zeaxanthin

The Carotenoid
Pigment
Zeaxanthin—A
Review
M.G. Sajilata, R.S. Singhal, and M.Y. Kamat
ABSTRACT: Scientific evidence linking several diseases with diet has brought to light the beneficial effects of a
number of natural food ingredients. Zeaxanthin is one such natural pigment emphasized for its critical role in
the prevention of age-related macular degeneration (AMD), the leading cause of blindness. The review highlights
zeaxanthin as a carotenoid pigment with promising nutraceutical implications, and enumerates the important plant
and microbial sources for its production, the absorptive pathway of zeaxanthin in human system, and methods to
assess its bioavailability besides other relevant aspects.
Introduction
Carotenoids are pigments naturally occurring in a number of
fruits and vegetables. They are synthesized by all photosynthetic
organisms and many nonphotosynthetic bacteria and fungi. They
are liposoluble tetraterpenes originating from the condensation of
isoprenyl units, which form a series of conjugated double bonds
constituting a chromophoric system (Britton 1995). There are 2
main classes of naturally occurring carotenoids: (1) carotenes
such as β-carotene and α-carotene, which are hydrocarbons, are
either linear or cyclized at one or both ends of the molecule,
and (2) xanthophylls, the oxygenated derivatives of carotenes.
All xanthophylls produced by higher plants, such as violaxanthin,
antheraxanthin, zeaxanthin, neoxanthin, and lutein, are also syn-
thesized by green algae (Eonseon and others 2003). Epidemiolog-
ical studies have established an inverse relationship between the
risk of laryngeal, lung, and colon cancers and the consumption of
foods containing carotenoids (Block and others 1992; Steinmetz
and Potter 1993).
The chemical name of zeaxanthin is (all-E)-1,1 -(3,7,12,16-
tetramethyl-1,3,5,7,9,11,13,15,17-octadecanonaene-1,18-diyl)
bis [2,6,6-trimethylcyclohexene-3-ol]. Synonyms are: 3R,
3 R-β,β-carotene-3,3 -diol; all-trans-β-carotene-3,3 -diol;
(3R,3 R)-dihydroxy-β-carotene; zeaxanthol; and anchovyx-
anthin. Zeaxanthin, the principal pigment of yellow corn,
Zeaxanthin mays L. (from which its name is derived), has a
molecular formula of C40H56O2 and a molecular weight of
568.88 daltons. Its CAS number is 144-68-3. It is composed of
40 carbon atoms, yellow in color, and naturally found in corn,
egg yolks, and some of the orange and yellow vegetables and
fruits such as alfalfa and marigold flowers (Nelis and DeLeenheer
1991; Handelman and others 1999; Humphries and Khachik
MS 20070403 Submitted 5/28/2007, Accepted 8/13/2007. Authors are with
Food Engineering and Technology Dept., Inst. of Chemical Technology, Univ.
of Mumbai, Matunga, Mumbai-400 019, India. Direct inquiries to author Singhal
(E-mail: rekha@udct.org).
2003). Zeaxanthin exhibits no vitamin A activity. Zeaxanthin
and its close relative lutein (Figure 1 and 2) play a critical role
in the prevention of age-related macular degeneration (AMD),
the leading cause of blindness (Snodderly 1995; Moeller and
others 2000). Zeaxanthin is isomeric with lutein; the 2 carotene
alcohols differ from each other just by the shift of a single double
bond so that in zeaxanthin all double bonds are conjugated.
Zeaxanthin is used as a feed additive and colorant in the food
industry for birds, swine, and fish (Hadden and others 1999).
The pigment imparts a yellow coloration to the skin and egg yolk
of birds, whereas in pigs and fish it is used for skin pigmentation
(Nelis and DeLeenheer 1991).
Stereoisomers of Zeaxanthin
Zeaxanthin has 2 chiral centers and, hence, 22
or 4 stereoiso-
meric forms. One chiral center is the number ‘3’ atom in the left
end ring, while the other chiral center is the number ‘3’ carbon
in the right end ring (Garnett and others 1998). One stereoiso-
mer is (3R, 3 R)-zeaxanthin; the other is (3S-3 S)-zeaxanthin. The
3rd stereoisomer is (3R, 3 S)-zeaxanthin and the 4th (3S-3 R)-
zeaxanthin. However, since zeaxanthin is a symmetric molecule,
the (3R, 3 S)—and (3S, 3 R)—stereoisomers are identical. There-
fore, zeaxanthin has only 3 stereoisomeric forms. The (3R,
3 S)—or (3S, 3 R)—stereoisomer is called meso-zeaxanthin. The
principal natural form of zeaxanthin is (3R, 3 R)-zeaxanthin. (3R,
3 R)-zeaxanthin and meso-zeaxanthin are found in the macula of
the retina, with much smaller amounts of (3S, 3 S)-zeaxanthin.
Meso-zeaxanthin is a rare isomer present in significant quantities
in commercially produced chickens and eggs in Mexico where
it is commonly added to the feed to achieve desirable coloration
in these products (Bone and others 2007).
Properties of Zeaxanthin
One gram of zeaxanthin dissolves in about 1.5 L of boiling
methanol. The pigment is almost insoluble in petroleum ether
C 2008 Institute of Food Technologists Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 29

CRFSFS: Comprehensive Reviews in Food Science and Food Safety
Figure 1 --- Structure of zeaxanthin.
Figure 2 --- Structure of lutein.
Figure 3 --- Resonance Raman spectra of zeaxanthin (ZX)
extracted from bacteria and dissolved in methanol and of
Flavobacterium multivorum culture broth (CB, plotted on
right Y axis (Bhosale and others 2003).
and hexane. Its solubility in ether, chloroform, carbon disulphide,
and pyridine is somewhat greater. Zeaxanthin dissolves in con-
centrated sulfuric acid with a fairly stable deep blue coloration.
On treating a solution of the pigment in chloroform with anti-
mony trichloride, a blue coloration is produced (Euler and others
1930).
Zeaxanthin is a polyene-like molecule, which contains 9 alter-
nating conjugated carbon double and single bonds. The carbon
backbone is terminated at each end by an ionone ring to which
a hydroxyl group is attached. When excited with monochro-
matic laser light, it exhibits characteristic wavelength shifts of
inelastically back-scattered light caused by vibrational modes
in its chemical structure. Two characteristic carotenoid peaks
shown in Figure 3 originate from rocking motions of the carbon–
carbon single bond stretch vibrations (1159 cm−1
) and from the
carbon–carbon double bond stretch vibrations (1525 cm−1
) of the
molecule backbone (Bhosale and others 2003).
The conjugated double-bond system constitutes the light-
absorbing chromophore that gives carotenoids their attractive
color and provides the visible absorption spectrum that serves as a
basis for their identification and quantification. Cis-isomerization
of a chromophore’s double bond causes a slight loss in color,
small hypsochromic shift, and hypochromic effect, accompanied
by the appearance of a cis peak in or near the ultraviolet region.
All-trans isomers absorb strongly in the visible region between
400 and 500 nm while cis-isomers exhibit absorption in the near-
UV region, around 320 nm (Rodriguez-Amaya 2001). The visible
Table 1 --- Ultraviolet and visible absorption data of zea-
xanthin (Davies 1976; Britton 1995).
Carotenoid Solvent λ max nma % III/IIb
Zeaxanthin Acetone (430) 452 479
Chloroform (433) 462 493
Ethanol (428) 450 478 26
Petroleum ether (424) 449 476 25
aParentheses indicate a shoulder.
bRatio of the height of the longest-wavelength absorption peak, designated III, and that of the
middle absorption peak, designated II, taking the minimum between the 2 peaks as baseline
multiplied by 100 (hni.ilsi.org/publications).
Figure 4 --- Calculation of % III/II as indication of spectral
ﬁne structure (% III/II × 100) (www.hni.ilsi.org).
spectrum of zeaxanthin, a derivative of β-carotene, resembles
that of β-carotene. The ultraviolet and visible absorption data
of zeaxanthin are shown in Table 1 with calculation of % III/II
as indication of spectral fine structure (% III/II × 100) illustrated
in Figure 4. Figure 5 shows the absorption spectra of isomers of
zeaxanthin.
Carotenoid molecules are strong Raman scatterers. Hence,
nondestructive resonance Raman spectroscopy could be an ex-
tremely valuable method for the rapid quantitative assessment
of carotenoids. There are reports of the detection of resonance
Raman scattering of laser radiation of the carotenoid pigments
30 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008

The carotenoid pigment zeaxanthin . . .
from intact plant samples and fruit juices (Gill and others
1970).
Biosynthetic Pathway and Genetic Manipulation
of the Pathway for Zeaxanthin Production
A paramount function of xanthophylls in all photosynthetic or-
ganisms, including cyanobacteria, is to provide protection against
photooxidation. It is proposed that zeaxanthin protects the mem-
brane directly against lipid peroxidation by reactive radicals that
have been created as toxic byproducts during photosynthetic re-
actions. Another mechanism suggests specific xanthophylls to be
involved in the de-excitation of singlet chlorophyll (1
Chl) that ac-
cumulates in the light-harvesting complexes (LHC) under condi-
tions of excessive illumination (Demmig-Adams 1990; Demmig-
Adams and Adams 1992; Demmig-Adams and others 1996).
Carotenoid biosynthesis leads to the all-trans-forms. Hence, all-
trans-lycopene, -lutein and -zeaxanthin predominantly occur in
fresh fruits and vegetables. All-trans-isomers have therefore been
assumed to be the thermodynamically stable form of carotenoids
(Miebach and Behsnilian 2006). Carotenoids are synthesized
from the basic C5-terpenoid precursor, isopentenyl diphosphate
(IPP) and dimethylallyl pyrophosphate (DMAPP). IPP is converted
into geranylgeranyl phosphate (GGPP) and the dimerization of
GGPP leads to phytoene (7,8,11,12,7 ,8 ,11 ,12 -octahydro-γ ,γ -
carotene) and the stepwise dehydrogenation via phytofluene
(15 Z, 7, 8,11,12, 7 ,8 -hexahydro-γ , γ - carotene), zeta
carotene (7,8,7 ,8 -tetrahydro-γ -γ -carotene), and neurosporene
(7,8-dihydro-γ , γ -carotene) gives lycopene. Subsequent cycliza-
tion, dehydrogenation, and oxidation lead to hundreds of dif-
Figure 5 --- Absorption spectra of the
isomers of zeaxanthin. Spectra were
recorded from pigment solution in
MTBE-methanol (5:95, by volume)
(Justyna and Gruszecki 2005).
ferent natural carotenoids (Figure 6). In Flavobacterium R1529,
nicotine blocks zeaxanthin biosynthesis by specifically inhibiting
the cyclization reaction (McDermott and others 1974). Lycopene
and rubixanthin replace zeaxanthin as the main carotenoid. In
the absence of nicotine, lycopene is converted to β-carotene un-
der anaerobic conditions and into zeaxanthin in the presence of
oxygen.
The biosynthesis of IPP and DMAPP from acetyl-CoA via
melavonate has been studied using animal cells and yeasts
(Bochar and others 1999). Three acetate units afford the 5 carbon
atoms of IPP from loss of 1 acetate carboxylic group as CO2.
DMAPP is obtained from IPP by an isomerase. A mevalonate-
independent 2nd pathway for the biosynthesis of IPP and
DMAPP via 1-deoxy-D-xylulose 5-phosphate has been discov-
ered in some eubacteria and plants (Rohmer and others 1993).
The nonmevalonate pathway starts with the formation of 1-
deoxyxylulose 5-phosphate from pyruvate and glyceraldehyde 3-
phosphate catalyzed by 1-deoxyxylulose 5-phsophate synthase.
The carbohydrate is converted into 2 C-methylerythritol 2, 4-
cyclodiphosphate by a series of 4-reaction steps (Eisenreich and
others 2002).
Genetic studies with Arabidopsis thalina (Rock and Zeevaart
1991; Rock and others 1994), Nicotiana plubaginifolia (Marin
and others 1996), and the green alga Chlamydomonas reinhardtii
(Niyogi and others 1997) revealed the presence of a single-gene
coding for the zeaxanthin epoxidase enzyme. Thus, a single-gene
product is apparently responsible for both the biosynthesis of vi-
olaxanthin during growth, and development and the epoxidation
reaction leading to the return of zeaxanthin via antheraxanthin
to violaxanthin following recovery after irradiance stress. Mutants
Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 31

