Vittorio Maria Moretti, Tiziana Mentasti, Federica Bellagamba, Umberto Luzzana, Fabio Caprino, Giovanni Turchini, Ivan Giani, Franco Valfrè

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1 Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchuss mykiss) as a tool to distinguish dietary pigmentation source Vittorio Maria Moretti, Tiziana Mentasti, Federica Bellagamba, Umberto Luzzana, Fabio Caprino, Giovanni Turchini, Ivan Giani, Franco Valfrè To cite this version: Vittorio Maria Moretti, Tiziana Mentasti, Federica Bellagamba, Umberto Luzzana, Fabio Caprino, et al.. Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchuss mykiss) as a tool to distinguish dietary pigmentation source. Food Additives and Contaminants, 00, (), pp.0-0. <0.00/00000>. <hal-00> HAL Id: hal-00 Submitted on Mar 0 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 Food Additives and Contaminants Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchuss mykiss) as a tool to distinguish dietary pigmentation source Journal: Food Additives and Contaminants Manuscript ID: TFAC-00-.R Manuscript Type: Original Research Paper Date Submitted by the Author: 0-Jun-00 Complete List of Authors: Moretti, Vittorio; University of Milan, VSA Mentasti, Tiziana; University of Milan, VSA Bellagamba, Federica; University of Milan, VSA Luzzana, Umberto; ASA - Naturalleva Caprino, Fabio; University of Milan, VSA Turchini, Giovanni; School of Ecology and Environment, Deakin University Giani, Ivan; University of Milan, VSA Valfrè, Franco; University of Milan, VSA Methods/Techniques: Chromatography - HPLC, Traceability Additives/Contaminants: Additives general, Colours, Feeding, Nutrition Food Types: Animal, Animal feed, Feeding, Fish

3 Page of Food Additives and Contaminants Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchus mykiss) as a tool to distinguish the dietary pigmentation source Vittorio Maria Moretti *, Tiziana Mentasti, Federica Bellagamba, Umberto Luzzana, Fabio Caprino, Giovanni Mario Turchini, Ivan Giani and Franco Valfrè Department of Veterinary Science and Technology for Food Safety, University of Milan. Via Trentacoste, 0 - Milan, Italy A.S.A. S.p.a. Naturalleva Viale del Lavoro,, 0 San Martino Buon Albergo (VR), Italy School of Ecology & Environment, Deakin University PO Box, Warrnambool, Victoria 0, Australia * To whom correspondence should be addressed: Phone: Fax: vittorio.moretti@unimi.it

4 Food Additives and Contaminants Page of Abstract The presence of carotenoids in animal tissue reflects their sources along the food chain. Astaxanthin, the main carotenoid used for salmonid pigmentation, is usually included in the feed as a synthetic product. However, other dietary sources of astaxanthin such as shrimp or krill wastes, algae meal or yeasts are also available on the market. Astaxanthin possesses two identical asymmetric atoms at C- and C- making possible three optical isomers with all-trans configuration of the chain: S, S ; R, S ; R, R. The distribution of the isomers in natural astaxanthin differs from that of the synthetic product. This latter is a racemic mixture, with a typical ratio of :: (S, S : R, S : R, R), while astaxanthin from natural sources has a variable distribution of the isomers deriving from the different biological organism that synthesised it. The high performance liquid chromatographic (HPLC) analysis of all-trans isomers of astaxanthin was performed in different pigment sources, such as red yeast Phaffia rhodozyma, alga meal Haematococcus pluvialis, krill meal and oil, and shrimp meal. With the aim to investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed for 0 days diets containing astaxanthin from synthetic source, Haematococcus pluvialis algae meal and Phaffia rhodozyma red yeast. Moreover, the distribution of optical isomers of astaxanthin in trout purchased on the Italian market was investigated. A characteristic distribution of astaxanthin stereoisomers was detected for each pigment sources and such distribution was reproduced in the flesh of trout fed with that source. Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources showed no significant differences. Similarly, different sources of pigment (natural or synthetic) produced colour values of fresh fillet with no relevant or significant differences. The coefficient of distance computed amongst the feed ingredient and the trout fillet astaxanthin stereoisomers was a useful tool to identify the origin of the pigment used on farm.

