AU695047B2

AU695047B2 - Method for extracting carotenoids, particularly astaxanthin, from a culture of microalgae

Info

Publication number: AU695047B2
Application number: AU65405/94A
Authority: AU
Inventors: Claude Gudin; Patrick Maillard; Claudine Trezzy
Original assignee: HELIOSYNTHESE SA
Current assignee: HELIOSYNTHESE
Priority date: 1993-04-07
Filing date: 1994-04-06
Publication date: 1998-08-06
Anticipated expiration: 2014-04-06
Also published as: ES2115229T3; DE69408129T2; EP0693133B1; FR2703692B1; FR2703692A1; AU6540594A; DE69408129D1; WO1994023057A1; IL109202A0; IL109202A; EP0693133A1; ZA942351B; GR3026686T3

Description

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 microalgal 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 -carotene, adonirubin, lutein, echinenone, astaxanthine, violaxanthine, 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 2 -2- 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 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 S-carotene from a culture of Dunaliella salina from US-A- 4,680,314 by adding an edible vegetable oil at the end of culturing the Dunaliella suspension. "This oil attracts to the surface the carotenoid-rich oily algae, which are harvested.

In order to separate the P -carotene from the microalgal cells, the pH is modified by acidifying the medium.

4 r1 15 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 microalgal cells burst and then the 20 carotenoids are dissolved in the organic solvent.

This solvent then contains carotenoids and microalgal waste and is then filtered under partial vacuum on a diatom powder.

The harvested filtrate contains approximately 1% by weight dry matter of P -carotene of microalgae, said 9 -carotene then being concentrated.

This known extraction processes suffer from the disadvantage of a low yield and therefore a high cost.

I

3 This knowri extraction processes suffer from the disadvantage of a low yield and therefore a high cost.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Disclosure of the Invention The present invention relates to a novel process for the extraction of carotenoids from microalgae having an at least 4 times 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, comprining: a) centrifuging the culture medium in order to form a paste rich in microalgae, b) diluting the paste in water and then adding a saline solution to bring about an osmotic shock on the cells of the microalgae, c) acidifying the solution to a pH of at most 4 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 25 microalgae.

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

The osmotic shock can be brought about with an acid salt and strong bases such as NaC1, KC1, NaSO 4 CaC12, KSO 4 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 ,/k -00 -4in particular their age, and as to whether or not said cells are encysted. In practise, this stage lasts between 10 and minutes.

The hot acid hydrolysis according to the invention serves to soften and render more hydrophilic the wall of the cells containing 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 halogenated 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 astaxanthine 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 harvest 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 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 to 100 g/l. 200 g/l of NaC1 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 1N HC1, 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 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 P -carotene, free astaxanthine, echinenone and free cantaxanthine.

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

i In order to concentrate the astaxanthine and its derivatives, the acetone is then evaporated at between 20 and 40 0 C for between 5 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 50C) and optionally under a nitrogen atmosphere, because SP 7655 LC

-I

6 it is photodestructible. By mass spectrometric identification, the extract also.contains red-coloured triglyceride and pheophytin residues.

The process according to the invention has been compared with a more conventional method consisting of grinding the lyophilized microalgae in acetone at between 5 and 7 0 C in a rotary grinder or mill rotating at 20,000 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 2.68% carotenoids using the process according to the invention.

The originality of the invention is the brutality of the extra- Sction (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 r i: IL r I II il~Il"rr -7-

TABLE

Sample No.

Conventional method carotenoids of dry matter weight.

Invention carotenoids of dry matter weight.

0.86 1.08 1.64 1.34 1 2 3 4 0.39 0.34 0.36 0.32 SP 7655 LC i 4

Claims

1. 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 water and then adding a saline solution to bring about an osmotic shock on the cells of the microalgae, c) acidifying the solution to a pH of at most 4 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, characterised in that the saline solution is a NaCl solution.

3. Process according to claim 2, characterised in that the saline solution contains 50 to 300 g/1 of NaC1.

4. Process according to any one of the claims 1 to 3, characterised in that Sthe solution is acidified to a pH of 2 to 4. 20 5. Process according to any one of the claims 1 to 4, characterised in that boiling takes place for between 10 and 60 minutes. Process according to any one of the claims 1 to 5, characterised in that o the organic solvent is acetone or dichloromethane. I Process according to any one of the claims 1 to 6, characterised in that 25 the carotenoids contained in the organic solvent are concentrated.

