IL109202A - Process for the extraction of carotenoids and in particular astaxanthine from a microalgal culture - Google Patents

Description

PROCESS FOR THE EXTRACTION OF CAROTENOIDS AND IN PARTICULAR ASTAXANTHINE FROM A MICROALGAL CULTURE HELIOSYNTHESE SOCIETE ANONYME C: 18992 Process for the extraction of carotenoids and in particular astaxanthine from a microalgal culture.

DESCRIPTION The present invention relates to a process for the extraction of carotenoids and more specifically astaxanthine from a micro-algal culture and in particular Haematococcus pluvialis. It is advantageously applied to the agroalimentary field and particularly in pisciculture for the production of food for fish and crustaceans, as well as in pharmacology.

The colouring of microalgae is due to pigments responsible for photochemical reactions of photosynthesis such as chlorophylls, phycobilins and carotenoids. In particular, astaxanthine ensures the pink pigmentation of the flesh of shrimps, trout, salmon, etc.

The carotenoids contained in Haematococcus pluvialis are β -carotene, adonirubin, lutein, echinenone, astaxanthine, yiolaxanthine, neoxanthine, cantaxanthine and more generally the mono-, di- and tri-ester derivatives of astaxanthine.

The process according to the invention is also applicable to microalgae of the Dunaliella bardawil and Dunaliella salina type containing B -carotene; Chlorella pyrenoidosa containing lutein; Vacularia virescens containing diadinoxanthine. In addition, astaxanthine is contained in Chlamydomonas nivalis, Euglena heliorobrescens and sanguinea, Balticola droebakensis.

The invention only relates to the extraction of carotenoids from a microalgal culture, the production of said carotenoids taking place in accordance with the prior art and in particular as described in FR-A-2,620, 131 and EP-A-329,754.

SP 7655 LC It is known from WO-A-90/05765 to extract carotenoids and in particular astaxanthine from the waste of plants, algae or crustaceans using a boiling alkaline solution ( >95°C) . The astaxanthine and the associated carotenoids are removed from the extracted product either by acid precipitation, or by cooling, removal and separation of phases and then subsequent purification.

It is also known to extract carotenoids and in particular β -carotene from a culture of Dunaliella salina from US-A- 4,680,314 by adding an edible vegetable oil at the end of cul - turing the Dunaliella suspension. This oil attracts to the surface the carotenoid-rich oily algae, which are harvested. In order to separate the & -carotene from the microalgai cells, the pH is modified by acidifying the medium.

Another process for the extraction of carotenoids from Dunaliella is described in AU-A-487 ,'018. This process consists of vigorously stirring the structure in the presence of an organic solvent, which is partly miscible with the aqueous medium of the culture, until the microalgai cells burst and then the carotenoids are dissolved in the organic solvent.

This solvent then contains carotenoids and microalgai waste and is then filtered under partial vacuum on a diatom powder. The harvested filtrate contains approximately 1% by weight dry matter of £ -carotene of microalgae, said 9 -carotene then being concentrated.

These known extraction processes suffer from the disadvantage of low yields and therefore high cost.

The present invention relates to a novel process for the extrac- ■ L-ion of carotenoids from microalgae having an at least 4 times SP 7655 LC higher yield than the prior art processes.

To this end, the invention relates to a process for the extraction of carotenoids from a microalgal culture, comprising: a) centrifuging the culture medium in order to form a paste rich in microalgae, b) diluting the paste in a saline solution to bring about an osmotic shock on the cells of the microalgae, c) acidifying the solution and boiling it in order to soften the wall of the cells, d) filtering the solution to separate the acidified cells from the solution, e) washing the cells obtained in d) with the aid of an organic solvent able to solubilize the carotenoids contained in the cells.

This extraction process is applicable to all the aforementioned microalgae.

The osmotic shock weakens the endoplasmic network of the microalgae, which retains the globules of carotenoids known as hema-tochromes.

The osmotic shock can be brought about with an acid salt and strong bases such as NaCl, KC1, NaSO^, CaCl2, SO^, etc., the concentration of said salt being between 50 and 300 g/1.

Acidification takes place with a strong mineral or organic acid, so as to obtain a pH of at the most 4. In particular, use is made of a mineral acid such as hydrochloric or sulphuric acid.

The boiling time is a function of the nature of the cells and SP 7655 LC in particular their age, and as to whether or not said cells are encysted. In practise, this stage lasts between 10 and 60 minutes.

The hot acid hydrolysis according to the invention serves to soften and render more hydrophilic the wall of the cells contain- dng the hematochromes and in particular the cysts containing these cells.

The organic solvent used for solubilizing the carotenoids is constituted by esters, alcohols, ketones or optionally halogen- ated alkanes. In particular, use is made of a ketone such as acetone, or dichloromethane.

Other features and advantages of the invention can be better gathered from the following illustrative and non-limitative description with reference to the single drawing illustrating the different stages of. the process according to the invention.

This process is completely suitable for the extraction of asta- xanthine and its esters derived from Haematococcus pluvialis.