5 Page of Food Additives and Contaminants Keywords:- Introduction Carotenoids are widely distributed in nature and present in many plants, algae, micro-organisms and animals. Although carotenoids are largely diffused in the plant and animal kingdoms, they are synthesized de novo only in higher plants and protists (Goodwin, ). Consequently, the traceability of carotenoids in animal reflects the presence of their sources along with the food chain. Animal carotenoids mainly result from metabolic transformation (oxidation and/or reduction) of those present in the food chain. Aquatic animals constitute a considerable rich source of carotenoids, which are in many cases responsible of organoleptic characteristics considered by the consumer (bright colours in lobster, shrimp, salmon, fish eggs) (Shahidi et al., Matsumo 00). Moreover, among the large number of pigments present in living organisms (i.e. chlorophylls, anthocyanins and porphyrins), carotenoids play an important role in protecting cells against photosensitised oxidation (Palozza and Krinsky ). In farmed salmonid, astaxanthin is mainly used for pigmentation and it is usually included in the feed as a synthetic product. However, other natural dietary sources of astaxanthin such as shrimp or krill wastes, algae meal or yeasts are also available on the market. All-trans astaxanthin may exists as three stereoisomers: two enantiomers (R, R and S, S) and a meso form (R, S; S, R). The distribution of the all-trans stereoisomers in natural astaxanthin differs from that of the synthetic product. This latter is a racemic mixture of three isomers, with a typical ratio of :: (S, S : R, S : R, R), while astaxanthin from natural sources has a

6 Food Additives and Contaminants Page of variable distribution of the isomers deriving from the different biological organism that synthesised it. Crustaceans belong to the class of marine animals that can oxidise carotenoids at the, and, position. Particularly, shrimp (Penaeus spp.) are able to convert by oxidative transformation dietary carotenoids such as ß-carotene, zeaxanthin and canthaxanthin into astaxanthin and they are also able to absorb astaxanthin (Matsumo, Schiedt et al., Sachindra et al. 00). Similarly, in krill (Euphausia spp.) astaxanthin and its esters represent the major carotenoid component. A metabolic transformation to astaxanthin stereoisomers from other carotenoid sources is hypothesized as well in zooplanktonic crustaceans (Takaichi et al. 00). The red yeast Phaffia rhodozyma produces and accumulates carotenoids in lipid droplets into the cytoplasmatic membranes. Andrewes et al., () reported that free astaxanthin was the most abundant carotenoid in Phaffia with -% of the total pigment mixture. Other carotenoids such as ß-carotene (-.% of total carotenoids), echinenone (-% of total carotenoids), - hydroxyechinenone (-.% of total carotenoids) and phoenicoxanthin (-% of total carotenoids) were also isolated red yeast culture. The predominant optical isomer of Phaffia rhodozyma unesterified astaxanthin is the R, R form. The Haematococcus pluvialis microalgae mainly contains astaxanthin monoesters linked to short chain fatty acids. The astaxanthin pool of encysted Haematococcus is approximately 0% monoesters, % diesters and % free (Lorenz and Cysewski 000). All of free astaxanthin and its mono and di- esters in Haematococcus pluvialis have predominantly the S, S form. Since commercially grown Haematococcus pluvialis can accumulate >0 g astaxanthin of dry matter,

7 Page of Food Additives and Contaminants the alga meal represents a good source of natural astaxanthin that found an increasing use in nutrition of farmed fish (Olaizola and Huntly 00). The distribution of astaxanthin stereoisomers has been investigated in different tissues of crustaceans (Renstrøm et al, Coral-Hinostroza and Bjerkeng 00), trout (Oncorhynchus mykiss) (Storebakken and No, Bjerkeng et al., Østerlie et al.,) and salmon (Oncorhynchus spp and Salmo salar) (Arai et al., Turujman et al. ). Astaxanthin deposition in white muscle of salmonid reflects the optical isomers ratio of dietary astaxanthin (Foss et al., Storebakken and No,, Storebakken at al. 00). On the contrary, the distribution of astaxanthin stereoisomers in skin and posterior kidney of rainbow trout has been observed to be different from that of dietary source, indicating some selectivity in the tissue metabolism of the optical isomers of astaxanthin. In particular Østerlie et al. () found in rainbow trout fed synthetic astaxanthin an higher percentage of S, S isomer and a lower percentage of R, R isomer in kidney and skin, if compared to the percentage of optical isomers in the diet, suggesting a degree of epimerization of (S, S)-astaxanthin in these tissues. High performance liquid chromatographic (HPLC) analysis of all-trans isomers of astaxanthin has been described in recent years by several authors (Vecchi and Müller, Maoka et al.,, Turujman, Turujman et al., Østerlie et al. ). The separation of optical isomers could be achieved on an achiral column after derivatization with a suitable derivatizing reagent in order to obtain the diasteroisomers, that could be easily resolved on an achiral phase column. The derivatizing reagent more used for this purpose in literature is camphenic acid chloride (Vecchi and Müller, Bowen et al. 00). Another approach is to perform direct HPLC analysis of underivatized astaxanthin on a chiral column. This latter method permits to

