Extraction of Astaxanthin from Plants of the Species Adonis Aestivalis

Europaisches Patentamt (19) European Patent Office Office europeenpeen des brevets EP 0 541 595 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) intci.6: C09B 61/00, A23L 1/27 of the grant of the patent: 12.02.1997 Bulletin 1997/07 (86) International application number: PCT/GB91/01182 (21) Application number: 91913052.6 (87) International publication number: (22) Date of filing: 17.07.1991 WO 92/01754 (06.02.1992 Gazette 1992/04)

(54) Extraction of astaxanthin from plants of the species Adonis aestivalis Extraktion von Astaxanthin aus Pflanzen der Spezies Adonis aestivalis Extraction d'astaxanthin des plants de I'espece Adonis aestivalis

(84) Designated Contracting States: (74) Representative: Dekker, Enno E.J. et al AT BE CH DE DK ES FR GB GR IT LI NL SE Unilever N.V. Patents Division (30) Priority: 20.07.1990 GB 9016012 P.O. Box 137 3130 AC Vlaardingen (NL) (43) Date of publication of application: 19.05.1993 Bulletin 1993/20 (56) References cited: WO-A-90/05765 (73) Proprietor: QUEST INTERNATIONAL B.V. 1411 GP Naarden (NL) • Biochemical Systematics and Ecology vol. 9, no. 4, 14 February 1981, Pergamon Press Ltd., GB, (72) Inventor: MAWSON, Rodney Fairlawn B. Renstrom et al. Thrapston Northants NN14 4NG (GB) • Nature vol. 184, 28 November 1959, London, GB, A. Seybold et al., pages 1714-1715

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LO Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice the Patent Office of the Notice of shall be filed in o to European opposition to European patent granted. opposition a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. a. 99(1) European Patent Convention). LU

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Description The invention particularly provides a process for obtaining astaxanthin, wherein plants of the species Adon- This invention relates to carotenoid pigments ex- is aestivalis having an average flower head petal tractable from natural sources. number of at least 1 6 are cultivated, harvested, and the There is a need for a cost-effective natural source 5 astaxanthin is extracted from the harvested flower of astaxanthin, which is the carotenoid pigment that con- heads or petals thereof. tributes the characteristic pink or red colour to the flesh A seed sample of the newly-discovered strain of of wild salmon. Salmon id fish are extensively farmed to- Adonis aestivalis has been deposited on 18 July 1990 day, and there is a commercial need to produce such with the National Collection of Industrial and Marine fish possessing a nature-identical flesh colour. 10 Bacteria Limited, Aberdeen, under Accession No. Astaxanthin occurs in various marine animals, such NCI MB 40309, in accordance with the provisions of the as Crustacea, but its extraction from such sources is un- Budapest Treaty. Plants of this deposited strain typically economic. It is also possible to synthesise astaxanthin, have an average of 18-22 petals per flower head, and but this is expensive, and moreover the use of such syn- the average amount of astaxanthin per flower head is thetic pigment does not convey the connotation of "nat- is 200-350u.g. ural" that many regard as desirable. A general proce- The invention provides a process for obtaining dure for extraction of astaxanthin from vegetable or an- astaxanthin, wherein the astaxanthin is extracted from imal sources has been described in WO 90/05765. the petals of plants having the essential characteristics Astaxanthin occurs in certain plants, especially cer- of the deposited strain. tain species belonging to the genus Adonis. It is known 20 The invention particularly provides an oral compo- from B. Renstrom et al, Biochem. System, and Ecology sition for administration to fish, comprising such extract- 9, 249 (1981) and from T.W. Goodwin, Nature184, 1714 ed astaxanthin, and a method of pigmenting the flesh of (1 959) that astaxanthin may be obtained from the petals fish, especially salmonid fish, involving the oral admin- of Adonis annua. Another species in which astaxanthin istration to the fish of such a composition. occurs is Adonis aestivalis, where it occurs predomi- 25 Preferably, the composition comprises the astaxan- nantly in the petals of the bright red flowers. However, thin mixed with edible feed material. Alternatively, the the reported wild strains of Adonis aestivalis possess astaxanthin can be in encapsulated form. only flower heads with very few petals, and the propor- Alternatively, pigmentation of the flesh of fish can tion of astaxanthin pigment relative to the total mass of be achieved by feeding astaxanthin-containing portions the plant is too small for it to be cultivated and extracted 30 of the plant to the fish. Preferably, the portion comprises on any sensible commercial scale. the flower petals and more preferably, consists entirely We have discovered a novel true-breeding strain of of such material. If desired, the plant material can be Adonis aestivalis having substantially heavier flower extracted with water or aqueous media in order to re- heads, and in particular having a substantially greater move water-soluble compounds such as glycosides number of petals. The proportion of astaxanthin pigment 35 which may be toxic to fish or other animals, without re- in the plant is sufficiently high to make cultivation of the moving significant quantities of the required astaxan- plant for the purposes of extracting the pigment com- thin. mercially attractive. In the pigmentation of farmed fish, the astaxanthin Described are plants of the genus Adonis having obtained by the invention can be administered orally to petals containing astaxanthin, the average number of 40 the fish in any manner analogous to the techniques al- petals per flower head being at least 10, more particu- ready used for astaxanthin derived from conventional larly at least 16. sources. Normally the pigment is included in a compo- The petals contain astaxanthin, wherein the amount sition, such as a pelleted compound feedstuff, that forms of astaxanthin per flower head is at least 100u.g, more all or part of the diet on which the fish are reared. The particularly at least 150u.g, and yet more particularly at 45 pigment is soluble in oil, and can be incorporated in the least 200u.g. diet in this form, either as free oil or as encapsulated oil. Described is the cultivation of such plants for the Alternatively, the petals or other plant material contain- purpose of obtaining astaxanthin, the extraction of ing the astaxanthin can be mixed (eg. in dried, ground astaxanthin from such plants, and the astaxanthin so form) with conventional fish feed ingredients. If desired, obtained. so the plant material can be partially extracted with aque- Extraction of the astaxanthin is preferably conduct- ous media (to remove water-soluble components such ed using an organic solvent, and more preferably using as glycosides) prior to being added to the feed. As a a mixed solvent comprising a water-miscible organic further alternative, the pigment can be added to the feed solvent (such as ethanol) and a non-water-missible or- in the form of an organic solution, eg. a solution obtained ganic solvent (such as hexane). 55 during extraction of the astaxanthin from the plant ma- Preferably, the harvested plant material is initially terial, if the organic solvent used is not toxic to the fish extracted with water or aqueous media to remove water in any amount that the fish are likely to ingest via the soluble compounds such as glycosides. completed feedstuff.

