Food Additives and Contaminants, November 2006; 23(11): 1056–1063

Determination of astaxanthin stereoisomers and colour attributes

in flesh of rainbow trout (Oncorhynchus mykiss) as a tool to
distinguish the dietary pigmentation source

V. M. MORETTI1, T. MENTASTI1, F. BELLAGAMBA1, U. LUZZANA2, F. CAPRINO1,

G. M. TURCHINI3, I. GIANI1, & F. VALFRÈ1
1
Department of Veterinary Science and Technology for Food Safety, University of Milan, Via Trentacoste 2, I-20134
Milan, Italy, 2A.S.A. S.p.a. — Naturalleva, Viale del Lavoro, 45, I-37036 San Martino Buon Albergo (VR), Italy,
and 3School of Ecology & Environment, Deakin University, PO Box 423, Warrnambool, Victoria 3280, Australia

(Received 30 November 2005; revised 1 June 2006; accepted 2 June 2006)

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-3 and C-30 making possible three optical isomers with all-trans configuration of the
chain: 3S,30 S, 3R,30 S, and 3R,30 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 1:2:1 (3S,30 S:3R,30 S:3R,30 R), while astaxanthin from
natural sources has a variable distribution of the isomers deriving from the different biological organism that synthesized 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 60 days diets containing astaxanthin from synthetic source, H. pluvialis algae meal and
P. 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.

Keywords: Carotenoids, rainbow trout, astaxanthin stereoisomers, flesh colour

Introduction result from metabolic transformation (oxidation and/

or reduction) of those present in the food chain.
Carotenoids are widely distributed in nature and Aquatic animals constitute a considerable rich
present in many plants, algae, micro-organisms, and source of carotenoids, which are in many cases
animals. Although carotenoids are largely diffused in responsible of organoleptic characteristics consid-
the plant and animal kingdoms, they are synthesized ered by the consumer (bright colours in lobster,
de novo only in higher plants and protists (Goodwin shrimp, salmon, fish eggs) (Shahidi et al. 1998;
1992). Consequently, the traceability of carotenoids Matsumo 2001). Moreover, among the large
in animals reflects the presence of their sources along number of pigments present in living organisms
with the food chain. Animal carotenoids mainly (i.e. chlorophylls, anthocyanins and porphyrins),

Correspondence: V. M. Moretti. E-mail: [email protected]

ISSN 0265–203X print/ISSN 1464–5122 online ß 2006 Taylor & Francis
DOI: 10.1080/02652030600838399
 Astaxanthin stereoisomers and colour attributes in O. mykiss flesh 1057

carotenoids play an important role in protecting The distribution of astaxanthin stereoisomers