with lesions in the zeaxanthin epoxidase gene not only are conse-
quently deficient in antheraxanthin and violaxanthin, but also fail
to synthesize neoxanthin (Niyogi and others 1997; Jin and oth-
ers 2003). In addition, these mutants accumulate large amounts of
zeaxanthin that are almost equivalent to the levels of violaxanthin
found in the wild type, even when grown under nonstressed con-
ditions. Mutations affecting zeaxanthin production exist in green
algae, Scenedemus obliquus (Bishop and others 1995), Chlamy-
Figure 6 --- Precursor
compounds and major
carotenes and xanthophylls
in the carotenoid
biosynthetic pathway in
plants. DMAPP =
dimethylallyl
pyrophosphate; GGPP =
geranylgeranyl
pyrophosphate; IPP =
isopentenyl pyrophosphate
(Kopsell and Kopsell 2006).
domonas reinhardtii (Niyogi and others 1997), and Dunaliella
salina (Jin and others 2003).
In microalgae, the zeaxanthin content is regulated by light ir-
radiance. When photosynthetic irradiance is greater than that
required for the saturation of photosynthesis in the chloro-
plasts of plants and green algae, a reversible violaxanthin de-
epoxidation reaction occurs to form antheraxanthin and, subse-
quently zeaxanthin, resulting in the accumulation of zeaxanthin

in the chloroplast thylakoids. The enzyme that catalyzes this re-
action, violaxanthin de-epoxidase, is localized in the lumen of
the chloroplast thylakoids (Hager and Holocher 1994). When the
absorbed irradiance is lower than that required for saturation of
photosynthesis, zeaxanthin is converted back into violaxanthin
by the enzyme zeaxanthin epoxidase with the monoepoxide an-
theraxanthin being an intermediate in this reversible oxidation–
reduction process (Hager 1980). The xanthophyll cycle is thought
to be essential for the protection of the photosynthetic appara-
tus from photooxidation, and zeaxanthin, antheraxanthin, and
violaxanthin are associated with the light-harvesting complexes
(Demmig-Adams and Adams 1992). In accordance with the role
of zeaxanthin and violaxanthin in photosynthesis, ABA2mRNA
(abscisic acid 2mRNA) is more abundant in photosynthetic than
in nonphotosynthetic tissues.
ABA, a breakdown product of xanthophyll carotenoids (C40)
via the C15 intermediate xanthoxin (Walton and Li 1995), mod-
ulates the growth and development of plants, particularly dur-
ing seed formation and also in response to environmental stress
(Zeevaart and Creelman 1988; Giraudat and others 1994). Mu-
tants blocked in the early steps of carotenoid synthesis, for ex-
ample, some viviparous mutants of maize (vp2, vp5, vp7, or
vp9), lack carotenoids essential for photosystem protection and,
therefore, exhibit photobleaching and ABA deficiency (Neill and
others 1986). In contrast, mutants impaired in the downstream
steps of carotenoid biosynthesis do not show photobleaching.
The aba1 mutant of Arabidopsis and the aba2 mutant of Nico-
tiana plumbaginifolia are impaired in the epoxidation of zeaxan-
thin and have been shown to be either slightly or not at all affected
in PSII photochemical efficiency (Rock and Zeevaart 1991; Rock
and others 1992; Marin and others 1996; Tardy and Havaux 1996;
Hurry and others 1997). Zeaxanthin is able to replace the miss-
ing epoxy-carotenoids, antheraxanthin, violaxanthin, and neo-
xanthin as a stabilizing component of the light-harvesting com-
plex II in the aba1 mutant of Arabidopsis.
The recent genetic elucidation of bacterial and plant carotenoid
biosynthetic pathways leading to the accumulation of zeaxan-
thin, canthaxanthin, and astaxanthin may offer interesting alter-
natives for their in vivo production (Misawa and others 1995a,
1995b; Misawa and Shimada 1998). For instance, blue-green al-
gae can be readily transformed with autonomously replicating
plasmids, while endogenous genes can be disrupted by homolo-
gous recombination. A number of commercial possibilities have
been proposed for recombinant blue-green algae (Lagarde and
others 2000). Recently, Synecocystis sp. strain PCC 6830 was
used as a transformation host to overproduce zeaxanthin in vivo.
Moreover, the system developed in the study allowed for gene
replacement without the introduction of antibiotic resistance cas-
settes in the final overexpressing strains. The absence of cas-
settes containing genes that confer antibiotic resistance in such
strains is a positive feature highlighting the increasing desire of
the biotechnology industry to avoid spreading antibiotic-resistant
cassettes, thereby respecting the concerns of consumers and
environmentalists.
A genetically engineered zeaxanthin-rich potato
In an attempt to provide a good supply of zeaxanthin in staple
crops such as potatoes (Solanum tuberosum L.), 2 different potato
varieties were genetically modified (R¨omer and others 2002).
By transformation with sense and antisense constructs encoding
zeaxanthin epoxidase, the conversion of zeaxanthin to violaxan-
thin was inhibited. Both antisense and cosuppression approaches
yielded potato tubers with high levels of zeaxanthin. Depending
on the transgenic lines and tuber development, zeaxanthin con-
tent was elevated by 4- to 130-fold, reaching values up to 40 µg/g
dry weight.
Localization and Sources of Zeaxanthin
Xanthophylls are relatively hydrophobic molecules. Therefore,
they are typically associated with membranes and/or noncova-
lently bound to specific proteins. In general, primary carotenoids
are localized in the thylakoid membrane, while secondary
carotenoids are found in lipid vesicles either in the plastid stroma
or the cytosol. Most xanthophylls that are found in cyanobacteria
and oxygenic photosynthetic bacteria are associated with chloro-
phyll (Chl)-binding polypeptides of the photosynthetic apparatus
(Grossman and others 1995). Among nonphotosynthetic bacte-
ria and, to a lesser extent, among photosynthetic bacteria and
cyanobacteria, xanthophylls and their glycosides can be found
in cytoplasmic and cell wall membranes where they are thought
to influence membrane fluidity (Armstrong 1997).
Plant sources
Green leafy vegetables are good dietary sources of lutein, but
poor sources of zeaxanthin. Dietary sources of zeaxanthin include
yellow corn, orange pepper, orange juice, honeydew, mango, and
chicken egg yolk. Jungalwala and Cama (1962) found zeaxanthin
to comprise about 90% of the total carotenoids in the anthers of
Delonix regia (Gul Mohr) flowers. Zeaxanthin is also the major
carotenoid in cold-pressed marionberry, boysenberry, red rasp-
berry, and blueberry seed oils, followed by β-carotene, lutein, and
cryptoxanthin (Parry and others 2005). Zeaxanthin has also been
identified in extracts from apricots, peaches, cantaloupe, and
a variety of pink grapefruit (Ruby seedless) among carotenoids
separated and quantified on C18 reversed-phase HPLC columns
with low and high carbon loading (Khachik and others 1989).
The major carotenoids of Viburnum tinus leaves are β-carotene
and lutein (38.5% and 47%, respectively) with neoxanthin and
zeaxanthin accounting for 7% and 6.8%, respectively (Chiralt
and others 1990). Carotenoids identified in persimmon fruits are
cis-mutatoxanthin, antheroxanthin, zeaxanthin, neolutein, cryp-
toxanthins, α-carotene, and β-carotene, and fatty acid esters of
cryptoxanthin and zeaxanthin (Daood and others 1992). Zeaxan-
thin has also been identified in the skin, flesh, and oil of avocado
(Ashton and others 2006). Lucerne contains 2% zeaxanthin and
40% lutein of the total xanthophyll content, while Fremontia (Ster-
culalareaceae) produces as much zeaxanthin as lutein (Goodwin
1976). The stereochemical correlation between capsanthin and
zeaxanthin and the cooccurrence of the 2 pigments in Capsicum
sp. have suggested a close biogenetic relationship between the
two. Formation of capsanthin from zeaxanthin via antheraxanthin
is indicated (Britton 1976). Of the total pigment content, zeaxan-
thin contributes to about 6.5%, 7.3%, and 15.9%, respectively,
in the red, orange, and yellow varieties of Capsicum annuum
(Goodwin 1976).
In corn, xanthophylls are mostly found in the horny endosperm.
The total xanthophyll content is estimated to be 11 to 30 mg/kg
(Zuber and Darrah 1987). In 1 study, yellow dent corn was found
to contain a total xanthophyll content of 21.97 µg/g with lutein
content of 15.7 µg/g, zeaxanthin content of 5.7 µg/g, and β-
cryptoxanthin of 0.57 µg/g (Moros and others 2002). Commercial
corn gluten meal has 7 times higher concentration of xanthophylls
(145 µg/g), and deoiled corn contains 18 µg/g, indicating that the
xanthophylls are probably bound to the zein fraction of corn pro-
teins. Wolfberry (Lycium chinese), a small fruit used to improve
vision in traditional Chinese medicine, contains concentrations
of zeaxanthin dipalmitate that can approach 1 g/kg wet weight
(Zhou and others 1999). The zeaxanthin concentration of fruits
and vegetables is shown in Table 2 (Khachik and others 1989).
Microbial sources
Recently, there has been much interest in the microbial produc-
tion of zeaxanthin (Ruther and others 1997; Jin and others 2003);