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When the astaxanthin is administered to fish via Typical starting material their feedstuff, the composition of the feedstuff need not be unconventional. The feedstuff formulation can con- Frozen block of flower heads, stored at -20°C in the tain any of the normal fish feed components, such as dark, or dried petals/flowerheads, finely ground, also fish meal and/or other protein, oil such as fish oil, cere- 5 stored at low temperature in the dark. als, vitamins, minerals, preservatives and medica- ments, in the various proportions that are normally used. Primary Extraction The extracted astaxanthin, or astaxanthin-containing portions of the plant, can also be used as a colouring Mix with 10 vols hexane/ethanol (50:50 v/v), allow agent in human foodstuffs, and also in poultry diets to 10 dispersion, and homogenise for 1 0 minutes in dark and enhance the colour of egg yolks. cool conditions using shear blender; or mill with a shear Specific embodiments of the invention will now be blender in ethanol, then add hexane for safety reasons. described in detail, by way of example only. Leave overnight in dark and cool conditions. Filter through filter (eg. muslin) on vibrating sieve, Novel strain of Adonis aestivalis is and wash plug with 50:50 ethanol:hexane. Retain orig- inal filtered liquid and washings as primary extract. This The essential characteristics of the newly-discov- primary extract contains water-soluble, ethanol-soluble ered strain are: and hexane-soluble material including pigments and Chromosome No. 2n = 32 glycoside. Erect, annual, leaves alternative, pinnately dissect- 20 ed into linear or filoform segments. Flowers bright red, Secondary Extraction actinomorphic, hypogynous, hermaphrodite, solitary and terminal on stem and branches. Add 1 part water to 2.5 parts primary extract, trans- Petals less than 1 5mm long, up to 2 times length of fer to phase separator and remove bottom layer of eth- sepals. Sepals 5-8 in number. Petals 18-22 in number. 25 anol. Wash upper hexane layer with 1:1 ethanol/water Values for these parameters, and for the astaxan- mixture allow to separate and discard lower layer. thin level per flower head, can be taken from analysis of Transfer to steam jacketed vacuum evaporator the first fully-open flower heads from 1000 plants. (with cyclone) and remove hexane at 45°C for 15-30 Ripe achenes 3-5mm, tooth on dorsal surface dis- minutes until a sludge is obtained. Wash with ethanol tant from the beak. Achenes having a transverse ridge 30 and evaporate, wash with hexane and evaporate (again passing around middle of achene. Achenes also having at low temperatures, under vacuum or under nitrogen) a dorsal hump a distance from the beak. to dryness. This yields a first concentrate of approximately 5% total pigment (80% astaxanthin) in dry mat- Cultivation ter. 35 Take up in hexane and apply to silica column (1:1 The plant can be grown under a wide range of tem- or 2:1 extract to silica). Wash column with hexane in perature conditions. Germination requires a degree of dark and cool conditions, and discard washings. Elute alternating temperatures of 10-20°C. Mature flowering with 2.5% ethanol in hexane until a red-orange band ap- plants are obtained four months after sowing at field pears. Collect the red-orange washings until the colour densities up to 150 plants/m2 in a variety of soil condi- 40 changes to orange-green. tions; flowering in the summer months (temperate cli- Dry eluent as before, take up in hexane or oil (fish mate). The plant prefers dry, well drained conditions. oil, vegetable oil). This yields a second concentrate of Seed can be harvested by combine, and flowers by approximately 20% total pigment containing approxi- hand. mately 80% astaxanthin. 45 Extraction of pigment Typical Salmon Grower Diet