8. Process for the extraction of astaxanthine and its ester derivatives from i a Haemotococcus pluvialis culture, characterised in that use is made of the process according to any one of the claims 1 to 7. Dated this 11th day of June 1998 HELIOSYNTHESE S A Patent Attorneys for the Applicant: F B RICE CO %A* I DESCRIPTIVE ABSTRACT Process for the extraction of carotenoids and in particular I astaxanthine from a microalgal culture. This process consists of centrifuging the culture medium in order to form a paste rich in microalgae, diluting (4,6) the paste in a saline solution in order to bring about an osmotic shock on the cells of the microalgae, acidifying (8) the solution and boiling (10) it in order to soften the wall of the cells, filtering (12) the solution in order to separate the acidified cells from the solution and-washing (14) the cells obtained with the aid of a organic solvent suitable for solubilizing the carotenoids contained in the cells. Single drawing. .i I SP 7655 LC ENTRIFUGK1 H 2 0 DILUTION NaCl OSMOTIC SHOCK HC1 iNAIIFCTO ~N G F ITRINGIZ .2 E WAS ZILG 14 FOCETIOEL ::if i i i i i t !1 i i j-ii i r:j ii i t i _r iI INTERNATIONAL SEARCH REPORT n ApplicatonNo PCT/FR 94/00383 A. CLASSIFICATION OF SUBJECT MATTER IPC 5 C12P23/00 C12N1/06 C12P1/00 //(C12P1/00,C12R1:89) According to International Patent Cassification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by clasfication symbols) IPC 5 C12P C12N C09B Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practical, search terms used) C. DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. A US,A,4 851 339 (HILLS, 25 July 1989 1-7 see column 3, line 57 line 59 see figure 1 A DATABASE WPI 1 Section Ch, Week 9317, Derwent Publications Ltd., London, GB; Class B05, AN 93-136904 JP,A,5 068 585 (HIGASHIMARU SHOYU KK) 23 March 1993 see abstract A WO,A,89 06910 (MICROBIO RESOURCES, INC.) 1-8 10 August 1989 see page 13, line 25 page 15, line 2 see claims SFurther documents are listed in the continuation of box C. Patent family members are listed in annex. Special categories of cited documents "T later document published after the international filing date or ionty de and nor in conflict with the application but document defining the general state of the art which is not cited to tderstad the principl or theory uncaderyin the considered to be of particular relevance invention earlier document but published on or after the international X. document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be cwsidered to document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken alone which is cited to estblish the publicaton date of another document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family Date of the actual completion of the international search Date of mailing of the international search report July 1994 2 2. 07, Name and mailing address of the ISA Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 NL 2280 HV Rijswijk Tel. (+31-70) 340-2040, Tx. 31 651 epon Ann Fax (+31.70) 340-3016 Andre Form PCT/ISA/210 (econd shet) (July 1992) page 1 of Lc- F. -RICE 00. ?\TN TFRNEYS ~IEI II II N6 INTERNATIONAL SEARCH REPORT Inwr ial Application No PCT/FR 94/00383 C4(Continutloo) DOCUMENTS CONSIDERED TO BE RELEVANT Category' Citaton of document, ith indicahon, where appropnie, of the relevant pame SReevant to claim No. A CHEMICAL ABSTRACTS, vol. 81, no. 15,1 13 October 1975, Columbus, Ohio, US; abstract no. 128497y, SIGALAT, C. DE KOUCHKOVSKY, Y. 'Fractionation and characterization of the photosynthetic apparatus of the unicellular blue-green alga Anacystis nidulans. I. Preparation of membrane fractions by osmotic lysis and pigment analysis' page 249 see abstract PHYSIOL.VEG., 13(2); 243-58, 1975 Form PCT/19AJ210 (continuation of second shoet) (July 1992) page 2 of 2 INTERNATIONAL SEARCH* REPORT .jfonnation on patent family mna C/RN PC/R94/00383 Patent document I Publication IPatetamy Publication cited in search report date mmber(s) date US-A-4851339 25-07-89 JP-A- 62232463 12-10-87 WO-A-8906910 10-08-89 US-A- 4871551 03-10-89 AU-B- 605082 03-01-91 AU-A- 3182389 25-08-89 EP-A,B 0356499 07-03-90 JP-T- 2503632 01-11-90 PT-B- 89656 31-12-93 Form PCT/ISA/210 (patant family annex) (July 19M) J RAPPORT DE RECHERCHE INTERNATIONALE e InratoleN