however, unlike β-carotene and astaxanthin, very few microbes
synthesize zeaxanthin as their predominant carotenoid (Johnson
and Schroeder 1996).
Flavobacterium sp. Among the sources of microbial xantho-
phylls, Flavobacterium sp. is reported to produce zeaxanthin as
essentially its only carotenoid. The pigment formed by Flavobac-
terium consists of 95% to 99% zeaxanthin. Flavobacterium-
produced zeaxanthin is identical to zeaxanthin from Zea mays
(Gierhart 1995). Beta-carotene along with β-cryptoxanthin is
known to act as precursors in the biochemical pathway of zeaxan-
thin production, and thus appreciable levels of these carotenoids
(about 5% to 10%) were observed during initial growth phases of
Flavobacterium sp. (Bhosale and others 2004). Hydroxylation of
β-carotene and β-cryptoxanthin ultimately leads to accumulation
of zeaxanthin.
However, for optimal industrial production, it is essential to
continue strain improvement and to select better producers in
order to maximize the yield. Currently, the only effective way to
screen for better producers is to extract carotenoids from microbes
and to perform HPLC analysis with ultraviolet/visible or photodi-
ode array detectors. Improved yields of zeaxanthin may be ob-
tained by culturing a microorganism of the genus Flavobacterium
under conditions whereby the amounts of carbon and nitrogen
present in the culture medium are maintained at a substantially
constant ratio (Gierhart 1995).
Cultures of Flavobacterium sp. in a nutrient medium contain-
ing glucose or sucrose, sulfur-containing amino acids, such as
methionine, cystine, or cysteine, pyridoxine, and bivalent metal
ions selected from the group consisting of Fe2+
, Co2+
, Mo2+
, or
Mn2+
were able to produce up to 190 mg zeaxanthin/L, with a
specific cell concentration of 16 mg/g dried cellular mass (Shep-
herd and others 1976). The optimized process claims to provide
up to 500 mg of zeaxanthin/L culture at lower costs and more
rapidly than known methods and microorganisms.
Several reports have shown different medium constituents
other than environmental factors to affect zeaxanthin production.
Among these factors, nitrogen and carbon sources play an impor-
tant role in zeaxanthin production (Gierhart 1994; Alcantara and
Sanchez 1999). For Flavobacterium, corn steep liquor is benefi-
cial for pigment synthesis because of its richness in amino acids,
Table 2 --- Zeaxanthin concentration in fruits and vegeta-
bles.
Zeaxanthin,
Food items µg per 100 g
Beans (drained, green, canned) 44
Broccoli (drained, cooked, boiled without salt) 23
Carrots (raw) 23
Celery (drained, cooked, boiled, without salt) 8
Celery (raw) 3
Collards (drained, cooked, boiled, without salt) 266
Corn (drained, sweet, yellow, canned, whole kernel) 528
Lettuce (raw) 187
Lettuce (raw) 70
Orange juice (frozen concentrate, unsweetened, diluted) 80
Oranges (all commercial varieties) 74
Papaya 20
Peaches (raw) 6
Peas (drained, green, canned) 58
Spinach (drained, cooked, boiled without salt) 179
Spinach (raw) 331
Squash, butternut 280
Tangerines, raw (mandarin oranges) 112
minerals, and other factors necessary for growth (Goodwin 1971;
Gierhart 1994). The effect on growth and production of zeaxan-
thin by Flavobacterium sp. was studied using different carbon
and nitrogen sources in a chemically defined medium by Al-
cantara and Sanchez (1999). The best growth was supported by
sucrose, and both asparagine and glutamine were found to stim-
ulate growth and pigment formation. Carotenoid production and
glucose consumption increased as a function of asparagine con-
centration. Flavobacterium sp. was found to utilize asparagine
primarily as a nitrogen source for growth and production of zeax-
anthin. In the presence of asparagine, high glucose concentra-
tions decreased pigment production without affecting biomass
formation. In the absence of glucose, asparagine could not sup-
port growth and zeaxanthin production (Table 3). Lactic acid and
palmitic acid methyl esters are reportedly pigment promoters of
zeaxanthin. With no special measures taken, fermentation of a
Flavobacterium sp. in a medium containing glucose and corn
steep liquor generates approximately 10 to 40 mg zeaxanthin/L
(Nelis and DeLeenheer 1989). The yield increased to 335 mg/L
by supplementation with palmitic esters, methionine, pyridoxine,
ferrous salts by continuous addition of the nutrients, and reduc-
tion of temperature.
Another important factor in the production of microbial pig-
ments is the oxygen provided to the culture medium (Goodwin
1971; Britton 1985). In general, an increase in oxygen supply to
a highly active culture can increase its productivity. The growth
of bacteria and fungi, measured in terms of dry weight, varies
directly with the efficiency of aeration above a predetermined
level depending on the available substrate. In addition to being
required for growth in Flavobacterium, oxygen is also required for
desaturation, cyclization, and oxygenation of carotenoids (Smith
and Johnson 1958; Edwards 1985; Han and Mudgett 1992; Brit-
ton 1995). To improve zeaxanthin production by Flavobacterium,
a fermentor with a high oxygen transfer rate is preferred. Agita-
tion speed and aeration rate are the 2 factors that strongly affect
the oxygen supply in stirred-tank fermentors. A proper combi-
nation of these factors can regulate the required oxygen supply
to a growing culture. However, any additional supply of oxygen
should be matched with the right availability of other nutrients
such as corn steep liquor. Some factors that have an unexpectedly
Table 3 --- Effect of different carbon and nitrogen sources
on growth and zeaxanthin production (Alcantara and
Sanchez 1999).
Growth Zeaxanthin
Conditions (OD at 540 nm) (µg/mL)
Carbon sourcesa
Yeast extract (1%) 2.38 ± 0.070 0.75 ± 0.025
D-glucose (55 mM) 1.55 ± 0.066 0.15 ± 0.020
Sucrose (55 mM) 1.82 ± 0.102 0.15 ± 0.027
Xylose (55 mM) 0.51 ± 0.032 0.07 ± 0.017
No carbohydrate 0.46 ± 0.055 0.07 ± 0.011
Nitrogen sources (7.5 mM)b
L-asparagine 3.74 ± 0.050 0.38 ± 0.016
L-glutamine 2.96 ± 0.119 0.36 ± 0.011
L-cysteine 1.89 ± 0.090 0.07 ± 0.0009
L-methionine 1.85 ± 0.015 0.08 ± 0.0095
Glycine 0.6 ± 0.020 0.03 ± 0.007
L-asparagine without glucose 0.44 ± 0.101 0.08 ± 0.009
L-asparagine without MgSO4 0.55 ± 0.030 0.03 ± 0.006
L-asparagine + L-cysteine 2.31 ± 0.076 0.28 ± 0.003
aFermentation was carried out for 48 h, 29 ◦C, and 180 rpm in CD medium supplemented
with 20 mM NH4Cl.
bFermentation was carried in CD medium supplemented with 100 mM glucose.

strong positive effect on β-carotene production at the expense
of zeaxanthin formation should also be considered. In a study
on carotenoid production by Flavobacterium multivorum ATCC
55238, urea and sodium carbonate were found to influence β-
carotene production, which represented 70% (w/w) of the total
carotenoid content (Bhosale and Bernstein 2004).
The nutrient media that are currently preferred for commercial-
scale fermentation have eliminated corn flour and several other
ingredients, and contain either high-maltose corn syrup of sugar
beet molasses at concentrations ranging from 1% to 10% (w/v),
along with corn steep liquor at 0.5% to 4% (w/v); ammonium sul-
fate heptahydrate at 0.5% (w/v); sodium chloride at 0.5% (w/v);
magnesium sulfate heptahydrate at 0.1% (w/v); sodium acetate at
0.1% (w/v); ferrous sulfate heptahydrate at 0.001% (w/v); yeast
extract at 0.2% (w/v); thiamine-HCl at 0.01% (w/v); between 1%
and 6% (w/v) hydrolyzed casein; and vegetable oil at 1% (v/v)
(Garnett and others 1998). Once the ingredients are mixed to-
gether, sufficient NaOH is added to raise the pH to 6.5. The cul-
ture medium is sterilized by autoclaving at 121 ◦
C for 30 min,
cooled to 27 ◦
C, and inoculated with 5% to 10% (v/v) of a “liq-
uid preculture” containing a strain of F. multivorum which pro-
duces the R-R isomer of zeaxanthin without producing S-S or S-R
isomers of zeaxanthin, or any other carotenoids, in significant
quantities.
Several strains of Flavobacterium have been taxonomically
reevaluated as Paracoccus zeaxanthinifaciens sp. (Berry and oth-
ers 2003). Metabolic engineering can be used to improve zeaxan-
thin production in the bacterium Paracoccus sp. strain PTA-3335,
a mutant derived from a zeaxanthin-producing bacterium origi-
nally classified as a species of Flavobacterium (Schocher and Wiss
1975).
Erwinia herbicola. Erwinia herbicola, a nonphotosynthetic
bacterium, is yellow-colored due to accumulation of polar
carotenoids, primarily mono- and diglucosides of zeaxanthin
(Hundle and others 1992).
Neospongiococcum. Among FDA-approved GRAS strains is
Neospongiococcum, the only alga presently designated as GRAS
for feeding poultry to enhance yellow pigmentation (21 C.F.R.
section 73,275). The green alga Neospongiococcum excentricum
is shown to produce up to 0.65% xanthophylls (dry mass basis)
(Liao and others 1995).
Dunaliella salina. A zeaxanthin-overproducing mutant strain
zea1 generated from Dunaliella salina may be considered for
commercial exploitation (Jin and others 2001, 2003). This mu-
tant strain has a defect in the zeaxanthin-epoxidation step. Thus,
the zea1 mutant lacks neoxanthin, violaxanthin, and antheraxan-
thin, but constitutively accumulates zeaxanthin in the thylakoid
membrane even under normal growth conditions. Under normal
growth conditions (low light), the mutant strain has 15-fold higher
zeaxanthin content than the wild type. Previous efforts to gener-
ate zeaxanthin-overproducing E. coli strains using metabolic en-
gineering have resulted in the production of 1.6 mg zeaxanthin/g
dry weight; however, this value is just one-third of that produced
by the zea1 strain of the photosynthetic microalga Dunaliella
salina (6 mg zeaxanthin/g dry weight) (Albrecht and others 1999;
Jin and others 2003).
Synechocystis sp. Zeaxanthin is a natural constituet of the outer
membrane of Synechocystis sp. PCC6714 (Jurgens and Weckesser
1985). In an attempt to increase zeaxanthin accumulation in a
photoautotrophic prokaryote, Synechocystis sp. strain PCC 6803,
a system designed to overexpress genes involved in carotenoid
synthesis in the organism employs the psbAII gene, which en-
codes the highly expressed D1 protein of photosystem II and has
a strong promoter in it. Synechocystis genome contains 3 genes
coding for the D1 protein, psbAI, psbAII, and psbAIII, the latter
two of which are expressed and can individually support nor-
mal photoautotrophic growth in the absence of the other two
psbA genes (Mohamed and Jansson 1989). Thus, the psbAII lo-
cus can be used as an integration platform to overexpress genes in
Synechocystis. Synechocystis sp. resulted in a 2.5-fold increase
in zeaxanthin accumulation in the mutant strain (Lagarde and
others 2000).
Microcystis aeruginosa. The microalgae Microcystis aeruginosa
is reported to produce the bioactive carotenoid zeaxanthin (Chen
and others 2005).
Spirulina. The blue-green alga Spirulina has zeaxanthin as one
of its carotenoids. A marked enhancement in carapace color, an
important quality parameter of cultured prawns, was observed
when prawns were fed with Spirulina-supplemented diets. Zea-
xanthin, one of the major carotenoids in Spirulina, was rapidly
converted to astaxanthin (Liao and others 1993). Spirulina, also
increases the yellowness and redness of broiled chicken due to
accumulation of zeaxanthin within the flesh (Toyomizu and oth-
ers 2001).
Phaffia rhodozyma. Among yeasts, asporogenous Phaffia
rhodozyma, although best known as an astaxanthin producer,
is also reported to be a zeaxanthin producer (Hoshino and others
2004).
Zeaxanthinibacter enoshimensis is a zeaxanthin-producing
marine bacterium of the family Flavobacteriaceae, isolated from
the seawater off Enoshima island in Japan (Asker and oth-
ers 2007a). Mesoflavibacter zeaxanthinifaciens is another novel
zeaxanthin-producing marine bacterium of the family Flavobac-
teriaceae (Asker and others 2007b). The carotenoids of the red
algae Corallina officinalis, C. elongate, and Jania sp. are report-
edly composed of β-carotene, zeaxanthin, fucoxanthin, 9 -cis-
fucoxanthin, fucoxanthinol, 9 -cis-fucoxanthinol, and 2 epimeric
mutatoxanthins (Palermo and others 1991). The symbiotic blue-
green algae Cyanophora paradoxa and Glaucocystis nostochin-
earum synthesize only β-carotene and zeaxanthin (Goodwin
1976). The thylakoid of the cyanobacteria Anacystis nidulans
is reported to have zeaxanthin as one of its major carotenoids
(Murata and others 1981). Dunaliella parva (Andrew and Brit-
ton 1990), Erythrotrichia carnea (Shlomai and others 1992),
Dunaliella bardawil (Ben-Amotz and others 1982), Prochloron
sp. (Withers and others 1978), and Pleurochloris commutata
(Goodwin 1976) are some of the other microbial sources of zeax-
anthin.
Extraction and Isolation of Zeaxanthin
The advantage of tetrahydrofuran (THF) in comparison to other
organic solvents of Class 2 as an extraction solvent is that the
carotenoid is highly soluble in the solvent, which allows for its
efficient extraction from plant matrices (Khachik 2005). Solvents
in Class 2 should be limited in pharmaceutical products because
of their inherent toxicity. The choice of extracting solvents is based
on a guideline set by the U.S. Dept. of Health and Human Ser-
vices, Food and Drug Administration (FDA) in Docket No. 97D-
0148 published in the Federal Register on May 2, 1997 (Vol 62,
Nr 85, p 24301–9). The draft guideline recommends acceptable
amounts of residual solvents in pharmaceuticals for the safety of
patients as well as the use of less toxic solvents in the manufacture
of drug substances and dosage forms.
Suitable modes of packaging and delivery for human ingestion
include various forms such as lyophilized coarse-grain powder,
viscous oily liquid, and micelles. An alternate form of packag-
ing for human ingestion can utilize tablets, provided that a solid
binder material is used which will hold the tablet together after
manufacture. Such tablets may be coated with an enteric coat-
ing that remains intact until after the tablet passes through the
stomach and enters the intestine. Zeaxanthin is believed to be