Pigment can be extracted by solvent extraction, eg. into mixed solvents such as ethanol/hexane, and further % inclusion by weight purified by partitioning a mixed solvent with water fol- so Fishmeal 75.0 lowed by column chromatography. The final extract, Vegetable protein 5.0 thus purified, is rich with respect to carotenoids, and pre- dominent is astaxanthin, present mainly as a racemic Cereal 7.8 mixture in the form of mono- and diesters, generally of Fish Oil 11.0 palmitic acid. 55 Minerals/Vitamins 1.0 Antioxidants/preservatives 0.2

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Pigment incorporation the astaxanthin is extracted from plants of the strain NCI MB 40309. Astaxanthin pigment from an Adonis strain of the invention can be added at levels ranging for example 8. A process according to claims 1 -7, wherein the ex- from 1 -1 00 ppm to the above type of diet in a variety of 5 traction of the astaxanthin is conducted using an or- ways: ganic solvent.

a) as extracted astaxanthin carried in a fish oil base, 9. A process according to claim 8 wherein the extrac- optionally containing antioxidants. tion is conducted using a mixed solvent comprising 10 a water-miscible organic solvent and a non-water- b) as extracted astaxanthin carried as a free-flowing miscible organic solvent. powder in wheatflour or any finely ground foodstuff for salmon. 10. A process according to claims 8 or 9, wherein the harvested plant material is initially extracted with c) as for (b) but encapsulated in alginate, gelatin or is water or aqueous media to remove water soluble xanthan gum, eg. by pan granulation or spray cool- compounds such as glycosides. ing. 11. A composition for use in the pigmentation of fish, d) as extracted astaxanthin carried in an encapsu- comprising astaxanthin produced by a process ac- lated lipid, eg. casein-protected lipid. 20 cording to claims 1-10 mixed with edible feed material. Pigmentation 12. A composition for use in the pigmentation of fish, The above typical salmon grower formulation con- comprising astaxanthin produced by a process ac- taining 50-100 ppm of astaxanthin extracted from the 25 cording to claims 1-10 in encapsulated form. deposited Adonis strain of the invention and carried in one of the product forms described above, is fed as pel- 13. A process for the pigmentation of fish, involving oral lets to fish of 300 g liveweight plus for aminimum period administration of a composition according to claim of 3 months. Typical values for pigmentation efficiency, 11 or claim 12. compared with commercially-available synthetic astax- 30 anthin, are at least about 80% of synthetic. 14. Harvested flower heads, or petals thereof, of the species Adonis aestivalis having an average of at least 10 petals per flower head. Claims 35 15. Harvested flower heads, or petals thereof, accord- 1. A process for the production of astaxanthin wherein ing to claim 1 4 having an average of at least 1 6 pet- the astaxanthin is extracted from plants of the spe- als per flower head. cies Adonis aestivalis having an average of at least 10 petals per flower head. 16. Harvested flower heads, or petals thereof, accord- 40 ing to claim 1 5 having an average of 1 8 to 22 petals 2. A process according to claim 1 , wherein the plants per flower head. have an average of at least 16 petals per flower head. 17. Harvested flower heads, or petals thereof, of the species Adonis aestivalis having at least 100u.g of 3. A process according to claim 2, wherein the plants 45 astaxanthin per flower head. have an average of 1 8 to 22 petals per flower head. 18. Harvested flower heads, or petals thereof, accord- 4. A process according to claims 1-3 wherein the ing to claim 1 7 having at least 1 50u.g of astaxanthin plants contain at least 1 00u.g astaxanthin per flower per flower head. head. 50 19. Harvested flower heads, or petals thereof, accord- 5. A process according to claim 4 wherein the plants ing to claim 1 8 having at least 200u.g of astaxanthin contain at least 150u.g astaxanthin per flower head. per flower head.