8 Food Additives and Contaminants Page of avoid various problems encountered during the derivatization step and to reduce the analysis time to less then minutes (Turujman, Turujman et al. ). This study was designed in order to investigate the ratios of optical isomers of astaxanthin from different synthetic and natural sources used for rainbow trout pigmentation. Different ingredients such as yeasts of the genus Phaffia (Phaffia rhodozyma), Haematococcus pluvialis algae meal, Antarctic krill meal, krill oil and shrimp meal were analysed. Afterwards, with the aim to investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed for 0 days diets containing 0 mg kg - of astaxanthin from synthetic source, Haematococcus pluvialis algae meal and Phaffia rhodozyma yeast. Moreover, in order to investigate the reliability of the optical isomers distribution to distinguish the type of pigmentation of commercially farmed trout available on the Italian market, nine specimens of pigmented rainbow trout farmed in different aquaculture plants of Northern Italy were also purchased in local retailers and analysed. Materials and methods Chemicals and reagents Standard astaxanthin (, -dihydroxy-ß,ß-carotene-, dione) was a gift from Hoffmann-La- Roche Inc. (Basel, Switzerland). Hexane, tetrahydrofuran (THF), methylene chloride, -propanol, acetonitrile, acetone, petroleum ether were LC grade and from Merck (Darmstadt, Germany); dimethyl sulfoxide (DMSO), triethylamine, sodium sulphate decahydrate and sodium sulphate anhydrous supplied by Sigma-Aldrich (St. Louis, MO, USA). Cholesterol esterase from Pseudomonas fluorescens

9 Page of Food Additives and Contaminants used for hydrolysis of carotenoids esters was purchased from Sigma (St. Louis, MO, USA). Sep- Pak silica gel cartridges were from Waters (Milford, MA, USA). Feed ingredients Unesterified astaxanthin (Carophyll pink) and canthaxanthin (Carophyll red) were from Hoffmann-La-Roche Inc. (Basel, Switzerland), microalgae meal from Haematococcus pluvialis (Naturose ) purchased from Cyanotech Corporation (Kailua-Koma, HI, USA) and red yeast meal (Astaxin ) (Phaffia rhodozyma) was from Igene Biotecnology Inc. (Columbia, MD, USA). Antarctic krill meal, krill oil and shrimp meal were supplied by Tepual SA (Santiago, Chile). Fish and experimental conditions Three outdoor raceways (L=0 X W= X H=.) were stocked each with 00 kg rainbow trout (average weight 0 g) and were reared under normal production scale for 0 days. During the trial, oxygen levels were always over 0% saturation and water temperature was ± C. Three pigmented diets were used in this study. Diet contained Carophyll Pink (CP), diet contained Haematococcus pluvialis algae meal (HP) and diet contained Phaffia rhodozyma red yeast (PR). The level of inclusion in all three diets was 0 mg kg - astaxanthin (free or esterified). The three groups of fish were fed with commercial extruded feed (% protein, % fat) coated with different synthetic source. The feed ratio was set at % of the biomass per day. No mortalities occurred during the experiment. At the end of the trial, ten fish per group were taken and transported to the laboratory for colour measurements and astaxanthin analysis. Parallelly, nine specimens of pigmented rainbow trout from unknown origin were purchased from local retailers and analysed.

