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 DO O) LO 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 Printed by Jouve, 75001 PARIS (FR) 1 EP 0 541 595 B1 2 Description The invention particularly provides a process for ob- taining 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. 2 3 EP 0 541 595 B1 4 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-contain- ing 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.