6. A process according to claim 5, wherein the flower- 55 20. A feedstuff for fish incorporating harvested material heads contain at least 200u.g astaxanthin. according to claims 1 4 - 19.

7. A process for the production of astaxanthin wherein

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Patentanspriiche 13. Verfahren zur Pigmentierung von Fisch, das das orale Verabreichen einer Zusammensetzung ge- 1. Verfahren zur Herstellung von Astaxanthin, wobei mal3 Anspruch 11 oder Anspruch 12 betrifft. das Astaxanthin aus Pflanzen der Spezies Adonis aestivalis extrahiert wird, die im Durchschnitt min- 5 14. Geerntete Blutenkopfe, oder Blutenblatter davon, destens 10 Blutenblatter pro Blutenkopf haben. der Spezies Adonis aestivalis, die im Durchschnitt mindestens 10 Blutenblatter pro Blutenkopf haben. 2. Verfahren gemal3 Anspruch 1, wobei die Pflanzen im Durchschnitt mindestens 16 Blutenblatter pro 15. Geerntete Blutenkopfe, oder Blutenblatter davon, Blutenkopf haben. 10 gemaB Anspruch 14, die im Durchschnitt mindestens 16 Blutenblatter pro Blutenkopf haben. 3. Verfahren gemaB Anspruch 2, wobei die Pflanzen im Durchschnitt 18 bis 22 Blutenblatter pro Bluten- 16. Geerntete Blutenkopfe, oder Blutenblatter davon, kopf haben. gemaB Anspruch 15, die im Durchschnitt 18 bis 22 is Blutenblatter pro Blutenkopf haben. 4. Verfahren gemaB den Anspruchen 1 bis 3, wobei die Pflanzen mindestens 100 u.g Astaxanthin pro 17. Geerntete Blutenkopfe, oder Blutenblatter davon, Blutenkopf enthalten. der Spezies Adonis aestivalis, die mindestens 100 u.g Astaxanthin pro Blutenkopf haben. 5. Verfahren gemaB Anspruch 4, wobei die Pflanzen 20 mindestens 150 u.g Astaxanthin pro Blutenkopf ent- 18. Geerntete Blutenkopfe, oder Blutenblatter davon, halten. gemaB Anspruch 17, die mindestens 150 u.g Asta- xanthin pro Blutenkopf haben. 6. Verfahren gemaB Anspruch 5, wobei die Blutenkop- fe mindestens 200 u.g Astaxanthin enthalten. 25 19. Geerntete Blutenkopfe, oder Blutenblatter davon, gemaB Anspruch 18, die mindestens 200 u.g Asta- 7. Verfahren zur Herstellung von Astaxanthin, wobei xanthin pro Blutenkopf haben. das Astaxanthin aus Pflanzen des Stammes NCIMB 40309 extrahiert wird. 20. Fischfuttermittel, dem das geerntete Material ge- 30 ma!3 einem der Anspruche 1 4 bis 1 9 einverleibt ist. 8. Verfahren gemaB den Anspruchen 1 bis 7, wobei die Extraktion des Astaxanthins unter Verwendung eines organischen Losungsmittels durchgefuhrt Revendications wird. 35 1 . Une methode de production d'astaxanthin, dans la- 9. Verfahren gemaB Anspruch 8, wobei die Extraktion quelle I'astaxanthin est extraite a partir de plants de unter Verwendung eines Losungsmittelgemischs I'espece Adonis aestivalis presentant une moyenne durchgefuhrt wird, das ein wassermischbares orga- d'au moins 10 petales par tete de fleur. nisches Losungsmittel und ein nicht wassermischbares organisches Losungsmittel umfaBt. 40 2. Une methode selon la Revendication 1, dans laquelle les plants presentent une moyenne d'au 10. Verfahren gemaB den Anspruchen 8 oder 9, wobei moins 16 petales par tete de fleur. das geerntete Pflanzenmaterial anfanglich mit Wasser oder waBrigen Medien extrahiert wird, urn 3. Une methode selon la Revendication 2, dans la- wasserlosliche Verbindungen, wie z.B. Glycoside, 45 quelle les plants presentent une moyenne d'au zu entfernen. moins 18 a 22 petales par tete de fleur.