cells against photosensitized oxidation (Palozza and has been investigated in different tissues of crusta-
Krinsky 1992). ceans (Renstrøm et al. 1981; Coral-Hinostroza
In farmed salmonid, astaxanthin is mainly used and Bjerkeng 2002), trout (Oncorhynchus mykiss)
for pigmentation and it is usually included in the (Storebakken and No 1992; Bjerkeng et al. 1997;
feed as a synthetic product. However, other natural Østerlie et al. 1999) and salmon (Oncorhynchus spp.
dietary sources of astaxanthin such as shrimp or and Salmo salar) (Arai et al. 1987; Turujman et al.
krill wastes, algae meal or yeasts are also available 1997). Astaxanthin deposition in white muscle of
on the market. All-trans astaxanthin may exists as salmonid reflects the optical isomers ratio of dietary
three stereoisomers: two enantiomers (3R,30 R and astaxanthin (Foss et al. 1984; Storebakken and No
3S,30 S) and a meso form (3R,30 S; 3S,30 R). The 1992; Storebakken et al. 2004). On the contrary,
distribution of the all-trans stereoisomers in the distribution of astaxanthin stereoisomers in the
natural astaxanthin differs from that of the skin and posterior kidney of rainbow trout has been
synthetic product. This latter is a racemic mixture observed to be different from that of dietary source,
of three isomers, with a typical ratio of 1:2:1 indicating some selectivity in the tissue metabolism
(3S,30 S:3R,30 S:3R,30 R), while astaxanthin from of the optical isomers of astaxanthin. In particular,
natural sources has a variable distribution of the Østerlie et al. (1999) found in rainbow trout fed
isomers deriving from the different biological synthetic astaxanthin a higher percentage of 3S,30 S
organism that synthesized it. isomer and a lower percentage of 3R,30 R isomer
Crustaceans belong to the class of marine animals in kidney and skin if compared with the percentage
that can oxidize carotenoids at the 3,30 and 4,40 of optical isomers in the diet, suggesting a degree
positions. Particularly, shrimp (Penaeus spp.) is able of epimerization of (3S,30 S)-astaxanthin in these
to convert by oxidative transformation dietary tissues.
carotenoids such as ß-carotene, zeaxanthin and High-performance liquid chromatographic
canthaxanthin into astaxanthin, and it is also able (HPLC) analysis of all-trans isomers of astaxanthin
to absorb astaxanthin (Matsumo 1992; Schiedt has been described in recent years by several authors
et al. 1993; Sachindra et al. 2005). Similarly, in (Vecchi and Müller 1979; Maoka et al. 1985;
krill (Euphausia spp.) astaxanthin and its esters Turujman 1993; Turujman et al. 1997; Østerlie
represent the major carotenoid component. A meta- et al. 1999). The separation of optical isomers could
bolic transformation to astaxanthin stereoisomers be achieved on an achiral column after derivatization
from other carotenoid sources is hypothesized as well with a suitable derivatizing reagent in order to
in zooplanktonic crustaceans (Takaichi et al. 2003). obtain the diasteroisomers that could be easily
The red yeast Phaffia rhodozyma produces and resolved on an achiral phase column. The derivatiz-
accumulates carotenoids in lipid droplets into the ing reagent used more for this purpose in the
cytoplasmatic membranes. Andrewes et al. (1976) literature is camphenic acid chloride (Vecchi and
reported that free astaxanthin was the most abun- Müller 1979; Bowen et al. 2002). Another approach
dant carotenoid in Phaffia with 83–87% of the total is to perform direct HPLC analysis of underivatized
pigment mixture. Other carotenoids such as astaxanthin on a chiral column. This latter method
ß-carotene (2.0–2.5% of total carotenoids), echine- permits one to avoid various problems encountered
none (2.0–4.0% of total carotenoids), 3-hydroxye- during the derivatization step and to reduce the
chinenone (3.0–4.5% of total carotenoids) and analysis time to less then 15 min (Turujman 1993;
phoenicoxanthin (5.0–7.0% of total carotenoids) Turujman et al. 1997).
were also isolated red yeast culture. The predomi- The present study was designed in order to
nant optical isomer of P. rhodozyma unesterified investigate the ratios of optical isomers of astax-
astaxanthin is the 3R,30 R form. anthin from different synthetic and natural sources
The Haematococcus pluvialis microalgae mainly used for rainbow trout pigmentation. Different
contains astaxanthin monoesters linked to short- ingredients such as yeasts of the genus Phaffia
chain fatty acids. The astaxanthin pool of encysted (P. rhodozyma), H. pluvialis algae meal, Antarctic
Haematococcus is approximately 70% monoesters, krill meal, krill oil, and shrimp meal were analysed.
25% di-esters and 5% free (Lorenz and Cysewsky Afterwards, with the aim to investigate astaxanthin
2000). All of the free astaxanthin and its mono- and isomer ratios in the flesh of fish fed different
di-esters in H. pluvialis have predominantly the carotenoid sources, three groups of rainbow trout
3S,30 S form. Since commercially grown H. pluvialis were fed for 60 days diets containing 60 mg kg
1 of
can accumulate 430 g astaxanthin of dry matter, the astaxanthin from synthetic source, H. pluvialis algae
alga meal represents a good source of natural meal and P. rhodozyma yeast. Moreover, in order to
astaxanthin that found an increasing use in nutrition investigate the reliability of the optical isomers
of farmed fish (Olaizola and Huntley 2003). distribution to distinguish the type of pigmentation
1058 V. M. Moretti et al.