10 Food Additives and Contaminants Page of Astaxanthin extraction from feed ingredients Carotenoids from algae and yeast pigments were extracted by suspending mg of sample in ml of dimethyl sulfoxyde (DMSO) and incubating in a thermostatic shaking bath at 0 C for 0 minutes. At the end, the sample was centrifuged at 00 rpm for 0 minutes and the supernatant collected. The obtained pellet was homogenized for min using an UltraTurrax (Janke & Kunkel, IKA, Staufen, Germany) in ml of acetone, then centrifuged at 00 rpm for minutes. Acetone extraction was repeated until the organic phase resulted colourless. Similarly, acetone extraction was used with Antarctic krill meal, krill oil, shrimp meal and with synthetic astaxanthin source. To avoid oxidation and isomerisation of astaxanthin, all manipulations were performed in diffuse light and conditioned room (0 C), under acid-free conditions and using pure peroxide-free solvents (Liaaen-Jensen ). Enzymatic hydrolysis of Haematococcus pluvialis extract ThreemL of Haematoccoccus acetone extract was added to ml of Tris HCl (0.0 M, ph.00) were added and put in a thermostatic bath at C for minutes. Then, 00 µl of cholesterol esterase solution (0 unit/ml) were added and incubated in a water bath at C for 0 minutes with gently shaking. After the incubation.0 g of sodium sulphate decahydrate and ml of petroleum ether were added and mixed vigorously by vortex. The upper petroleum ether phase was collected in a clean tube and the procedure repeated two times to make the organic phase clear. Petroleum ether phase containing astaxanthin was added with sodium sulphate anhydrous mixing by vortex for 0 seconds and the ethereal phase transferred in a clean tube, then dried under a stream of nitrogen and reconstituted in 00 µl of hexane.

11 Page of Food Additives and Contaminants Colour measurements in fillet The fish were decapitated, gutted, filleted and the right fillet used for colour analysis. Colour measurements of the flesh samples were performed in the following sites: posterior to the head (neck), at the center below the dorsal fin (center) and anterior to the caudal fin (tail). Colour was measured using a Minolta Chroma Meter II Reflectance instrument (Minolta Camera Co. Ltd., Osaka, Japan). Results were recorded as a*, b*, L* values where a* describes redness, b* yellowness and L* lightness. Chroma values (brightness, C*) were calculated according to the following equation: C* = a * b * +. Two measurements were taken on each site and the measuring head was rotated 0 between each measurement. The left fillet was packed under vacuum and stored at -0 C until astaxanthin analysis, which was performed within a few days. Astaxanthin extraction from fish fillets Astaxanthin was extracted from flesh according to the method of Turujman et al. (). Briefly, 0 g of tissue were sampled at the centre of the fillet and homogenized using an Ultra Turrax for min with 0x ml of hexane, to remove most of lipids. The homogenate was centrifuged at 00 rpm for 0 minutes and hexane phase was discharged. Astaxanthin was extracted from the pellet resulted after hexane extraction with 0x ml of acetone, following the procedure mentioned above. The acetone phase was evaporated with a rotary evaporator (Rotavapor R 0, Büchi, Switzerland) and the aqueous residue (approximately ml) reconstituted in 0 ml of methylene chloride and vigorous swirled to dissolve astaxanthin. The residual water was removed with a separatory funnel and the organic phase was dried over approximately g of anhydrous sodium sulfate. The methylene chloride phase containing astaxanthin was transferred in a clean tube. The amount of astaxanthin extracted was determined

12 Food Additives and Contaminants Page 0 of recording the volume and measuring the absorbance of the methylene chloride extract a nm using a Jasco model V-0 spectrophotometer (Ishikawa-cho, Japan). HPLC analysis of astaxanthin The astaxanthin was purified by loading the methylene chloride phase onto a silica gel Sep-Pak cartridge (Waters, Milford, MA, USA) pretreated with hexane. Astaxanthin was eluted from the cartridge with ml of chloroform and the eluate dried under a stream of nitrogen. Dry residue was dissolved in 00 µl of hexane and 0 µl were injected into the HPLC system. The HPLC system consisted of a Jasco model 0-PU pump (Ishikawa-cho, Japan), a Jasco model LG-0-0 gradient unit, a Jasco model AS-0 autosampler and a Waters model Photodiode Array Detector (Waters, Milford, MA, USA). Chromatograms were recorded and integrated on a Millennium Chromatography Software (Waters, Milford, MA, USA). Analyses were performed with a Pirkle L-leucine chiral column (Regis Technologies, Morton Grove, IL, USA). Mobile phase was hexane-tetrahydrofuran-triethylamine-acetonitrile (::: v/v) at a flow rate of. ml min. Isocratic condition and ambient temperature were used, and the monitoring wavelength was nm. In these conditions the retention times of the S, S, meso and R, R forms were approximately 0.,. and. minutes, respectively. Each astaxanthin extract was analyzed in duplicate. Statistical analysis Data are reported as mean value ± standard error of the mean. Homogeneity of variance was confirmed and comparison between means was by one-way ANOVA. Significance was accepted at probabilities of 0.0 or less. 0