susceptible to degradation at high pressures (Garnett and others
1998). Thus, it is assumed that compressed tablets will be less
preferred or tablet binders that ensure good cohesion at lower
pressures could be used.
Flavobacterium sp. Flavobacterium multivorum, a nonfastidi-
ous and nonpathogenic bacterium that rapidly accumulates zeax-
anthin, is considered to be a potential source of the carotenoid
(Dasek and others 1973; Pasamontes and others 1997; Alcan-
tara and Sanchez 1999; Masetto and others 2001). Synthesis of
a pure form of the R-R isomer of zeaxanthin involves ferment-
ing bacterial cells from a strain of Flavobacterium multivorum
(ATCC 55238) (Garnett and others 1998). The bacterial strain can
synthesize zeaxanthin as a sole carotenoid with virtually no sub-
stantial amounts of other carotenoids under proper fermentation
conditions. Therefore, the extremely difficult task of purifying zea-
xanthin by separating it from closely related carotenoids can be
completely avoided if this bacterial strain or its descendants are
used.
In the isolation and pure cultivation of the yellow Flavobac-
terium microorganisms, material from the natural source is sus-
pended in physiological saline (Gierhart 1994). Streak cultures
are applied to Petri dishes and the yellow colonies growing
on the agar are examined for carotenoid content. Colonies of
Flavobacterium are identified by comparing the cells to the tax-
onomic description provided in Bergey’s Manual (1984 edition).
The microorganisms are identified by their zeaxanthin content
confirmed by analytical procedures such as high-performance
liquid chromatography (HPLC). A method of extracting zeaxan-
thin involves carefully drying the cell mass, pulverizing the dried
cell mass, digesting the pulverized material with an inert organic
solvent, filtering the solution, and isolating the pure zeaxanthin
by elution of the filtration residue with an inert organic solvent
such as ethanol, acetone, or chloroform.
Lyophilized coarse-grain zeaxanthin powder
In an attempt to isolate zeaxanthin, Flavobacterium multivo-
rum cells (ATCC 55238) were fermented to produce zeaxanthin
and separated from the liquid broth by centrifugation. Acetone
was added to the cells to extract a majority of the zeaxanthin
into the solvent phase. The cells were then placed in a filter press
to remove the cell solids and acetone was evaporated from the
liquid under warm conditions (about 33 to 43 ◦
C) and mild vac-
uum (Guerro-Santos and others 2005). The oily residue contained
zeaxanthin in a large crystalline form and some cell residues. Wa-
ter was added and the mixture was processed using a standard
mechanical mixer, and forced through a Teflon filter at 15 psig
pressure. The zeaxanthin and the solids that stayed on the filter
were washed with hexane, a mixture of 95% hexane and 5%
acetone, and then a mixture of 90% hexane and 5% acetone to
remove impurities. Subsequent washings with pure acetone re-
sulting in an acetone/zeaxanthin mixture were passed through
a silica gel column. Since zeaxanthin would not cling to silica
as tightly as various other impurities will, the solvent passing
through the silica gel eluted the zeaxanthin from the gel. The
resulting zeaxanthin, after removal of acetone, was 95% to 99%
pure. Lyophilization at −70 ◦
C and less than 100 mbar resulted
in free-flowing powders at room temperature.
Zeaxanthin in viscous oily liquid
The R-R isomer produced by Flavobacterium multivorum
(ATCC 55238) can be concentrated in large quantities and at low
cost into a viscous oily fluid containing about 5% to 20% zeaxan-
thin by means of a simple solvent extraction process (Garnett and
others 1998). The oily fluid may be mixed with a carrier such as
vegetable oil and enclosed within a digestible capsule, compara-
ble to a conventional capsule containing vitamin E. Alternately,
a zeaxanthin fluid can be added to various types of foods such
as margarine, dairy products, syrup, cookie dough, and certain
types of meat preparations which are not subjected to harsh cook-
ing. Additional purification steps can be used to produce granular
zeaxanthin containing nearly pure zeaxanthin. Such processing
methods may be used to create formulations such as ingestible
tablets to be added to soups, salads, drinks, or other foods.
Micelles containing zeaxanthin
Zeaxanthin-containing “micelles,” which are less than 1 µm in
diameter, are obtained from either the solvent extract of biomass
or the oily fluid by using certain types of bile salts (Garnett and
others 1998). An oily fluid may be mixed with a suitable bile salt
such as the phosphate salts of glyco- or taurocholate or by use of
gall bladder extracts containing mixtures of bile salts. The bile ma-
terial mixed with either the solvent extract or oily mass and with
salts such as sodium chloride, calcium chloride, or potassium
chloride is processed in a mechanical homogenizer to produce
micelles that are dried and diluted to a desired concentration
using a carrier or diluent fluid such as vegetable oil. The mix-
ture may be enclosed within a capsule or other device that will
aid in swallowing and help protect the resultant micelles against
degradation by stomach acid.
Microcystis aeruginosa. High-speed counter-current chro-
matography has been successfully applied in the isolation and
purification of zeaxanthin from the cyanobacterium Microcystis
aeruginosa. Preparative high-speed counter-current chromatog-
raphy with a 2-phase solvent system composed of n-hexane-ethyl
acetate–ethanol–water (8:2:7:3, [v/v/v/v]) resulted in zeaxanthin
of 96.2% purity in a 1-step separation (Chen and others 2005).
Chinese wolfberries and marigold flowers
Briefly, the commercial extraction of xanthophylls (oleoresin)
from marigold flowers involves the following stages: ensilage,
pressing, drying, hexane extraction, and saponification. The en-
silage is considered critical in determining the efficiency of the
overall process. Isolation of lutein and zeaxanthin esters from
marigold flowers and Chinese wolfberries may involve the use
of hexane as a solvent for extraction at room temperature fol-
lowed by evaporation of hexane at 60 ◦
C. The isolated oleoresin
may be mixed with an alcohol to remove some of the by-product
impurities.
Enzymatic treatment can enhance xanthophyll extraction from
marigold flowers (Delgado-Vargas and Paredes-López 1997;
Bárzana and others 2002). Petals treated with 0.1% (w/w)
Econase-cep (Enzyme Development Corp., N.Y., U.S.A.) for a pe-
riod of 120 h produced a significant increment in xanthophyll
yield (24.7 g/kg dry weight) that compared favorably with the
yield from the untreated control (11.4 g/kg dry weight) (Delgado-
Vargas and Paredes-López 1997). Enhanced yields (>85%) of
recovered carotenoids were also obtained from fresh flowers
macerated with 0.3% (v/w flower) Viscozyme/Neutrase (Novo-
Nordisk, Bagsvaerd, Denmark) and simultaneously treated with
hexane (Bárzana and others 2002). However, enzymatic treat-
ments present practical limitations owing to the high cost of com-
mercial enzymes. Supercritical fluid extraction (SFE) is a viable
alternative for many commercial separation applications to ob-
tain carotene, lutein, and oleoresin from marigold flowers (Favati
and others 1988; Naranjo-Modad and others 2000).
Lutein and zeaxanthin may also be isolated by simultaneous
extraction and saponification at room temperature using tetrahy-
drofuran and alcoholic potassium or sodium hydroxide. To obtain
pure zeaxanthin from Chinese wolfberries, zeaxanthin dipalmi-
tate was saponified by dissolving in THF and treated with 10%
KOH in methanol or ethanol (Khachik 2005). The mixture was
stirred at room temperature for 1 h to complete the hydrolysis

of zeaxanthin dipalmitate to zeaxanthin. The solvents were co-
distilled under reduced pressure and zeaxanthin crystallized as
a dark yellow-orange solid. The crystals were centrifuged and
vacuum-dried at 60 ◦
C. The yield of zeaxanthin was 16 mg from
36 mg of zeaxanthin dipalmitate (Figure 7).
Corn
Supercritical fluid extraction (SFE) can extract yellow pigments
from corn gluten meal, a by-product of corn starch processing
(Jing 2004); optimum conditions are a temperature of 40 ◦
C, a
pressure of 20 MPa, and a time of 120 min, with 20% absolute
ethyl alcohol as a cosolvent. Yields of corn pigment extracted by
SFE were 2.2 times that obtained by solvent extraction. However,
the pigment was found to be sensitive to sunlight and, therefore,
should be stored in the dark at low temperatures.
Lipophilic carotenoids, notably (all-E)-lutein and (all-E)-
zeaxanthin, and also neoxanthin, violaxanthin, β-carotene, and
chlorophylls a and b from spinach and sweet corn were iso-
lated and analyzed by high-speed counter-current chromatog-
raphy (HSCCC) (Aman and others 2005a). Pretreatment with pro-
teinase can increase the extraction efficiency of lutein, zeaxan-
thin, and total carotenoids from corn gluten meal (Lu and others
2005).
Orange juice
Carotenoid pigments from Brazilian Valencia orange juice
were isolated by open-column chromatography (OCC) after
extraction using acetone and saponification with 10% methano-
lic KOH (Gama and Sylos 2005). The pigments were identi-
fied as α-carotene, zeta-carotene, β-carotene, α-cryptoxanthin,
β-cryptoxanthin, lutein-5,6-epoxide, violaxanthin, lutein, anther-
axanthin, zeaxanthin, luteoxanthin A, luteoxanthin B, mutatox-
anthin A, mutatoxanthin B, auroxanthin B, and trollichrome B.
Leaves of Physalis cups
In the leaves of Physalis, zeaxanthin occurs in the form of the
palmitic acid ester physalien, from which the pigment is obtained
by saponification. Physalien may also be isolated from Physalis
berries (Kuhn and Wiegund 1929).
Determination of Zeaxanthin
Standards for all trans-zeaxanthin, 12 apo-zeaxanthinal, and
parasiloxanthin are available from DSM (DSM Nutritional Prod-
ucts Ltd., Kaiseraugst, Switzerland). All-trans-zeaxanthin is also
available from Fluka (Buchs, St Gallen, Switzerland).
HPLC, MS
The most common method for analysis of carotenoids is
HPLC employing various detection techniques. Both normal-
and reversed-phase systems, either in isocratic or gradient elu-
tion modes, are employed with reversed-phase systems being
more preferred. Antioxidants are added to the mobile phase,
and the column temperature is usually maintained at around
Figure 7 --- Chemical structure of zeaxanthin dipalmitate (Peng and others 2005).
20 ◦
C to prevent decomposition of the carotenoids during the
analysis for better reproducibility of the results. A normal phase
LC-atmospheric pressure chemical ionization (LC-APCI) MS-MS
technique can simultaneously quantify β-carotene, α-tocopherol,
β-cryptoxanthin, zeaxanthin, and lutein in biological materials
(Hao and others 2005). In reversed-phase HPLC, where partition
is the major chromatographic mode, the order is more or less the
reverse of that encountered in normal-phase adsorption open-
column chromatography. Polymeric C18 phases have excellent
selectivity for structurally similar carotenoids such as the geo-
metric isomers of β-carotene (Bushway 1985; Quackenbush and
Smallidge 1986; Lesellier and others 1989; Craft and others 1990)
and of lutein and zeaxanthin (Epler and others 1992). However,
the total carbon load is lower in the wide-pore polymeric phases,
resulting in weak retention of the carotenoids (Craft 1992). The
peaks also tend to be broader and columns from different produc-
tion lots are more variable than with monomeric columns. The
C30 column provides an excellent resolution of photoisomerized
standards of lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-
carotene, and lycopene (Emenhiser and others 1995, 1996).
Temperature regulation is recommended to maintain day-to-
day reproducibility. Variations in column temperature result in
substantial fluctuation of the carotenoids’ retention times. With
a monomeric C18 column and acetonitrile–dichloromethane–
methanol (70:20:10) as mobile phase, no separation of lutein and
zeaxanthin, and 9-cis- and trans-β-carotene occurred at ambient
(30 ◦
C) temperature (Sander and Craft 1990). At subambient tem-
perature (−13 ◦
C), good separation of lutein and zeaxanthin and
baseline separation of 9-cis- and trans-β-carotene were achieved.
In a Vydac 201TP54 (polymeric) column with acetonitrile–
methanol–dichloromethane (75:20:5) as mobile phase, optimum
resolution of lutein, zeaxanthin, β-cryptoxanthin, lycopene, α-
carotene, and β-carotene was achieved at 20 to 22.5 ◦
C (Scott and
Hart 1993). Also, with a Vydac 201TP column and 5% tetrahy-
drofuran in methanol as mobile phase, resolution of lutein and
zeaxanthin and of β-carotene and lycopene was better at lower
temperature (Craft and others 1992).
Cereal and cereal products. A rapid procedure for the extrac-
tion and determination of carotenoids from cereals and cereal
by-products involves sample saponification and extraction fol-
lowed by normal-phase HPLC, allowing separation of the major
carotenoids of cereals, particularly lutein and zeaxanthin (Panfili
and others 2004). Among the cereals analyzed, the highest lev-
els of carotenoids were found in corn (11.14 mg/kg dry weight),
which contained β-cryptoxanthin (2.40 mg/kg dry weight), zeax-
anthin (6.43 mg/kg dry weight), and α + β-carotenes (1.44 mg/kg
dry weight).
Plant pigments. A procedure for the simultaneous determina-
tion of lutein and zeaxanthin stereoisomers in thermally pro-
cessed vegetables by HPLC with diode array detection was
developed by Aman and others (2005b). (Z)-isomers of lutein
and zeaxanthin were prepared by iodine-catalyzed photoiso-
merization. Their structures were analyzed by 1D- and 2D-LC-
NMR spectroscopy, APCI-MS in the positive mode, and UV/Vis