of commercially farmed trout available on the Italian colour measurements and astaxanthin analysis.
market, nine specimens of pigmented rainbow trout Simultaneuously, nine specimens of pigmented rain-
farmed in different aquaculture plants of Northern bow trout from unknown origin were purchased
Italy were also purchased in local retailers and from local retailers and analysed.
analysed.
Astaxanthin extraction from feed ingredients

Materials and methods Carotenoids from algae and yeast pigments were
extracted by suspending 25 mg of sample in 5 ml of
Chemicals and reagents DMSO and incubating in a thermostatic shaking
Standard astaxanthin (3,30 -dihydroxy-ß,ß-carotene- bath at 50 C for 30 min. At the end the sample was
4,40 dione) was a gift from Hoffmann-La-Roche, Inc. centrifuged at 4000 rpm for 10 min and the super-
(Basle, Switzerland). natant collected. The obtained pellet was homo-
Hexane, tetrahydrofuran (THF), methylene genized for 2 min using an UltraTurrax (Janke and
chloride, 2-propanol, acetonitrile, acetone, Kunkel, IKA, Staufen, Germany) in 5 ml of acetone,
petroleum ether were liquid chromatographic (LC) then centrifuged at 4000 rpm for 15 min. Acetone
grade and from Merck (Darmstadt, Germany); extraction was repeated until the organic phase
dimethyl sulfoxide (DMSO), triethylamine, sodium resulted colourless. Similarly, acetone extraction
sulphate decahydrate, and sodium sulphate was used with Antarctic krill meal, krill oil, shrimp
anhydrous supplied by Sigma-Aldrich (St Louis, meal and with synthetic astaxanthin source. To
MO, USA). Cholesterol esterase from Pseudomonas avoid oxidation and isomerization of astaxanthin,
fluorescens used for hydrolysis of carotenoids esters all manipulations were performed in diffuse light
was purchased from Sigma Chemical Co. and conditioned room (20 C), under acid-free
(St Louis, MO, USA). Sep-Pak silica gel cartridges conditions and using pure peroxide-free solvents
were from Waters (Milford, MA, USA). (Liaaen-Jensen 1971).

Feed ingredients Enzymatic hydrolysis of Haematococcus pluvialis

extract
Unesterified astaxanthin (CarophyllÕ pink) and
canthaxanthin (CarophyllÕ red) were from A total of 3 ml of Haematoccoccus acetone extract was
Hoffmann-La-Roche; microalgae meal from added to 2 ml of Tris HCl (0.05 M, pH 7.00) were
H. pluvialis (NaturoseÕ ) was purchased added and put in a thermostatic bath at 37 C for
from Cyanotech Corporation (Kailua-Koma, HI, 2 min. Then, 100 ml of cholesterol esterase solution
USA); and red yeast meal (AstaxinÕ ) (P. rhodozyma) (50 unit ml
1) were added and incubated in a water
was from Igene Biotecnology, Inc. (Columbia, MD, bath at 37 C for 30 min with gently shaking. After
USA). Antarctic krill meal, krill oil and shrimp meal the incubation, 1.0 g of sodium sulphate decahydrate
were supplied by Tepual SA (Santiago, Chile). and 2 ml of petroleum ether were added and mixed
vigorously by vortex. The upper petroleum ether
Fish and experimental conditions phase was collected in a clean tube and the procedure
repeated two times to make the organic phase clear.
Three outdoor raceways (length ¼ 50  width ¼ Petroleum ether phase containing astaxanthin was
4  height ¼ 1.5) were stocked each with 1900 kg added with sodium sulphate anhydrous mixing by
rainbow trout (average weight 350 g) and were vortex for 30 s and the ethereal phase transferred in a
reared under normal production scale for 60 days. clean tube, then dried under a stream of nitrogen and
During the trial, oxygen levels were always over 80% reconstituted in 200 ml of hexane.
saturation and water temperature was 11  1 C.
Three pigmented diets were used in this study.
Colour measurements in fillet
Diet 1 contained Carophyll pink (CP), diet 2
contained H. pluvialis algae meal (HP) and diet 3 The fish were decapitated, gutted, filleted, and the
contained P. rhodozyma red yeast (PR). The level right fillet used for colour analysis. Colour measure-
of inclusion in all three diets was 60 mg kg
1 ments of the flesh samples were performed in the
astaxanthin (free or esterified). The three groups of following sites: posterior to the head (neck), at the
fish were fed with commercial extruded feed (41% centre below the dorsal fin (centre) and anterior
protein, 26% fat) coated with different synthetic to the caudal fin (tail). Colour was measured using
source. The feed ratio was set at 1% of the biomass a Minolta Chroma Meter II Reflectance instrument
per day. No mortalities occurred during the experi- (Minolta Camera Co. Ltd, Osaka, Japan). Results
ment. At the end of the trial, ten fish per group were recorded as a*, b*, L* values, where a* is
were taken and transported to the laboratory for redness, b* is yellowness and L* is lightness.
 Astaxanthin stereoisomers and colour attributes in O. mykiss flesh 1059