11. Zusammensetzung zur Verwendung zur Pigmentie- 4. Une methode selon les Revendications 1 a 3, dans rung von Fisch, die Astaxanthin umfaBt, das durch laquelle les plants contiennent au moins 100 u.g ein Verfahren gemaB einem der Anspruche 1 bis 1 0 50 d'astaxanthin par tete de fleur. hergestellt wurde und mit eBbarem Futtermaterial gemischt ist. 5. Une methode selon la Revendication 4, dans laquelle les plants contiennent au moins 1 50 u.g d'as- 12. Zusammensetzung zur Verwendung zur Pigmentie- taxanthin par tete de fleur. rung von Fisch, die Astaxanthin, das durch ein Ver- fahren gemaB einem der Anspruche 1 bis 10 her- 6. Une methode selon la Revendication 5, dans la- gestellt wurde, in eingekapselter Form umfaBt. quelle les tetes de fleurs contiennent au moins 200 u.g d'astaxanthin.

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7. Une methode de production d'astaxanthin, dans la- 19. Tetes de fleurs recoltees, ou petales de celles-ci, quelle I'astaxanthin est extraite a partir de plants is- selon la Revendication 1 8, presentant au moins 200 sus de la variete NCI MB 40309. u.g d'astaxanthin par tete de fleur.

8. Une methode selon les Revendications 1 a 7, dans 5 20. Une matiere alimentaire pour poisson incorporant laquelle I'extraction d'astaxanthin est realisee en de la matiere recoltee selon les Revendications 14 utilisant un solvant organique. a 19.

9. Une methode selon la Revendication 8, dans laquelle I'extraction est realisee en utilisant un solvant melange contenant un solvant organique miscible dans I'eau et un solvant organique non miscible dans I'eau.

10. Une methode selon la Revendication 8 ou 9, dans 15 laquelle la matiere de plant recoltee est au depart extraite avec de I'eau ou un milieu aqueux afin d'eli- miner les composes solubles dans I'eau tels que des glycosides. 20 11 . Une composition destinee a etre utilisee pour la pigmentation du poisson, comprenant de I'astaxanthin produite selon une methode en accord avec les Re- vendications 1 a 10, que Ton melange a de la matiere d'alimentation comestible. 25

12. Une composition destinee a etre utilisee pour la pigmentation du poisson, comprenant de I'astaxanthin produite selon une methode en accord avec les Re- vendications 1 a 10, se presentant sous forme en- 30 capsulee.

13. Une methode destinee a etre utilisee pour la pigmentation du poisson, impliquant I'administration orale d'une composition selon la Revendication 11 35 ou 12.

14. Tetes de fleurs recoltees, ou petales de celles-ci, provenantde I'espece Adonis aestivalis, presentant une moyenne d'au moins 10 petales par tete de 40 fleur

15. Tetes de fleurs recoltees, ou petales de celles-ci, selon la Revendication 14, presentant une moyenne d'au moins 1 6 petales par tete de fleur. 45

16. Tetes de fleurs recoltees, ou petales de celles-ci, selon la Revendication 15, presentant une moyenne de 1 8 a 22 petales par tete de fleur. 50 17. Tetes de fleurs recoltees, ou petales de celles-ci, provenant de I'espece Adonis aestivalis presentant au moins 100 u.g d'astaxanthin par tete de fleur.

18. Tetes de fleurs recoltees, ou petales de celles-ci, 55 selon la Revendication 1 7, presentant au moins 1 50 u.g d'astaxanthin par tete de fleur.