spectroscopy. Near-baseline separation was observed for (13-Z)-
lutein, (13 -Z)-lutein, (all-E)-lutein, (9-Z)-lutein, (9 -Z)-lutein, (13-
Z)-zeaxanthin, (all-E)-zeaxanthin, and (9-Z)-zeaxanthin.
A reversed phase-HPLC method may be used to analyze
the full complement of higher plant photosynthetic pigments
(cis-neoxanthin, neoxanthin, violaxanthin, anteraxanthin, lutein,
zeaxanthin, cis-lutein, chlorophyll b, chlorophyll a, and α- and
β-carotene) (Rivas and others 1989). The separation on a C18
column takes about 10 min, using a single high-pressure pump
and 3 different mobile phases in 3 isocratic steps. The method
introduces a major improvement in higher plant photosynthetic
pigment analysis, resolving in all photosynthetic pigments while
achieving good separation of lutein from its isomer zeaxanthin.
Positive-ion fast-atom bombardment tandem MS (FAB MS-MS)
using a double-focusing mass spectrometer with linked scanning
at constant B/E and high-energy collisionally activated dissocia-
tion (CAD) was used to differentiate 17 different carotenoids, in-
cluding zeaxanthin (Breemen and others 1995). Carotenoids were
either synthetic or isolated from plant tissues, including carrots
and tomatoes. Both polar xanthophylls and nonpolar carotenes
formed molecular ions during FAB ionization. Following colli-
sionally activated dissociation, fragment ions of selected molec-
ular ion precursors showed structural features indicative of the
presence of hydroxyl groups, ring systems, ester groups, and alde-
hydes groups, and the extent of aliphatic polyene conjugation. It
is suggested that the fragmentation patterns observed in the mass
spectra may be used as a reference for structural determination
of carotenoids isolated from plant and animal tissues.
Fruits, fruit juices. An HPLC-diode array detector (HPLC-DAD)
method was used to quantify the content of zeaxanthin dipalmi-
tate, a major carotenoid in Fructus lycii, on a C18 column with
the mobile phase consisting of acetonitrile and dichloromethane
(42:58). Zeaxanthin dipalmitate was the predominant carotenoid,
comprising 31% to 56% of the total carotenoids (Peng and others
2005).
An isocratic RP-HPLC method was developed for routine anal-
ysis of the main carotenoids related to the color of orange
juice, using a more selective wavelength (486 nm) in which
the absorption in the red-orange region of the visible spec-
trum is maximum. Separation was carried out using a mixture
of methanol:acetonitrile:methylene chloride:water (50:30:15:5
[v/v/v/v]) to which small amounts of BHT and triethylamine were
added (0.1%) as the mobile phase. Application of the method to
Valencia ultrafrozen orange juices showed the major carotenoids
to be lutein + zeaxanthin (36%), lutein 5, 6-epoxide (16%), an-
theraxanthin (14%), and β-cryptoxanthin (12%).
An HPLC-MS technique was developed for identification and
determination of carotenoids and their fatty acid esters in citrus
fruit juices (orange and tangerine juice concentrates) (Wingerath
and others 1996). Gradient and isocratic HPLC separated as
many as 38 carotenoid components in extracts from fruit juices.
Structural elucidation was based on UV/vis spectroscopy, matrix-
assisted laser desorption ionization (MALDI) post-source-decay
(PSD) MS, and comparison with synthetic reference compounds.
The xanthophylls lutein, zeaxanthin, α-cryptoxanthin, and β-
cryptoxanthin were detected in extracts of saponified tangerine
concentrate.
Poultry products. An HPLC-DAD with a C30 phase was used
for the simultaneous separation of xanthophylls in egg yolks. Peak
identification was carried out by LC-(APCI) MS (Schlatterer and
Breithaupt 2006).
Fish. An HPLC procedure was developed to separate 2 retinol
forms (retinol1 and retinol2 or dehydroretinol), their correspond-
ing retinal forms, and carotenoid pigments commonly found in
fish tissues (Guillou and others 1993). A reversed-phase C18
column was used with an isocratic solvent system (acetonitrile/
dichloromethane/methanol/water/propionic acid, 71:22:4:2:1
[v/v/v/v/v]). Prior to HPLC analysis, fatty tissues were defatted
with acetone/methanol (1:1 [v/v]) at −80 ◦
C or on a silica gel
column. The method gave satisfactory resolution of polar com-
pounds (retinol and retinal forms, astaxanthin and phoenicox-
anthin or zeaxanthin) and an acceptable elution time for less
polar molecules (retinyl palmitate, α- and β-carotene). Levels of
retinoid forms and carotenoids were determined in eyes, blood,
and eggs of mature rainbow trout.
TLC
Carotenoids are so strongly colored that use of special reagents
for the detection is normally unnecessary (Bolliger and Konig
1969). A number of precautions must be taken to prevent loss
of pigments. These include applying the sample quickly and run-
ning the chromatogram without delay, applying the sample under
subdued illumination and running the chromatogram in the dark,
and using an atmosphere of nitrogen if possible. The rapid fad-
ing of carotenoids on developed thin layers may be delayed by
spraying the chromatogram with a solution of liquid paraffin in
light petroleum (Bolliger and Konig 1969).
Zeaxanthin can be separated from other carotenoids using a
silica gel G (activated) plate and a solvent system consisting of
dichloromethane:ethyl acetate (4:1 [v/v]) where lutein and zeax-
anthin separate with Rf values of 0.35 and 0.24, respectively
(Bolliger and Konig 1969). On silica gel G (activated) plate de-
veloped with benzene:ethyl acetate:methanol (75: 20: 5, by vol.),
the Rf values of lutein and zeaxanthin are reportedly 0.57 and
0.53, respectively (Davies and others 1970a, 1970b).
Kieselguhr paper can separate carotenoids of many types and
is particularly recommended as being superior to other micro-
scale methods for the resolution of cis-trans isomeric xanthophylls
(Liaaen-Jensen 1971). The Rf values of lutein and zeaxanthin are
0.72 and 0.59, respectively, when developed in 10% acetone in
petroleum ether, and 0.91 and 0.87, respectively, in 20% acetone
in petroleum ether.
NIR reﬂectance spectroscopy
NIR reflectance spectroscopy may be used for screening large
numbers of corn samples for breeding programs aimed at produc-
ing corn hybrids with increased carotenoid concentration (Be-
rardo and others 2004). Genotypic variation in corn carotenoid
concentration was investigated in 64 genotypes, including vari-
eties of different geographical origin, commercial hybrids, lines in
selection, cornflakes, popcorn, and sweet corn. Carotenoid con-
centration in meals from each genotype was determined using
NIR reflectance spectroscopy and HPLC. Comparison of the 2 an-
alytical techniques revealed similar results for each; correlations
of between 0.84 (lutein) and 0.94 (zeaxanthin) were identified.
Isomerization and Oxidation of Zeaxanthin
The highly unsaturated carotenoid is prone to isomeriza-
tion and oxidation (www.hni.ilsi.org). Oxidative degradation, the
principal cause of extensive losses of carotenoids, depends on
the availability of oxygen and is stimulated by light, enzymes,
metals, and co-oxidation with lipid hydroperoxides. Carotenoids
appear to have different susceptibilities to oxidation, with ζ-
carotene, lutein, and violaxanthin being cited as more labile
(Rodriguez-Amaya 2001). Formation of epoxides and apoc-
arotenoids (carotenoids with shortened carbon skeleton) appears
to be the initial step (Figure 8). Subsequent fragmentations yield a
series of low-molecular-weight compounds similar to those pro-
duced in fatty acid oxidation. Thus, total loss of color and bi-
ologic activities are the final consequences. Heat, light, acids,
and adsorption on an active surface (such as alumina) promote