Chroma values (brightness, C*) were calculated hexane–tetrahydrofuran–triethylamine–acetonitrile

according to the following equation: (77:19:2:2 v/v) at a flow rate of 1.5 ml min
1.
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Isocratic condition and ambient temperature were
C ? ¼ a?2 þ b?2 : used, and the monitoring wavelength was 474 nm.
Two measurements were taken on each site and In these conditions the retention times of the 3S,30 S,
the measuring head was rotated 90 between each meso and 3R,30 R forms were approximately 10.5,
measurement. The left fillet was packed under 11.5 and 12.5 min, respectively. Each astaxanthin
vacuum and stored at
20 C until astaxanthin extract was analysed in duplicate.
analysis, which was performed within a few days.
Statistical analysis
Astaxanthin extraction from fish fillets Data are reported as mean  standard error of
the mean (SEM). Homogeneity of variance was
Astaxanthin was extracted from flesh according to
confirmed and comparison between means was by
the method of Turujman et al. (1997). Briefly, 10 g
one-way analysis of variance (ANOVA). Significance
of tissue were sampled at the centre of the fillet and
was accepted at probabilities (p) of 0.05 or less.
homogenized using an Ultra Turrax for 2 min with
For comparison of whole astaxanthin stereoi-
10  2 ml of hexane, to remove most of lipids. The
somers profile of trout fillet and the pigment
homogenate was centrifuged at 4000 rpm for 10 min
ingredients of the diet, the coefficient of distance,
and hexane phase was discharged. Astaxanthin was
D (McIntire et al. 1969), between ‘reference’ and
extracted from the pellet resulted after hexane
‘test’ samples (pigment source and trout fillet,
extraction with 10  2 ml of acetone, following the
respectively) was computed. This coefficient was
procedure mentioned above. The acetone phase was
originally developed to estimate the similitude of
evaporated with a rotary evaporator (Rotavapor R
aquatic animals on the base of their fatty acids
110, Büchi, Switzerland) and the aqueous residue
composition. Therefore, the smaller the value of the
(approximately 4 ml) reconstituted in 20 ml of
coefficient of distance, D, the higher the probability
methylene chloride and vigorous swirled to dissolve
that trout have been fed with a diet containing that
astaxanthin. The residual water was removed with
specific pigment source.
a separatory funnel and the organic phase was dried
For comparison of the whole astaxanthin stereo-
over approximately 1 g of anhydrous sodium sulfate. isomers profile of ‘reference’ (feed ingredient) and
The methylene chloride phase containing astax- ‘test’ (trout fillet), the coefficient of distance, D, was
anthin was transferred in a clean tube. The amount computed using the following equation:
of astaxanthin extracted was determined recording
" #1=2
the volume and measuring the absorbance of the X
n
2
methylene chloride extract a 474 nm using a Jasco Djh ¼ ðPij
Pih Þ ,
model V-530 spectrophotometer (Ishikawa-cho, i¼1