13 Page of Food Additives and Contaminants For comparison of whole astaxanthin stereoisomers profile of trout fillet and the pigment ingredients of the diet, the coefficient of distance D (McIntire et al. ) between reference and test samples (pigment source and trout fillet, respectively) was computed. This coefficient was originally developed to estimate the similitude of aquatic animals on the base of their fatty acids composition. Therefore, the smaller the value of the coefficient of distance D the higher the probability that trout have been fed with a diet containing that specific pigment source. For comparison of the whole astaxanthin stereoisomers profile of reference (feed ingredient) and test (trout fillet), the coefficient of distance D (McIntire et al., ) was computed using the following equation: D jh = n i= ( ) Pij Pih / where, D jh is the degree of difference (coefficient of distance) between samples j ( reference diet) and h ( test trout fillet), P ij and P ih are percentage of astaxanthin stereoisomers i in sample j and h, for each i stereoisomers. All the statistical analysis were performed by SPSS.0 (SPSS Inc. Chicago, Illinois). Results and Discussion In Figure the chromatograms of a standard solution ( µg ml - ) of syntetic astaxanthin and of the extracts obtained from different sources are presented. The three peaks corresponding to S, S isomer, meso form and R, R isomer were eluted at 0.,. and. minutes, respectively.

14 Food Additives and Contaminants Page of Percentage distribution of stereoisomers was calculated from chromatograms of different pigment extracts by means of relative peak area measurements. The results are presented in Figure. A characteristic distribution of astaxanthin optical isomers was detected for each pigment sources and such distribution was reproduced in trout fed with that source. Haematococcus pluvialis algae meal contained.% of the S, S enantiomer, 0.% of the meso form and.% of the R, R enantiomer, while the red yeast Phaffia rhodozyma contained.% of the meso form and.% of the R, R enantiomer. The values of optical isomers in krill meal and krill oil were similar. In fact,.% of S, S enantiomer,.% of the meso form and 0.% of R, R enantiomer were found in the krill meal whilst.% of S, S enantiomer,.% of the meso form and 0.% of R, R enantiomer were found in krill oil. On the other hand, shrimps meal contained.% of S, S enantiomer,.% of the meso form and.% of R, R enantiomer. These results were consistent with literature values. In particular, Foss et al. () analysed the content of astaxanthin stereoisomers in several species of crustaceans and demonstated that in Euphausia superba (krill) the R, R isomer predominated, whereas in Thysanoessa inermis and in Calanun finmarchicus, the S, S isomer was dominant. These latter two small crustaceans are important ingredient of diets of wilds salmonids, and their isomeric composition has been demonstrated similar to the isomeric composition encountered in wild salmon, in which the S-form predominated (Turujman et al. ). Astaxanthin isomer ratios in the flesh of fish fed different carotenoid sources is reported in Table. Dietary treatment influenced significantly the distribution of the optical isomers in the fillets, and the average values of the optical isomers in the flesh reflected the isomeric ratio of the dietary astaxanthin source. The astaxanthin isomers distribution in microalgae and in the corresponding trout flesh were similar and ranged from 0.0% to.%, from.% to 0.% and from.% to.% for the S, S isomer, meso form and R, R isomer, respectively. In the