isomerization of trans-carotenoids, their usual configuration, to
the cis forms (Figure 9).
Oxygen, especially in combination with light and heat, is
highly destructive. The presence of even traces of oxygen in stored
samples (even at deep-freeze temperatures) and of peroxides in
solvents (for example, diethyl ether and tetrahydrofuran) or of any
oxidizing agent, even in crude extracts of carotenoids, can rapidly
Figure 8 --- Possible scheme for the degradation of
carotenoids (www.hni.ilsi.org).
Figure 9 --- Chemical structure of zeaxanthin in the confor-
mation: all-trans, 9-cis, 13-cis, and 15-cis.
lead to bleaching and the formation of artifacts such as epoxy
carotenoids and apocarotenals (Britton 1991). Oxygen can be ex-
cluded at several steps during analysis and storage with the use of
vacuum and a nitrogen or argon atmosphere. Antioxidants such
as butylated hydroxytoluene, pyrogallol, and ascorbyl palmitate
may also be used, especially when the analysis is prolonged. They
can be added during sample disintegration or saponification or
added to solvents (for example, tetrahydrofuran), standard solu-
tions, and isolates. Exposure to light, especially direct sunlight or
ultraviolet light, induces trans-cis photoisomerization and pho-
todestruction of carotenoids. Thus, work on carotenoids must be
performed under subdued light. Open columns and vessels con-
taining carotenoids should be wrapped with aluminum foil, and
thin-layer chromatography development tanks should be kept in
the dark or covered with dark material or aluminum foil. Polycar-
bonate shields are available for fluorescent lights, which are no-
torious for emission of high-energy, short-wavelength radiation.
Inﬂuence of storage and thermal processing
on the stability of zeaxanthin
Pure crystalline zeaxanthin degrades under the influence of
atmospheric oxygen and light. Concentrated zeaxanthin stored
in darkness under the inert gas argon remains stable for a month
at a temperature of 40 ◦
C, but for at least 3 y at a temperature
between −3 and 5 ◦
C. The longest storage period that has been
evaluated involved zeaxanthin in a gelatin matrix, which was kept
at 15 ◦
C for 2 y and exhibited no measurable signs of degeneration
(www.cbg-meb.nl).
Processing of foods may cause all-trans carotenoids to change
into cis-isomers. Cis-isomers differ from trans-isomers in terms
of bioavailability and antioxidant capacity (Scheiber and Carle
2005). In 1 study, all-trans zeaxanthin was incubated at 75 ◦
C for
different time intervals to study the effect of prolonged incubation
at an elevated temperature on isomerization of the xanthophylls.
Partial isomerization of the xanthophyll to the 13-cis conforma-
tion was found to depend on time of reaction and temperature.
Formation of small amounts of the isomers 9-cis and 15-cis was
shown to occur at relatively high temperatures (above 75 ◦
C) but
the rate of the formation of the 13-cis isomer was much higher
(Milanowska and Gruszecki 2005). Updike and Schwartz (2003)
studied the formation of cis isomers of zeaxanthin in canned corn,
kale, green peas, and spinach and microwaved broccoli. Ther-
mal processing was found to increase the cis- isomers of zea-
xanthin by 17%. In zeaxanthin-rich potatoes, 70% of the initial
zeaxanthin content was detected after treatment at 120 ◦
C for
2 h (Behsnilian and others 2006). At all temperatures studied, the
reduction of all-trans carotenoids was accompanied by the gen-
eration of cis-isomers, 9-cis- and 13-cis zeaxanthin in the potato
matrix. However, during pasteurization of orange juice at 90 ◦
C
for 30 s, zeaxanthin content remained unaffected (Lee and Coates
2003).
A slow drying process with an irregular temperature profile
(where the temperature fluctuates with the following mean val-
ues: from 22 to 34 ◦
C in 24 h; 5 d at 44 ◦
C; finally lowered to 5 ◦
C
in 48 h) was applied to whole pods of a Spanish pepper variety
(Perez-Galvez and others 2004). The zeaxanthin losses during
the first 24 h (38%) were attributed to enhanced activity of oxida-
tive enzymes. During the temperature-holding and the cooling-
down periods, only fluctuations in the zeaxanthin content were
observed. Zeaxanthin losses over 80% have been reported on
drying by a traditional sun-drying method for the production of
paprika (Topuz and Ozdemir 2003). The zeaxanthin content of
dried red pepper (pods, cut, or whole) remained stable for up to
4 mo storage at 0 ◦
C. Increasing the storage temperature to 20 ◦
C
or the specific area of the material (powder) yielded significant
zeaxanthin losses (Kim and others 2004).

Food products such as fruit juices, soy drinks, yoghurt, ice
cream, biscuits, cereals and cereal bars, margarine, and soft can-
dies with added zeaxanthin (and lutein) remain stable for periods
of up to 12 mo. “Jelly bears” and extruded cereals showed losses
of zeaxanthin of 25% and 78% after 9 and 12 mo, respectively
(Table 4) (Koenig-Grillo 2002). Encapsulation of carotenoids with
lipids, dextrins, and polyvinylpyrrolidone is 1 approach being
evaluated in order to reduce carotenoid degradation in foods.
Decrease in degradation during storage is reported for encapsu-
lated carotenoids (Selim and others 2000; Basu and Del Vecchio
2001; Rodriguez-Huezo and others 2004).
The small red berry, wolfberry (Fructus barbarum L.), is one of
the richest natural sources of zeaxanthin. Enhanced bioavailabil-
ity of zeaxanthin in a milk-based formulation of wolfberries was
obtained when homogenization of the berries was performed in
hot-skimmed milk (80 ◦
C) compared with the hot water (80 ◦
C)
and warm-skimmed milk treatment of the berries (40 ◦
C) (Benzie
and others 2006).
Chemical Synthesis of Zeaxanthin
Synthetic zeaxanthin is an orange-red crystalline powder with
little or no odor. It is practically insoluble in water and ethanol,
Table 4 --- Stability of zeaxanthin and lutein in foods.
Results
Physical and Retention of Retention of
Product Storage time visual stability lutein (%) zeaxanthin (%)
Soft drinks without juice 6 mo Physically stable without pectin; No chemical stability evaluated
not stable with pectin and 10 ppm of lutein
Juice drinks 6 mo No visible influence 86 92
Health eye drink 3 mo No visible influence 100 100
Yogurt Processing No visible influence 96 100
Yogurt 3 wk No visible influence 98 100
Ice cream Processing No visible influence 94 94
Ice cream 6 mo No visible influence 100 100
Soy drink Processing No visible influence 95 92
Soy drink 6 mo., ambient temperature Tiny yellow ring, flaky sediment from juice pulp 95 92
Soy drink 6 mo, 5 ◦C No ring, flaky sediment from juice pulp 100 100
Biscuits 6 mo No visible influence 100 96
Extruded cereals 12 mo No visible influence 21 22
Cereal bars 6 mo No visible influence 99 78
Margarine 6 mo No visible influence 93 Not tested
Jelly bears 9 mo No visible influence 70 75
Figure 10 --- The Wittiz reaction used
in the production of zeaxanthin
(synthetic).
and slightly soluble in chloroform giving a clear intensive orange-
red solution. It is composed of trans-zeaxanthin and minor quan-
tities of cis-zeaxanthin, 12 -apo-zeaxanthinal, diatoxanthin, and
parasiloxanthin. Nonfermentation processes suffer from several
disadvantages; they typically require numerous reaction steps,
and each step has a less than 100% yield, so that the final yield
of zeaxanthin at the end of the multistep processing tends to be
relatively poor. In addition, chemical synthesis tends to yield un-
desirable S-S and S-R stereoisomers of zeaxanthin, as well as
various conversion products such as oxidized zeaxanthin, and
zeaxanthin molecules that have lost one or more of the double
bonds in the straight portion or end rings.
Zeaxanthin synthesis follows a sequence of reactions in which
the final step is a double Wittig condensation of a symmetri-
cal C10-dialdehyde as the central building block with 2 equiva-
lents of the appropriate C15-phosphonium salt (Figure 10). The
1st step in the process is the production of an enantiopure C9-
hydroxyketone either by an enantioselective catalytic hydrogena-
tion or by a biocatalytic process combined with a chemical re-
duction. The enantiopure C9-hydroxyketone is then converted
into the C15-phosphonium salt employed in the Wittig condensa-
tion. The C10-dialdehyde is commercially available. A similar ap-
proach is used for the synthesis of other symmetrical carotenoids,

such as lycopene, β-carotene, and astaxanthin, also commer-
cially available (Soukup and others 1996; Ernst 2002).
In the method described by Loeber and others (1971), a mix-
ture of Wittig salt (146 mg, 0.26 mmol), 2, 7-dimethylocta-2,4,6-
trienedial (16 mg, 0.1 mmol), and 1.2-epoxybutane (0.5 g) was
heated for 1 h at 100 ◦
C under nitrogen in a sealed tube, and
cooled. The crude product was chromatographed on alumina
(gradient elution with light petroleum, benzene, and ethyl ac-
etate); the main red band yielded an orange solid (35 mg, 62%).
Crystallization from methanol gave zeaxanthin, m.p, 211 ◦
C; λmax
(C6H6) 589, 462, and 439; V max, 3610, 1035, and 965 cm−l
. The
product did not separate from natural zeaxanthin on mixed TLC.
[Kieselgel HF 254; 30% acetone in light petroleum (b.p. 60 to
80 ◦
C)].
Zeaxanthin and its dipalmitate and physalien have been syn-
thesized by Isler and others (1956a, 1956b). Starting from the
optically pure hydroxyketone 1, an efficient synthesis of (3R, 3 R)-
zeaxanthin is described by Widmer and others (1990) (Figure 11).
Creemers and others (2002) synthesized pure all-E zeaxanthin
as follows: TiCl3 (0.22 g, 1.4 mmol) was added to dry THF un-
der argon; LiAlH4 (0.04 g, 1.1 mmol) was added to the suspen-
sion and the mixture was stirred for 2 h at RT. 3-Hydroxyretinal
(0.21 g, 0.7 mmol) in THF was slowly added and the mixture
was stirred overnight at RT. HCl (1 M) was added and the mix-
ture was extracted twice with diethyl ether. The combined or-
ganic layers were washed with NaCl, dried with MgSO4, and
concentrated in vacuo. After purification on a silica-gel column
(50% diethyl ether/petroleum ether), and subsequent recrystal-
lization (CH2Cl2/EtOH, −20 ◦
C), pure all-E zeaxanthin (0.02 g,
0.04 mmol, 10%) was obtained.
In vitro, the conversion of lutein to meso-zeaxanthin is readily
achieved in a base-catalyzed reaction (Bone and others 1993),
and this is the basis of an industrial process for its synthesis and
use in poultry feeds. The conversion hypothesis is supported by
the distribution of the individual carotenoids in the retina (Bone
and others 2007).
Figure 11 --- Synthesis of (3R, 3 R)-zeaxanthin as described by Widmer and others (1990).
Acceptable Daily Intake of Zeaxanthin
The safety of zeaxanthin in supplements has been confirmed
by the New Dietary Ingredient Notification for dietary 3R, 3R
zeaxanthin. The notification was filed by Roche Vitamins and
was reviewed by FDA without objection in 2001 (Docket Nr
95S-0316: 75-Day Premarket Notifications for New Dietary In-
gredients, Report Nr 96). In addition, the Joint Food and Agricul-
ture Organization/World Health Organization (FAO/WHO) Ex-
pert Committee on Food Additives (JECFA) recently completed a
toxicological evaluation of zeaxanthin. JECFA established a group
Acceptable Daily Intake (ADI) for lutein and zeaxanthin of 0 to
2 mg/kg body weight for zeaxanthin (JECFA, Summary and Con-
clusions, 63rd meeting, Geneva, June 2006). There are also di-
rect animal data demonstrating retinal protective effects of dietary
zeaxanthin (Thomson and others 2002).
Functional Properties of Zeaxanthin
The intake of a carotenoid-rich diet is epidemiologically related
to a lower risk of some types of cancer and AMD (Perez-Galvez
and others 2003). Zeaxanthin is used in the prevention of human
macular degeneration and also for poultry pigmentation (Berry
and others 2003). For infants, human milk is the main source
of lutein and zeaxanthin until weaning occurs. The carotenoids
appear to be important as protective factors in the retinal pigment
epithelium of the newborn infants (Jewell and others 2001, 2004).
The evaluation of zeaxanthin in the cosmetic area, especially in
solar protection, is under way owing to its natural function as a
protectant of cells from photosensitization.
Nutraceutical value of zeaxanthin
Age-related macular degeneration (AMD). AMD, the leading
cause of irreversible blindness in adults, is the result of degen-
erative changes that occur in the central region of the retina and
the macula, eventually leading to loss of vision. “Dry” macu-
lar degeneration is caused by a thinning of the macula’s layers,

