. The process according to claim 1, characterised in that the fraction obtained in step e) containing purified diatoxanthin is evaporated under nitrogen and stored at -80°C, when protected from light.

13. The process according to claim 1, characterised in that the fraction obtained in step e) contaimng purified diadinoxanthin is evaporated under nitrogen and stored at -80°C, when protected from light.