15 Page of Food Additives and Contaminants red yeast and in the corresponding trout flesh the R, R isomer predominated, ranging from.% to.%, while the meso form and the S, S isomer ranged from.% to 0.% and from 0% to.%, respectively. In the flesh of trout fed synthetic astaxanthin, the averages of the distribution of optical isomer were.%,.% and.0% for the S, S isomer, meso form and R, R isomer, respectively. Also in this case the distribution resembles that of the diet and these results were in ageement with those of Østerlie et al. (), who found in rainbow trout fed syntetic astaxanthin a distribution of isomers in muscle of.%,.0% and.% for S, S, meso and R, R isomers, respectively. Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources are presented in table. No significant differences were registered in different parts of the flesh. Similarly, different sources of pigment (natural or synthetic), used with the three different diets, produced colour values of fresh fillet (table ) with no significant differences, although there was a tendency for the CP diet to give an higher value of redness (a*) and brightness (C*) when compared to HP diet. This confirms the better pigmenting efficacy of free astaxanthin in comparison with esterified astaxanthin from microalgae. (White et al. 00). Among the specimens purchased on the market (table ), two samples showed the algal astaxhantin isomeric profile (trout and ), and three (trout,, ) were found to contain a different pigment source that on the basis of its retention time and UV/VIS absorption spectra was reasonably recognized as canthaxanthin. Three fish samples showed to contain only synthetic astaxanthin (trout,, ) and one (trout ) both synthetic carotenoids: astaxanthin and canthaxantin. The coefficient of distance (table ) computed amongst the feed ingredient and the trout fillet astaxanthin stereoisomers is a simple geometrical tool which can be useful to tentatively identify the origin of the pigment used on farm.

16 Food Additives and Contaminants Page of The smallest D values registered in trout fed the Carophyll pink and trout fed microalgae were. and. when computed using the synthetic astaxanthin and the microalgae as reference, respectively. The other D values, computed using the astaxanthin stereoisomers composition of the other analysed ingredients, were considerably higher. In trout fed the read yeast, D value (.) was similar to those calculated for krill meal (.) and krill oil (.). This information suggests that the coefficient of distance can be used as a general tool to explain pigment source only amongst some sources and it is particularly useful to distinguish amongst synthetic and natural sources whilst is not sensitive enough to distinguish amongst different natural astaxanthin sources, or amongst mixtures of synthetic and natural sources. Using the coefficient of distance to estimate the potential origin of the astaxanthin found in the market purchased trout, it is possible to point out that samples trout, trout and trout were with high probability fed with a diet containing a synthetic source of astaxanthin. At the best of our knowledge, this is the first time the coefficient of distance was used as a tool for the identification of the astaxanthin origin in salmonids and further information, particularly as regard the inter- and intra- population variability and the dose-response effects are requested. References Andrewes, A.G., Phaff, H.J.,and Starr, M.P.,, Carotenoids of Phaffia rhodozyma a red pigmented fermenting yeast. Phytochemistry,, Arai, S., Mori, T., Miki, W., Yamaguchi, K., Konosu, S., Satake, M., and Fujita, T.,, Pigmentation of juvenile coho salmon with carotenoid oil extracted from Antarctic krill. Aquaculture,, -.

17 Page of Food Additives and Contaminants Bjerkeng, B., Følling, M., Lagocki, S.; Storebakken, T., Olli, J.J., Alsted, N.,, Bioavailability of all-e-astaxanthin and Z-isomers of astaxanthin in rainbow trout (Oncorhynchus mykiss). Aquaculture,, -. Bowen, J., Soutar, C., Serwata, R.D., Lagocki, S., White, D.A., Davies, S.J., and Young, A.J., 00, Utilization of (S, S)-astaxanthin acyl esters in pigmentation of rainbow trout (Oncorhynchus mykiss). Aquaculture Nutrition,, -. Coral-Hinostroza, G.N., and Bjerkeng, B., 00, Astaxanthin from the red crab langostilla (Pleuroncodes planipes): optical R/S isomers and fatty acid moieties of astaxanthin esters. Comparative Biochemistry and Physiology Part B,, -. Foss, P., Storebakken, T., Schiedt, K., Liaaen-Jensen, S., Austreng, E., and Streiff, K.,, Carotenoids in diets for salmonids. I. Pigmentation of rainbow trout with the individual optical isomers of astaxanthin in comparison with canthaxanthin. Aquaculture,,. Foss, P., Renstrøm, B., Liaaen-Jensen, S.,, Natural occurence of enantiomeric and meso astaxanthin:. Crustaceans including zooplankton. Comparative Biochemistry and Physiology B,, -. Goodwin, T.W.,, Distribution of carotenoids. Methods in Enzymology, Carotenoids, Part A, edited by L. Packer (Academic Press, Inc), vol., pp. -. Liaaen-Jensen S.,, III. Isolations, reactions. Carotenoids, edited by O. Isler (Basel: Birkhäuser Verlag), pp. -. Lorenz, R.T., and Cysewsky, G.R., 000, Commercial potential for Heamatococcus microalgae as natural source of astaxanthin. Trends in Biotecnology,, 0- Maoka, T., Komori, T., and Matsuno,T.,, Direct diastereomeric resolution of carotenoids: I. -hydroxy--ocyβ-end group. Journal of Chromatography,, -.