and vision loss typically is gradual. However, tiny, fragile blood
vessels can develop underneath the macula. “Wet” macular de-
generation can result when these blood vessels hemorrhage, and
blood and other fluid can further destroy macular tissue, even
causing scarring. In this case, vision loss can be rapid, over
months or even weeks. Lutein and zeaxanthin may play a ma-
jor role in the prevention of AMD. They are not metabolized to
vitamin A, and they accumulate in the macular region of the hu-
man retina, where they prevent damage by absorbing high-energy
blue light through their antioxidant properties (Handelman and
others 1988; Britton 1995; Snodderly 1995). It is suggested that
people supplement their diets with 4 mg of lutein/zeaxanthin
daily. Dietary studies have confirmed the association between
frequent consumption of spinach or collard greens, particularly
good sources of lutein and zeaxanthin, and lower AMD risk
(www.aoa.org). Foods provide other phytochemicals that might
act in a synergistic way to help the absorption and utilization
of the nutrient or to aid in the way it protects the body from
damage.
Zeaxanthin and lutein are specifically accumulated in the mac-
ular region of the retina where they bind to the retinal pro-
tein, tubulin (Handelman and others 1988, 1991). Zeaxanthin
is specifically concentrated at the macula, whereas lutein is dis-
tributed throughout the retina. Within the central macula, zea-
xanthin is the dominant component, up to 75% of the total,
whereas in the peripheral retina, lutein predominates, usually
being 67% or greater. Khachik and others (2002) found the ra-
tio of lutein to dietary 3R, 3R –zeaxanthin in the lens to be ap-
proximately 1.5:1, which suggests a preferential uptake for di-
etary 3R, 3R –zeaxanthin from the diet and plasma to the lens.
The human retina accumulates lutein and zeaxanthin together
with nondietary metabolites of lutein and zeaxanthin includ-
ing meso-zeaxanthin [(3R, 3 S)-β, β-carotene-3, 3 diol] and 3 -
oxolutein [(3-hydroxy-3 -oxo-β-ε-carotene)] (Bernstein and oth-
ers 2001). A significant fraction of lutein in the central retina is
converted into meso-zeaxanthin. In the center of the retina, the
ratio of meso-zeaxanthin to lutein is the highest and approaches
1:1. Data from autopsy eyes indicate that the (lutein + meso-
zeaxanthin):zeaxanthin ratio varies throughout the retina from
2:1 in the center, where most of the conversion occurs, to 3:1
in the periphery (Bone and others 1997). Thus there may be
an advantage in providing meso-zeaxanthin in supplements at
the expense of lutein if the goal is to raise the overall zeaxan-
thin (Bone and others 2007). Lycium Chinese Mill, also known
as Chinese boxthorn used in traditional Chinese medicine for
the treatment of a number of disorders, including visual prob-
lems, has zeaxanthin in its fatty acid ester form as the principal
carotenoid.
Cataract. Zeaxanthin and lutein may also be protective against
age-related increase in lens density and cataract formation.
Cataracts are the leading cause of impaired vision, with a large
percentage of the geriatric population exhibiting signs of the le-
sion. Cataracts are developmental or degenerative opacities of
the lens that result in a gradual, painless loss of vision. Studies
examining lutein and zeaxanthin levels in extracted cataractous
lenses have found up to 3-fold higher levels in the newer ep-
ithelial tissue of the lens than in the older inner cortex portion.
The epithelial cortex layer comprises 50% of the tissue, yet it has
been found to contain 74% of the total lens lutein and zeaxan-
thin, supporting the hypothesis that these nutrients are protective
against the oxidative damage causing cataract formation (Yeum
and others 1999).
Conjugated double bonds are particularly effective at absorb-
ing harmful UV rays and also quenching singlet oxygen that
produces reactive oxygen species (ROS). Zeaxanthin, which is
fully conjugated, may offer better protection than lutein against
oxidative damage of the lipid matrix caused by the blue and
near-ultraviolet light radiation in macular membranes. A pro-
tective effect of lutein and zeaxanthin against the oxidative
damage of egg yolk lecithin liposomal membranes induced by
exposure to UV radiation and incubation with 2,2 -azobis(2-
methypropionamidine)dihydrochloride, a water-soluble peroxi-
dation initiator, is shown by Sujak and others (1999). How-
ever, both lutein and zeaxanthin were found to protect lipid
membranes against free radical attack with almost the same
efficacy. As a free-radical scavenger, β-carotene and zeaxan-
thin, with the same chromophore, are less effective than ly-
copene. Lycopene is considered to be the most effective free
radical scavenger owing to its 11 conjugated coplanar double
bonds.
Cancer. Zeaxanthin has cancer-preventive properties. Sponta-
neous liver carcinogenesis in C3H/He male mice was suppressed
when fed for 40 wk with zeaxanthin at a concentration of 0.005%
and mixed as an emulsion in drinking water (Nishino and others
1999).
Inhibition of LDL oxidation. The carotenoid plays an impor-
tant role in the inhibition of macrophage-mediated LDL ox-
idation (Keri and others 1997). Zeaxanthin when incubated
with human monocyte-macrophages for 24 h with human low-
density lipoprotein (LDL) was found to inhibit LDL oxidation in
a concentration-dependent manner, suggesting that zeaxanthin
might help in slowing atherosclerosis progression.
Pigmentation of poultry and ﬁsh
Zeaxanthin is preferred over other carotenoids for enhancing
pigmentation in poultry and fish due to its potency to provide a
true color and ability to deposit evenly in the flesh and eggs (Orn-
dorff and others 1994). Zeaxanthin is at least 1.5 times as potent
as lutein. When administered to poultry in high doses, carotenoid
or carotenoid-containing compounds such as canthaxanthin, al-
falfa, and cayenne pepper cause abnormal red or purple colors in
the flesh and color striations in yolks. High doses of lutein in the
feed have been shown to impart a greenish hue to poultry flesh
and egg yolks. Beta-carotene is not deposited well in the flesh
of poultry, and canthaxanthin apparently deposits in the iris of
the eye. Zeaxanthin, in contrast, imparts a yellow-red color even
at high doses and deposits well in poultry flesh, based on high
zeaxanthin-containing corn feed studies. Labeled zeaxanthin fed
to laying hens undergoes conversion to (6S,6 S)-ε, ε-carotene-3,3
dione and the intermediate (3R,3 S)-3-hydroxy-β, ε-carotene-3 -
one, both of which appear in the egg yolks (Schiedt and others
1981). Some fish and crustaceans, such as shrimp, goldfish, and
carp, apparently can convert zeaxanthin into the red-colored pig-
ment astaxanthin, suggesting that feeding of zeaxanthin to such
fish and crustaceans will enhance desirable red coloration. The
pigment-containing biomass can be used to color foodstuffs with-
out the necessity for isolating pure zeaxanthin (Gierhart 1994).
For poultry products, there are problems with stability and bio-
logical availability, particularly with xanthophylls from marigold
and alfalfa. Most marigold products must be solvent-extracted
and saponified, and may require the inclusion of antioxidants in
the extraction process (Gierhart 1994).
Absorption of Zeaxanthin
Humans cannot synthesize carotenoids and, therefore, must
rely on dietary sources to provide sufficient levels. The human
body absorbs zeaxanthin in the same way as other carotenoids

and fat-soluble vitamins. There is no difference between syn-
thetic and natural zeaxanthin in terms of the way they are pro-
cessed by the human system. The average concentration (ng/g)
of zeaxanthin in human tissues and skin is reported to be liver,
591; lung, 90; breast, 14; prostate, 35; colon, 32; and skin,
6 (Khachik and others 1998; Scholz and others 2000). Aver-
age carotenoid concentration (ng/tissue) in human ciliary and
RPE/choroid is 2.54 and 4.85, respectively (Bernstein and others
2001).
The biological availability of zeaxanthin depends not only on
the composition of the matrix containing it, but also on other
foods consumed at around the same time. The major factors lim-
iting the bioavailability include molecular structure, interactions
of xanthophylls with other nutrients (mainly dietary fat), the man-
ner in which the food is processed, the physical form, speciation,
molecular linkage of the carotenoid in the plant tissue, the nutri-
tional status and physiological state of the subject, and genetic
background and the physical disposition of xanthophylls in the
food matrix (Yeum and Russell 2002). Carotenoids are best ab-
sorbed in the presence of fat, but as little as 3 to 5 g in a meal
appear to ensure carotenoid absorption (van Het Hof and oth-
ers 2000a). Zeaxanthin in egg yolks might be highly bioavailable
because of its association with the lipid matrix of the egg yolk
(Handelman and others 1999). The carotenoids in egg yolk are in
a digestible lipid matrix consisting of cholesterol (200 mg/yolk),
phospholipids (1 g/yolk), and triacylglycerols (4 g/yolk) (Human
Nutrition Information Service 1989). Such a lipid matrix may be
optimal for carotenoid absorption from the diet. Only about 5%
of the carotenoids in whole, raw vegetables are absorbed by the
Figure 12 --- Pathway of carotenoid
absorption and metabolism: C =
carotene; X = xanthophylls; LPL
= lipoprotein lipase; HDL =
high-density lipoprotein; VLDL =
very low-density lipoprotein;
LDL=low-density lipoprotein.
intestine, whereas 50% or more of the carotenoid is absorbed
from micellar solutions. Thus, the physical form in which the
carotenoid is presented to intestinal mucosal cells is of crucial
importance (Olson 1994).
The 1st step in carotenoid bioavailability is release from
the food matrix. Food processing activities such as ther-
mal processing, mincing, or liquefying result in changes to
carotenoid chemistry, probably through isomerization or oxi-
dation reactions (Kopsell and Kopsell 2006). However, freez-
ing or low-temperature storage generally preserves carotenoid
concentration by reducing potential enzymatic oxidation. Pro-
cessing activities usually increase bioavailability through in-
creased release of bound carotenoids from the food ma-
trix; however, thermal degradation in carotenoid chem-
istry might adversely affect bioavailability in some food
crops.
Carotenoids are lipid-soluble molecules that follow the absorp-
tion pathway of dietary fat (Figure 12). Early in the digestive pro-
cess, carotenoids are partially released from the food matrix by
mastication, gastric action, and digestive enzymes (Deming and
Erdman 1999). Carotenoids released from the food matrix mi-
grate and solubilize into lipid globules of varying sizes in the
stomach, where they are eventually transformed into smaller
lipid emulsion particles by normal digestive motility. Solubiliza-
tion of individual carotenoid molecules into lipid emulsions is
thought to be a selective process related to the specific polar-
ity of each molecule. Nonpolar carotenes most likely migrate to
the triacylglycerol-rich core of the particle, while the more polar
xanthophylls orient at the surface monolayer along with proteins,