Japan). where Djh is the degree of difference (coefficient of

distance) between samples j (‘reference’ diet) and h
HPLC analysis of astaxanthin (‘test’ trout fillet), and Pij and Pih are percentage of
astaxanthin stereoisomers i in sample j and h, for
The astaxanthin was purified by loading the methy-
each i stereoisomers. All the statistical analyses
lene chloride phase onto a silica gel Sep-Pak
were performed by SPSS 12.0 (SPSS, Inc.
cartridge (Waters, Milford, MA, USA) pretreated
Chicago, IL, USA).
with hexane. Astaxanthin was eluted from the
cartridge with 5 ml of chloroform and the eluate
dried under a stream of nitrogen. Dry residue was
Results and discussion
dissolved in 200 ml of hexane and 20 ml were injected
into the HPLC system. Figure 1 shows the chromatograms of a standard
The HPLC system consisted of a Jasco model solution (32 mg ml
1) of synthetic astaxanthin and
980-PU pump (Ishikawa-cho), a Jasco model of the extracts obtained from different sources. The
LG-980-02 gradient unit, a Jasco model AS-950 three peaks corresponding to 3S,30 S isomer, meso
autosampler and a Waters model 996 Photodiode form and 3R,30 R isomer were eluted at 10.5, 11.5
Array Detector. Chromatograms were recorded and and 12.6 min, respectively.
integrated on a Millennium 32 Chromatography The percentage distribution of stereoisomers
Software (Waters). was calculated from chromatograms of different
Analyses were performed with a Pirkle L-leucine pigment extracts by means of relative peak area
chiral column (Regis Technologies, Morton measurements. The results are presented in
Grove, IL, USA). Mobile phase was Figure 2. A characteristic distribution of astaxanthin
1060 V. M. Moretti et al.

(a)
11,523 (meso)

(3S,3′S)
10,539
0.10
12,623 (3R,3′R)
AU
0.05

0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

minutes

(b) 0.14
0.12 10,909 (3S,3′S)

0.10

11,663 (meso)

12,781 (3R,3′R)
0.08
AU
0.06
0.04
0.02
0.00
2.00 4.00 6.00 8.00 10.0 12.0 14.0 16.0 18.0

(c)
12,503
0.15
(3R,3′R)
11,450 (meso)

0.10
AU

0.05

0.00
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Figure 1. Chromatograms of stereoisomers composition of all-trans astaxanthin of: (a) a standard solution of synthetic astaxanthin
(32 mg ml
1); (b) a Haematococcus pluvialis extract; and (c) a Phaffia rhodozyma extract. High-performance liquid chromatography (HPLC)
conditions: Pirkle L-leucine chiral column; mobile phase hexane–tetrahydrofuran–triethylamine–acetonitrile (77:19:2:2 v/v); flow rate of
1 ml min
1; monitoring wavelength 474 nm.

optical isomers was detected for each pigment of 3S,30 S enantiomer, 27.9% of the meso form
sources and such distribution was reproduced in and 6.7% of 3R,30 R enantiomer. These results were
trout fed with that source. Haematococcus pluvialis consistent with literature values. In particular,
algae meal contained 87.6% of the 3S,30 S enantio- Foss et al. (1987) analysed the content of astax-
mer, 10.1% of the meso form and 2.3% of the anthin stereoisomers in several species of
3R,30 R enantiomer, while the red yeast Phaffia crustaceans and demonstrated that in Euphausia
rhodozyma contained 1.5% of the meso form and superba (krill) the 3R,30 R isomer predominated,
98.5% of the 3R,30 R enantiomer. The values of whereas in Thysanoessa inermis and in Calanun
optical isomers in krill meal and krill oil were similar. finmarchicus, the 3S,30 S isomer was dominant.
In fact, 27.4% of 3S,30 S enantiomer, 21.8% of the These latter two small crustaceans are important
meso form and 50.8% of 3R,30 R enantiomer ingredient of diets of wilds salmonids, and their
were found in the krill meal whilst 35.8% of 3S,30 S isomeric composition has been demonstrated
enantiomer, 13.6% of the meso form and 50.6% similar to the isomeric composition encountered in
of 3R,30 R enantiomer were found in krill oil. wild salmon, in which the S-form predominated
On the other hand, shrimps meal contained 65.4% (Turujman et al. 1997).
 Astaxanthin stereoisomers and colour attributes in O. mykiss flesh 1061