A. CLASSEMENT DE LOBJET DE LA DEMANDE C/R9 038 CIB 5 C12P23/00 C12N1/06 C12P1/0O //(C12P1/00,C12R1:89) Salon Iii classification intemnaionale des brevets (CIB) cit Ala fat. salon la classification nationale et la Cifi B. DOMAINES SUR. LESQUELS LA RECHERCH-E A P'ORTE Dcumentation minimaic consultee (systeme de classification suivi des symboles de classement) CIB 5 C12P C12N C09B Documentation consultee autre qua la documentation minimala danh I& mesure oi ces documents releant des domaines sur lesquela a porte Iii rderdse Base do doies tlectronique cosssultte au cows do la recherche internationalC (nomn de la base de donnees, et at cola est rtalisablc, termnes do recherce utilizes) C. DOCUMENTS CONSIDERES COMME PERTINENTS__________ Cattgorie Identification des documents cites, avec, le cas echeant, l'indication des passages pertinents no. des revendicafioms vistes A US,A,4 851 339 (HILLS, 25 Juillet 1-7 1989 voir colonne 3, ligne 57 ligne 59 voir figure 1 A DATABASE WPI 1 Section Ch, Week 9317, Derwent Publications Ltd., London, GB; Class B05, AN 93-136904 JP,A,5 068 585 (HIGASHIMARU SHOYU KK) 23 Mars 1993 voir abrdg6 A WO,A,89 06910 (MICROBIO RESOURCES, INC.) 1-8 AoUt 1989 voir page 13, ligne 25 page 15, ligne 2 voir revendications Vi Voir Ia suite du cadre C pour Ia fin de Ia liste des documents MxV Les documents de families de brevets sont indliques an annexe Caegores atoals d docmant cits:r docusnent ultieur publie aprtg I& date de dtpft international cii I& document dtfinssant I't&t general de Ia technique, non techde isOt at i'Aanfa pa A mretade lepInc considtrt comnre particultrement pertinent tebIii ertine t base o lctonr cpicp document anfteiur, mais publie A I& date de dhp~t international X douetarcuirnntprne inninreniqec et ou a"r~ cette date ttecnzeracmenouvl oticmme ixnpliquant mie activitt 'L document pouvant jeter un doute sur une revendication do inventive par rapport au document considere isoltsnent pionite ci cite pour dtamincr la date de publication d'une 'Y document particulitremant pertinent; I'invention reventdiqute sutre citation cii pour tne raison speciale (telle qu'indiquee) e t re consmderee corma impliquant une activite inventive document se referant A wia diviulgation orate, Aim usage, A losuel documant est associt Ai i plses ute une exposition cii tows sues moyens documnents de Mine nature, cette .obssio etAt ident. document y~ibUC avant la date de depOft international, mais powr ue personne dui mttier postneuiement A I& date de pniornt6revendiquee W document qwi fait partie de I& meme famille de brevets Date A laquelle: I a recherche intemnationale a ett effectivesnent achevee Date d'expidition du prtsent rapport de rechecmhe mnternataonale Juillet 1994 22. 07. 9 Nom Ct drg se postale de l'adiistration chargte de I& recherche intemnationale Fonctionnaire autorise Office European des Brevets, P.B. 5818 Patentlaan 2 NL 2280 HY Rijswijk Tel. 31-70) 340.2040, Tx 31651 npiI,An es S Faxc +31-70) 340-3016Anrs Fesmulius PCT/ISA/21 0 (damsrn foulla) (juillat 1992) page 1lde 2 %A.3 w 0 ~NT O~ RAPPORT DE RECHERCHE INTERNATIONALEDe nmaoa N PCT/FR 94/00383 C.4uge) DOCUMENTS CONSIPERES COMME PERTINENTS Cattgone Id ufcation des docints citts, avec, le cas fthtant, Vindicaaon des ponagei peruncas no. des rcveccons vsta A CHEMICAL ABSTRACTS, vol. 83, no. 15,1 13 Octobre 1975, Columbus, Ohio, US; abrdgd No. 128497y, SIGALAT, C. DE KOUCHKOVSKY, Y. 'Fractionation and characterization of the photosynthetic apparatus of the V unicellular blue-green alga Anacystis nidulans. I. Preparation of membrane fractions by osmotic lysis and pigment analysis' page 249 voir abrdgd PHYSIOL.VEG., 13(2); 243-58, 1975 Pormulalto PCThISAIMlO (sufts do 1A dsuA~NsM feuffi) (juwat 1IM2 page 2 de 2 1111 ','Vroe~ I RAPPORT DE RECHERCHE INTERNATIONALE Renaewwgnms relatifs au. emrcs de familes de brcvets Dem, lntea~ic No PCT/FR 94/00383 Document brevet cit6 I Date de IMembre(s) de a I Date de au rapport de recherche publication famille de brevet(s) I publication US-A-4851339 25-07-89 JP-A- 62232463 12-10-87 WO-A-8906910 10-08-89 US-A- 4871551 03-10-89 AU-B- 605082 03-01-91 AU-A- 3182389 25-08-89 EP-A,B 0356499 07-03-90 JP-T- 2503632 01-11-90 PT-B- 89656 31-12-93 14 Feiniulairs PCTflSA/21O (annexs faniin do brvaeu) audlet 1"92)