18 Food Additives and Contaminants Page of Matsuno, T.,, Structure and characterization of carotenoids from various habitats and natural sources. Methods in Enzymology, Carotenoids, Part A, edited by L. Packer (Academic Press, Inc), vol., pp. -. Matsumo, T., 00, Aquatic animal carotenoids. Fisheries Science,, -. McIntire, C.D., Tinsley, I.J. and Lowry, R.R.,, Fatty acid in lotic periphyton: another measure of community structure. Journal of Phycology,, -. Olaizola, M.,and Huntley, M.E.,00, Recent advances in commercial production of astaxanthin from microalgae. Biomaterials and Bioprocessing, edited by Fingerman M., Nagabhushanam R. (Science Publishers). Østerlie, M., Bjerkeng, B., and Liaaen-Jensen, S.,, Accumulation of astaxanthin all-e, Z and Z geometrical isomers and and ' RS optical isomers in rainbow trout (Oncorhynchus mykiss) is selective. Journal of Nutrition,, -. Palozza, P., and Krinsky, N.I.,, Antioxidant effects of carotenoids in vivo and in vitro: an overview. Methods in Enzymology, Carotenoids, Part A, edited by L. Packer (Academic Press, Inc), vol., pp. -. Renstrøm, B., Borch, G., and Liaaen-Jensen, S.,, Natural occurrence of enantiomeric and meso-astaxanthin. Ex shrimps (Pandalus borealis). Comparative Biochemistry and Physiology Part B,, -. Sachindra, N.M., Bhaskar, N., and Mahendrakar, N.S., 00, Carotenoids in different body components of Indian shrimps. Journal of the Science of Food and Agriculture,, -. Schiedt, K., Bischof, S., and Glinz, E.,, Metabolism of carotenoids and in vivo racemization of (S, S)-astaxanthin in the crustacean Penaeus. Methods in Enzymology, Carotenoids, Part B, edited by L. Packer (Academic Press, Inc), vol., pp. -.

19 Page of Food Additives and Contaminants Shahidi, F., Metusalach, and Brown, J., A., Carotenoid pigments in seafoods and aquaculture,, Critical Reviews in Food Science,, -. Storebakken, T., Foss, P., Scheidt, K., Austreng, E., Jensen, S.-L. and, Manz, U.,, Carotenoids in diets for salmonids IV. Pigmentation of Atlantic salmon with astaxanthin, astaxanthin dipalmitate and canthaxanthin. Aquaculture,,. Storebakken, T and No, H.K.,, Pigmentation of rainbow trout. Aquaculture, 00, 0. Storebakken, T., Sørensen, M., Bjerkeng, B., and Hiu, S., 00, Utilization of astaxanthin from yeast, Xanthophyllomyces dendrorhous, in rainbow trout, Oncorhynchus mykiss: effect of enzymatic cell wall disruption and feed extrusion temperature. Aquaculture, -. Takaichi, S, Matsui, K., Nakamura, M., Muramatsu, M., and Hanada, S., 00, Fatty acids of astaxanthin esters in krill determined by mild mass spectrometry. Comparative Biochemistry and Physiology Part B,, -. Turujman, S.A.,, Rapid direct resolution of the stereoisomers of all-trans astaxanthin on a Pirkle covalent L-leucine column. Journal of Chromatographt,, -. Turujman, S.A., Wamer, W.G., Wie, R.R, and Albert, R.H.,, Rapid liquid chromatographic method to distinguish wild salmon from aquacultures salmon fed synthetic astaxanthin. Journal of AOAC International, 0, -. Vecchi, M., and Müller, R.K.,, Separation of (S,'S)-, (R,'R)- and (S,'R)-astaxanthin via (-)-camphanic acid esters. Journal of High Resolution Chromatography,, -. White, D.A., Moody, A.J., Serwata, R.D., Bowen, J., Soutar, C., Young, A.J.,and Davies, S.J., 00, The degree of carotenoid esterification influences the absorption of astaxanthin in rainbow trout, Oncorhynchus mykiss (Walbaum), Aquaculture Nutrition,, -.