phospholipids, and partially ionized fatty acids. Borel and oth-
ers (1996) reported this selective orientation of carotenoids in
phospholipid-triacylglycerol droplets using an in vitro biological
emulsion model. Beta-carotene, a nonpolar carotene, was shown
to solubilize in the core of the droplets, while zeaxanthin, a more
polar xanthophyll, accumulated at the surface. Selective orienta-
tion of carotenoids in membranes, micelles, and lipoproteins is
most likely similar to that of other lipid molecules and is based
upon the polarity, length, and structure of the molecule (Dem-
ing and Erdman 1999). Carotenoids solubilized in lipid emulsion
particles are transported from the stomach to the duodenum of
the small intestine (Deming and Erdman 1999). Dietary fat in the
duodenum triggers the release of bile acids from the gall blad-
der and regulates levels of pancreatic lipase. Bile acids aid in
the reduction of lipid particle size and stabilization into mixed
micelles. Dietary fat increases the synthesis and secretion of pan-
creatic cholesteryl esterase (CEL) and chylomicrons. CEL is ca-
pable of hydrolyzing triacylglycerols, phospholipids, cholesteryl
esters, and vitamin A and E esters in the presence of bile salts.
Carotenoids are solubilized into mixed micelles along with di-
etary triacylglycerols, their hydrolysis products, phospholipids,
cholesterol esters, and bile acids (Deming and Erdman 1999).
Studies on the capacity of CEL to hydrolyze zeaxanthin esters
incorporated into micelles showed mixed micelles containing
zeaxanthin esters extracted from wolfberry and incubated with
CEL (1000 unit/L) decreased by 84% after 4 h (Chitchumroon-
chokchai and Failla 2006). The decrease in zeaxanthin diesters
was majorly associated with the appearance of free zeaxanthin
in the micelles. In contrast, free zeaxanthin was not detected in
the medium without CEL.
Uptake of carotenoids into the intestinal mucosa occurs by
passive diffusion (Deming and Erdman 1999). The process re-
quires solubilization of the mixed micelle in the unstirred water
layer surrounding the microvillus cell membrane of the entero-
cyte. The mixed micelles collide and diffuse into the membrane,
releasing carotenoids and other lipid components into the cytosol
of the cell. Parker (1996) suggested a concentration gradient be-
tween the micelle and the cell membrane to most likely deter-
mine the rate of diffusion. High doses of carotenoid may saturate
uptake leading to insufficient removal from the plasma mem-
brane, thereby reducing this concentration gradient. Carotenoid
uptake into the enterocyte does not ensure its metabolism or ab-
sorption in the body. Carotenoids in the enterocyte may be lost
in the lumen of the gastrointestinal tract due to normal physio-
logical turnover of the mucosal cells (Erdman and others 1993).
Selective surface orientation of more polar xanthophylls into mi-
celles suggests that their uptake into the enterocyte and incor-
poration into chylomicrons may occur before that of nonpolar
carotenes. Gartner and others (1996) reported the preferential
uptake of lutein and zeaxanthin from the intestinal lumen into
chylomicrons in humans, even in the presence of high amounts
of β-carotene. Interestingly, while most xanthophylls are present
in fruits as esters (Khachik and others 1991), only free xantho-
phylls have been found in the chylomicrons and serum of humans
(Wingerath and others 1995), suggesting a requirement for hydrol-
ysis of carotenoid esters prior to uptake and incorporation into
chylomicrons. Several human studies show that free xanthophylls
are present in the triglyceride-rich fraction of plasma after inges-
tion of a meal enriched with esterified zeaxanthin (Wingerath
and others 1995; Breithaupt and others 2004) and cryptoxanthin
(Breithaupt and others 2003). Wingerath and others (1995) also
reported an increase in the concentration of free cryptoxanthin,
zeaxanthin, and lutein in chylomicrons and sera of human sub-
jects after they ingested tangerine juice concentrate that was rich
in the esterified forms of the xanthophylls. These observations
suggest that carotenoid esters are hydrolyzed before incorpora-
tion into nascent chylomicrons synthesized in the absorptive in-
testinal epithelial cells.
Chylomicrons are secreted from the enterocyte into the lym-
phatic circulation for transport to the liver. Prior to hepatic up-
take, chylomicrons in the bloodstream are rapidly degraded by
lipoprotein lipase associated with tissue endothelium and trans-
formed into chlylomicron remnants. During this process, some
carotenoids may be taken up by extrahepatic tissues (Deming
and Erdman 1999). However, most chylomicron remnants deliver
carotenoids to the liver where they are stored or resecreted into
the bloodstream in very low density lipoproteins (VLDL) (John-
son and Russell 1992). Circulating VLDLs are subsequently delipi-
dated to low-density lipoproteins (LDL). Similar to other nonpolar
lipids, carotenes such as β-carotene and lycopene are thought to
migrate to the hydrophobic core of lipoproteins, while the more
polar xanthophylls reside closer to the surface where the likeli-
hood of exchange among lipoproteins is enhanced (Romanchik
and others 1995). In the fasting state, LDLs are the main carriers of
nonpolar carotenoids, β-carotene, α-carotene, and lycopene, in
human serum (Pateau and others 1998). The more polar xantho-
phylls are evenly distributed between high-density lipoproteins
(HDL) and LDL, and to a lesser extent VLDL. Carotenoids released
from lipoproteins, especially LDL, are taken up by extrahepatic
tissues.
Red pepper (Capsicum annuum L.) and its dietary prod-
ucts containing a variety of carotenoids may contribute to the
carotenoid pattern of human blood and tissues. The avail-
ability of carotenoids from paprika oleoresin, including zeax-
anthin, β-cryptoxanthin, β-carotene, and the paprika-specific
oxocarotenoids, capsanthin, and capsorubin, was assessed in
human volunteers (Perez-Galvez and others 2003). At different
time points, the carotenoid pattern in the chylomicron fraction
was analyzed to evaluate carotenoid absorption. Of the ma-
jor carotenoids present in paprika oleoresin, only zeaxanthin,
β-cryptoxanthin, and β-carotene were detectable in consider-
able amounts. Although the xanthophylls were mainly present
as mono- or diesters, only free zeaxanthin and β-cryptoxanthin
were found in the human samples. Although the bioavailability of
the pepper-specific carotenoids, capsanthin, and capsorubin from
paprika oleoresin is very low, the oleoresin is a suitable source of
the provitamin A-carotenoids, β-carotene, and β-cryptoxanthin,
and the macular pigment zeaxanthin.
Assessment of Bioavailability of Zeaxanthin
In vivo methods
The in vivo bioavailability studies imply the consumption of a
certain dose of a nutrient and changes of its concentration (mea-
sured by standard analytical procedures) in the blood plasma
compared with time (such as postprandial period, or the time
after the meal) (Parada and Aguilera 2007). Three parameters
are derived from the kinetics: the area under the curve (AUC),
the maximal plasma concentration (Cmax), and the time to reach
Cmax, tmax. AUC is a measure of the absorption intensity, whereas
Cmax and tmax give an idea of the rate of absorption (Heacock
and others 2004; Manach and others 2005). Another method to
assess bioavailability is to measure the plasma concentration of
a nutrient through an extended period (days, weeks) of constant
consumption of a specific food (van het Hof and others 1999;
Richelle and others 2002). Relevant parameters in this case are
Csat (value at which the concentration remains constant in the
time) and tsat (time at which Csat is attained).
A single-blinded, placebo-controlled, human intervention trial
was used to provide data on the effect of dietary supplemen-
tation with whole wolfberries on fasting plasma zeaxanthin

concentration (Cheng and others 2005). A total of 14 human
subjects took 15 g/d wolfberry (estimated to contain almost 3 mg
zeaxanthin) for 28 d. Age- and sex-matched controls (n = 13) took
no wolfberry in their diet. On supplementation, plasma zeaxan-
thin concentration increased 2.5-fold, indicating zeaxanthin in
whole wolfberries to be bioavailable and that intake of a modest
daily amount could markedly increase fasting plasma zeaxanthin
levels.
The influence of saponification on the deposition in egg yolks
of carotenoids from the oleoresin of red pepper (Capsicum an-
nuum) was studied by Hamilton and others (1990). Four repli-
cates of 10 hens depleted of carotenoid stores were fed for 3
wk with a basal diet of white corn and soybean meal, amended
with (1) saponified or (2) nonsaponified oleoresin. Trans-lutein,
trans-zeaxanthin, and trans-capsanthin accounted for >85% of
the total carotenoids deposited in the yolks. From the ratios of
total carotenoid concentration in the yolks to those in the feed,
the carotenoids from (1) were deposited twice as efficiently as
those from (2).
The main drawbacks of in vivo data are the variability in phys-
iological state of individuals and the possible interaction of the
nutrient with other components in the diet (Boileau and others
1999). Whole plasma pharmacokinetics may not be the most
practical way to measure carotenoid status as plasma concentra-
tions are not only a measure of the absorption of carotenoids
from the diet, but also a measure of exchange from tissue
storage, bioconversion, and excretion (Castenmiller and West
1998). Also, ethical restrictions to abide by severe protocols
when humans and/or animals are used in biological research
severely limit these types of studies (van het Hof and others
2000b).
In vitro methods/gastrointestinal models (GIMs)
In vitro methods are being extensively used since they are rapid,
safe, and do not have the ethical restrictions of in vivo methods.
They are used as a suitable alternative to in vivo assays to deter-
mine bioavailability. In vitro methods simulate either the diges-
tion and absorption processes (for bioavailability) or only the di-
gestion process (for bioaccessibility), and the response measured
is the concentration of a nutrient in the final extract. The digestion
Figure 13 --- In vitro method to determine bioavailability in-
volving a digestion absorption step using a Caco-2 cell
culture (Parada and Aguilera 2007).
process is simulated under controlled conditions using commer-
cial digestive enzymes (for example, pepsin, pancreatin, and so
on) while the final absorption process is commonly assessed us-
ing “Caco-2 cells” cultures. “Caco-2 cells” is the short name for
polarized human colon carcinoma cells line (Verwei and oth-
ers 2005). Methods that simulate under laboratory conditions the
gastrointestinal digestion process are known as gastrointestinal
models (GIMs) (Parada and Aguilera 2007).
The in vitro digestion/Caco-2 cell culture procedure is a rapid
and cost-effective model for screening the bioavailability of
carotenoids from plant foods (fresh spinach, fresh carrots, and
tomato paste) (Figure 13). Chitchumroonchokchai and others
(2004) studied lutein bioavailability by simulating the gastric and
small intestinal phases of digestion, followed by a Caco-2 cell
assay. According to Chitchumroonchokchai and Failla (2006),
ingested xanthophyll esters are processed in a manner similar
to dietary cholesteryl esters in the lumen of the small intestine.
With differentiated cultures of Caco-2 intestinal cells and CEL-null
mice, CEL enhanced intestinal uptake and transport of cholesterol
by cleaving cholesteryl esters both before and after their partition-
ing in micelles to increase the extracellular concentration of free
cholesterol. In contrast, CEL does not affect apical uptake of either
free cholesterol or free zeaxanthin. Once taken up by the entero-
cyte, the metabolism of cholesterol and the xanthophylls differs.
Cholesterol is esterified by acyl-CoA:cholesterol acyltransferase
and incorporated into nascent chylomicrons for secretion into
lymph. In contrast, the concentration of free zeaxanthin remain-
ing relatively constant in Caco-2 cells suggests that enterocytes
do not esterify xanthophylls before their transfer to nascent chy-
lomicrons. The cells preferentially took up free zeaxanthin from
micelles and the extent of transport (4.5%) in chylomicrons was
similar to that reported for lutein in Caco-2 cells (Chitchumroon-
chokchai and others 2004). This low amount is consistent with
the estimated absorption of 3.3% of 5 mg zeaxanthin adminis-
tered to human subjects. Studies are now required to determine
whether, as expected, zeaxanthin uptake occurs in a manner sim-
ilar to that of lutein and to examine possible interactions between
zeaxanthin and the other dietary carotenoids that affect their
absorption.
Commercial Zeaxanthin
Naturally occurring carotenoids are of commercial interest as
coloring agents for foods, pharmaceuticals, cosmetics, and ani-
mal feeds. Owing to their antioxidant properties, most of them
have been proposed in the prevention of chronic diseases (Rao
and Agarwal 1999). Although lutein is almost always accom-
panied by zeaxanthin, the reverse is not true. Pure commercial
dietary 3R, 3R zeaxanthin contains no lutein.
As a supplement, lutein and zeaxanthin are available in ei-
ther the free or esterified forms, which appear to have compa-
rable bioavailability (Bowen and others 2002). Very few com-
mercial carotenoid mixtures contain more than extremely small
or trace quantities of zeaxanthin (Garnett and others 1998). A
commercial carotenoid mixture that lists zeaxanthin as one of
the carotenoids contained in their carotenoid mixtures is the “β-
carotene formula preparation,” sold by General Nutrition Corp.
The great majority of carotenoids in the carotenoid mixtures are
β-carotene and vitamin A. Xangold 10% beadlets, a dark, reddish-
brown tablet grade powder containing natural mixed carotenoid
esters isolated from marigold flowers, comprise mainly lutein es-
ters with very small amounts of zeaxanthin esters. Although com-
mercial lutein/zeaxanthin supplements often contain significantly
more lutein than zeaxanthin, new products are being developed
with higher amounts of zeaxanthin. A new dietary supplement,
with zeaxanthin and lutein in the same 2:1 ratio as found in a

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