Thus, the. following description will relate to the extraction of carotenoids from a Haematococcus pluvialis culture, although the invention is also applicable to all microalgae containing carotenoids and in particular those referred' to hereinbefore.

At the end of the culturing of ' Haematococcus microalgae, e.g. in accordance with EP-A-329 ,754, in the manner shown at 2 in the attached drawing centrifuging takes place in order to har -vest a red paste containing the total carotenoids in a concentration of 1 to 6% of the dry matter weight. This centrifuging is carried out at between 5000 and 40,000 g and preferably between '20,000 and 35,000 g. The paste collected contains 30 to 70% by weight carotenoids.

SP 7655 LC This paste is then diluted with water, as illustrated at 4, so as to arrive at a biomass dry matter concentration of 10 to 100 g/1. 200 g/1 of NaCl is then added to this solution in order to bring about an osmotic shock on the Haematococcus cells, weakening the endoplasmic network of said cells, this stage being designated 6.

The solution is then acidified, as indicated in 8, by the addition of IN HCl, so as to obtain a pH of 2. The solution is then heated to boiling for between 10 and 60 minutes, as a function of the age of the cells and their encystation. This stage is designated 10.

The solution is then cooled and then filtered 12, e.g. on a glass filter and at ambient temperature. This gives on the one hand a clear, colourless filtrate and on the other the red, hydrolyzed biomass on the filter.

The concentrate is washed with acetone, as indicated at 14, which gradually decolourizes in order to give a red filtrate containing astaxanthine, its mono-, di- and tri-ester derivatives, as well as traces of β -carotene, free astaxanthine, echinenon¾ and free cantaxanthine.

As soon as it leaves the cell, this acetone extract is no longer photostable, i.e. its colour is unstable in natural light.

In order to concentrate the astaxanthine and its derivatives, the acetone is then evaporated at between 20 and 40°C for between and 30 minutes, which leads to a red oily residue which can then be redissolved in any solvent compatible with astaxanthine, such as dichloromethane. This astaxanthine-concentrated extract must be kept in the dark and at low temperature (0 to 5°C) and optionally under a nitrogen atmosphere, because SP 7655 LC - - it is photodestructible. By mass spectrometric identification, the extract. also contains red-coloured triglyceride and pheophy- tin residues.

The process according to the invention has. been compared with a more conventional method consisting of grinding the lyophi-* izsd microalgae in acetone at between 5 and 7°C in a rotary grinder or mill rotating at 20,000 r.p.m., under an argon atmosphere and in the dark. The process according to the invention and the conventional process were carried out on four identical samples. The results are given in the following table. The table makes it clear that the extraction yield of the process according to the invention is approximately 4 times higher than that of the presently used, conventional methods.

In addition, extraction tests were performed on samples of red cysts obtained from a natural efflorescence of Haematococcus pluvialis using the . process- according to the invention and that known under the name Bligh Dyers described in the document Can. J. Biochem. Physiol. 31, 911, 1959 by E.G. Bligh and W.J. Dyer. This known process uses a mixture of chloroform, methanol and water for the extraction of the carotenoids.

On a same sample and using the Bligh Dyers method, 0.64% of total carotenoids based on .the dry matter weight is obtained instead of 2.6S carotenoids using the process according to the invention.

The originality of the invention is the brutality of the extraction (osmotic shock) on pigments considered to be extremely fragile and unstable with respect to photooxidation when in the pure state. The aim of this osmotic shock is to gradually break the stable forms in which these fragile compounds occur, in order to make them available and usable.

SP 7655 LC TABLE Conventional method Invention Sample No. % carotenoids of dry matter % carotenoids of dry weight. matter weight. 1 0.39 0.86 2 0.34 1.08 3 0.36 1.64 4 0.32 1.34 SP 7655 LC 109202/3 - 8 -

Claims (8)

1. Process for the extraction of carotenoids from a microalgal culture, comprising: a) centifuging the culture medium in order to form a paste rich in microalgae, b) diluting the paste in a saline solution to bring about an osmotic shock on the cells of the microalgae, c) acidifying the solution and boiling it in order to soften the wall of the cells, d) filtering the solution to separate the acidified cells from the solution, e) washing the cells obtained in d) with the aid of an organic solvent able to solubilize the carotenoids contained in the cells.

2. Process according to claim 1, characterized in that the saline solution is a NaCl solution.

3. Process according to claim 2, characterized i that Che saline solution contains 50 to 300 g/1 of NaCl.

4. Process according to any one of the claims 1 to 3, charact-erized in that the solution is acidified to a.pH of 2 to 4.

5. Process according to any one of the claims 1' to 4, characterized in that boiling takes place for between 10 and 60 minutes.

6. Process according to any one of the claims 1 to 5, charact-erized in that the organic solvent is acetone or dichlorc methane- SP 7655 LC 109202/2 - 9 -

7. Process according to any one of the claims 1-6, characterized in that the carotenoids contained in the organic solvent are concentrated.

8. Process for the extraction of astaxanthine and its esters according to any one of claims 1 to 7, in which the microalgal culture is a Haematococcus pluvialis culture.