98.5
100 3S,3′S

3R,3′S
87.6
90
3R,3′R

80

70 65.4

60
50.6 50.8 50.6

% 50

40 35.8

27.4 27.9
30 25.2 24.3
21.8

20
13.6
10.1
6.7
10
2.4 1.5

0
Astaxanthin Microalgae Red Yeast Krill meal Krill oil Shrimp meal

Figure 2. Percentage distribution of astaxanthin stereoisomers in pigment sources.

Table I. Percentage distribution of astaxanthin stereoisomers in fillet of rainbow trout fed with different pigment sources.

Samples (n ¼ 10) 3S,30 S enantiomer (%) 3R,30 S meso (%) 3R,30 R enantiomer (%)

CarophyllÕ pink-fed trout 29.6  3.62 48.4  4.26 22.0  7.50

Microalgae (Haematococcus pluvialis)-fed trout 80.0  2.01 11.7  1.14 8.3  1.03
Red yeast (Phaffia rhodozyma)-fed trout 14.4  2.33 10.7  2.28 74.9  4.52
Values are means  standard error.

Astaxanthin isomer ratios in the flesh of fish fed Table II. Colour values measured in three different part of fillet of
different carotenoid sources is reported in Table I. rainbow trout fed with different pigment sources.
Dietary treatment influenced significantly the Colour
distribution of the optical isomers in the fillets, and value Neck Centre Tail p1
the average values of the optical isomers in the flesh
a* 14.8  0.74 14.5  0.66 15.8  0.33 0.28
reflected the isomeric ratio of the dietary astaxanthin
b* 18.9  1.13 17.3  1.17 19.3  0.87 0.40
source. The astaxanthin isomers distribution in L* 44.8  1.31 42.3  1.42 43.8  1.60 0.48
microalgae and in the corresponding trout flesh C* 24.1  1.31 22.6  1.28 25.0  0.86 0.37
were similar and ranged from 80.0 to 87.6%, from 1
Significance accepted at probabilities of 0.05 or less.
11.7 to 10.1% and from 2.4 to 8.3% for the 3S,30 S Values are means  standard deviation (n ¼ 10).
isomer, meso form and 3R,30 R isomer, respectively.
In the red yeast and in the corresponding trout flesh
the 3R,30 R isomer predominated, ranging from 98.5
to 74.9%, while the meso form and the 3S,30 S distribution of optical isomer were 29.6, 48.4 and
isomer ranged from 1.5 to 10.7% and from 0 to 22.0% for the 3S,30 S isomer, meso form and 3R,30 R
14.4%, respectively. In the flesh of trout fed isomer, respectively. Also in this case the distribution
synthetic astaxanthin, the averages of the resembles that of the diet and these results were in
1062 V. M. Moretti et al.