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21 Page of Food Additives and Contaminants Figure : Chromatograms of stereoisomers composition of all-trans astaxanthin of: (a) a standard solution of syntetic astaxanthin ( µg ml - ); (b) an Haematococcus pluvialis extract; (c) a Phaffia rhodozyma extract. HPLC conditions: Pirkle L-leucine chiral column; mobile phase hexane-tetrahydrofuran-triethylamine-acetonitrile (::: v/v); flow rate of ml min ; monitoring wavelength nm. (a) AU (b) A U (c) AU 0,0 0,0 0,00 0, 0, 0, 0 0,0 0,0 0,0 0,0 0,0 0 0, 0,0 0,0 0,00 Astaxantina standard g/ml 0, (S, S, (meso), (R, R),00,00,00,00 0,00,00,00,00,00 minutes 0, 0 ( S, S ),0 0,0 0,0 0,0 0 0,0,0,0,0,0 minutes Crom at. Astaxin,00,00,00,00 0,00,00,00,00,00,0 (m eso), (m e s, ( R,,0 (R, R)

22 Food Additives and Contaminants Page 0 of Figure. Percentage distribution of astaxanthin stereoisomers in pigment sources % S, S R, S R, R Astaxanthin Microalgae Red yeast Krill meal Krill oil Shrimp meal 0

23 Page of Food Additives and Contaminants Table. Percentage distribution of astaxanthin stereoisomers in fillet of rainbow trout fed with different pigment sources (means±s.e.) Samples (n=0) S, S enantiomer (%) R, S meso (%) R, R enantiomer (%) Carophyll pink fed trout.±..±..0±.0 Microalgae (Haematococcus pluvialis) fed trout Red yeast (Phaffia rhodozyma) fed trout 0.0±.0.±..±.0.±. 0.±..±.

24 Food Additives and Contaminants Page of Table. Colour values measured in three different part of fillet of rainbow trout fed with different pigment sources (means±s.d., n=0) Color value Neck Center Tail P a*.±0..±0..±0. 0. b*.±..±..±0. 0. L*.±..±..±.0 0. C*.±..±..0±0. 0. Significance accepted at probabilities of 0.0 or less

25 Page of Food Additives and Contaminants Table. Colour values measured in fillet of rainbow trout fed with different pigment sources (means±s.d., n=0) Color value Synthetic astaxanthin Phaffia rhodozyma Haematococcus pluvialis a*.±0..±0..±0. 0. b*.±.0.±.0.±.0 0. L*.±..±.0.±.0 0. C*.±..±..±. 0. Significance accepted at probabilities of 0.0 or less P

26 Food Additives and Contaminants Page of Table. Percentage distribution of astaxanthin stereoisomers in flesh of rainbow trout purchased on the market. Samples S, S enantiomer (%) R, S meso (%) R, R enantiomer (%) Trout no Trout nd nd nd yes Trout... no Trout... no Trout nd nd nd yes Trout... yes Trout nd nd nd yes Trout... no Trout... no nd = not detected Presence canthaxanthin of

27 Page of Food Additives and Contaminants Table. The coefficient of distance (D value) of the astaxanthin stereoisomers amongst known and unknown trout samples and the analysed astaxanthin sources Trout fed Carophyll pink Trout fed microalagae (H.plivualis) Trout fed red yeast (X. dendrorhous) Synthetic astaxanthin Microalage (H.pluvialis) Red yeast (X. dendrorhous) Antartic Krill meal Krill oil Shrimps meal Trout Trout Trout Trout Trout Trout In bold font, the actual coefficient of distance amongst pigment source and trout fillet of the known samples; in italics the possible coefficient of distance amongst pigment source and trout fillet of the unknown samples.