agreement with those of Østerlie et al. (1999), who and ultraviolet-visible light (UV-VIS) absorption
found in rainbow trout fed synthetic astaxanthin a spectra was reasonably recognized as canthaxanthin.
distribution of isomers in muscle of 28.5, 49.0 and Three fish samples showed to contain only synthetic
22.5% for 3S,30 S, meso and 3R,30 R isomers, astaxanthin (trouts 1, 8, 9) and one (trout 6) both
respectively. synthetic carotenoids: astaxanthin and canthaxantin.
Colour values measured in different sites of fillet The coefficient of distance (Table V) computed
of rainbow trout fed with different pigment sources amongst the feed ingredient and the trout fillet
are presented in Table II. No significant differences astaxanthin stereoisomers is a simple geometrical
were registered in different parts of the flesh. tool which can be useful to tentatively identify the
Similarly, different sources of pigment (natural or origin of the pigment used on farm.
synthetic), used with the three different diets, The smallest D values registered in trout fed the
produced colour values of fresh fillet (Table III) Carophyll pink and trout fed microalgae were 5.4
with no significant differences, although there was a and 9.8 when computed using the synthetic astax-
tendency for the CP diet to give an higher value of anthin and the microalgae as ‘reference’, respec-
redness (a*) and brightness (C*) when compared tively. The other D values, computed using the
with HP diet. This confirms the better pigmenting astaxanthin stereoisomers composition of the other
efficacy of free astaxanthin in comparison with analysed ingredients, were considerably higher.
esterified astaxanthin from microalgae (White et al. In trout fed the read yeast, D value (29.1) was
2003). similar to those calculated for krill meal (29.5) and
Among the specimens purchased on the market krill oil (32.5).
(Table IV), two samples showed the algal astaxhan-
tin isomeric profile (trouts 3 and 4), and three
(trouts 2, 5, 7) were found to contain a different Table IV. Percentage distribution of astaxanthin stereoisomers in
flesh of rainbow trout purchased on the market.
pigment source that on the basis of its retention time
3S,30 S 3R,30 S 3R,30 R
enantiomer meso enantiomer Presence of
Table III. Colour values measured in fillet of rainbow trout fed Samples (%) (%) (%) canthaxanthin
with different pigment sources.
Trout 1 30.7 48.0 21.3 No
Colour Synthetic Phaffia Haematococcus Trout 2 n.d. n.d. n.d. Yes
value astaxanthin rhodozyma pluvialis p1 Trout 3 71.2 19.9 8.9 No
Trout 4 68.4 21.8 9.8 No
a* 15.3  0.58 15.7  0.56 14.2  0.64 0.19 Trout 5 n.d. n.d. n.d. Yes
b* 18.7  1.06 18.6  1.04 18.2  1.20 0.95 Trout 6 40.5 26.9 32.6 Yes
L* 42.9  1.40 43.4  1.50 44.7  1.50 0.64 Trout 7 n.d. n.d. n.d. Yes
C* 24.1  1.18 24.4  1.14 23.1  1.28 0.74 Trout 8 28.2 49.1 22.7 No
1 Trout 9 29.8 48.3 21.9 No
Significance accepted at probabilities of 0.05 or less.
Values are means  standard deviation (n ¼ 10). n.d., Not detected.

Table V. Coefficient of distance (D) of the astaxanthin stereoisomers amongst known and unknown trout samples and the analysed
astaxanthin sources.

Microalgae Red yeast

Synthetic (Haematococcus (Xanthophyllomyces Antarctic Krill Shrimp
astaxanthin pluvialis) dendrorhous) krill meal oil meal

Trout-fed 5.4 72.2 94.5 39.3 45.5 44.0

CarophyllÕ pink
Trout-fed 69.1 9.8 121.0 68.4 61.2 21.9
microalgae
(H. pluvialis)
Trout-fed 65.3 103.0 29.1 29.5 32.5 86.9
red yeast
(X. dendrorhous)
Trout 1 6.8 70.9 95.2 39.6 45.5 42.7
Trout 3 57.4 20.2 115.9 60.6 55.1 10.1
Trout 4 53.9 23.7 113.8 58.0 52.9 7.5
Trout 6 29.4 58.4 81.4 23.0 22.9 35.9
Trout 8 3.7 73.9 93.8 39.2 45.8 45.7
Trout 9 5.7 72.0 94.6 39.3 45.4 43.8
A value in bold font is the actual coefficient of distance amongst pigment source and trout fillet of the known samples; a value in italics is the
possible coefficient of distance amongst pigment source and trout fillet of the unknown samples.
 Astaxanthin stereoisomers and colour attributes in O. mykiss flesh 1063

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