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 2003). Zeaxanthin exhibits no vitamin A activity. Zeaxanthin Carotenoids are pigments naturally occurring in a number of and its close relative lutein (Figure 1 and 2) play a critical role fruits and vegetables. They are synthesized by all photosynthetic in the prevention of age-related macular degeneration (AMD), organisms and many nonphotosynthetic bacteria and fungi. They the leading cause of blindness (Snodderly 1995; Moeller and are liposoluble tetraterpenes originating from the condensation of others 2000). Zeaxanthin is isomeric with lutein; the 2 carotene isoprenyl units, which form a series of conjugated double bonds alcohols differ from each other just by the shift of a single double constituting a chromophoric system (Britton 1995). There are 2 bond so that in zeaxanthin all double bonds are conjugated. main classes of naturally occurring carotenoids: (1) carotenes Zeaxanthin is used as a feed additive and colorant in the food such as β-carotene and α-carotene, which are hydrocarbons, are industry for birds, swine, and fish (Hadden and others 1999). either linear or cyclized at one or both ends of the molecule, The pigment imparts a yellow coloration to the skin and egg yolk and (2) xanthophylls, the oxygenated derivatives of carotenes. of birds, whereas in pigs and fish it is used for skin pigmentation All xanthophylls produced by higher plants, such as violaxanthin, (Nelis and DeLeenheer 1991). 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 Stereoisomers of Zeaxanthin risk of laryngeal, lung, and colon cancers and the consumption of Zeaxanthin has 2 chiral centers and, hence, 22 or 4 stereoiso- foods containing carotenoids (Block and others 1992; Steinmetz meric forms. One chiral center is the number ‘3’ atom in the left and Potter 1993). end ring, while the other chiral center is the number ‘3’ carbon The chemical name of zeaxanthin is (all-E)-1,1-(3,7,12,16- in the right end ring (Garnett and others 1998). One stereoiso- tetramethyl-1,3,5,7,9,11,13,15,17-octadecanonaene-1,18-diyl) mer is (3R, 3R)-zeaxanthin; the other is (3S-3S)-zeaxanthin. The bis [2,6,6-trimethylcyclohexene-3-ol]. Synonyms are: 3R, 3rd stereoisomer is (3R, 3S)-zeaxanthin and the 4th (3S-3R)- 3R-β,β-carotene-3,3-diol; all-trans-β-carotene-3,3-diol; zeaxanthin. However, since zeaxanthin is a symmetric molecule, (3R,3R)-dihydroxy-β-carotene; zeaxanthol; and anchovyx- the (3R, 3S)—and (3S, 3R)—stereoisomers are identical. There- anthin. Zeaxanthin, the principal pigment of yellow corn, fore, zeaxanthin has only 3 stereoisomeric forms. The (3R, Zeaxanthin mays L. (from which its name is derived), has a 3S)—or (3S, 3R)—stereoisomer is called meso-zeaxanthin. The  molecular formula of C40H56O2 and a molecular weight of principal natural form of zeaxanthin is (3R, 3 R)-zeaxanthin. (3R, 568.88 daltons. Its CAS number is 144-68-3. It is composed of 3R)-zeaxanthin and meso-zeaxanthin are found in the macula of 40 carbon atoms, yellow in color, and naturally found in corn, the retina, with much smaller amounts of (3S, 3S)-zeaxanthin. egg yolks, and some of the orange and yellow vegetables and Meso-zeaxanthin is a rare isomer present in significant quantities fruits such as alfalfa and marigold flowers (Nelis and DeLeenheer in commercially produced chickens and eggs in Mexico where 1991; Handelman and others 1999; Humphries and Khachik it is commonly added to the feed to achieve desirable coloration in these products (Bone and others 2007).

MS 20070403 Submitted 5/28/2007, Accepted 8/13/2007. Authors are with Food Engineering and Technology Dept., Inst. of Chemical Technology, Univ. Properties of Zeaxanthin of Mumbai, Matunga, Mumbai-400 019, India. Direct inquiries to author Singhal (E-mail: [email protected]). 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.

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 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- Figure 4 --- Calculation of % III/II as indication of spectral matic laser light, it exhibits characteristic wavelength shifts of fine structure (% III/II × 100) (www.hni.ilsi.org). 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). spectrum of zeaxanthin, a derivative of β-carotene, resembles The conjugated double-bond system constitutes the light- that of β-carotene. The ultraviolet and visible absorption data absorbing chromophore that gives carotenoids their attractive of zeaxanthin are shown in Table 1 with calculation of % III/II color and provides the visible absorption spectrum that serves as a as indication of spectral fine structure (% III/II × 100) illustrated basis for their identification and quantification. Cis-isomerization in Figure 4. Figure 5 shows the absorption spectra of isomers of of a chromophore’s double bond causes a slight loss in color, zeaxanthin. small hypsochromic shift, and hypochromic effect, accompanied Carotenoid molecules are strong Raman scatterers. Hence, by the appearance of a cis peak in or near the ultraviolet region. nondestructive resonance Raman spectroscopy could be an ex- All-trans isomers absorb strongly in the visible region between tremely valuable method for the rapid quantitative assessment 400 and 500 nm while cis-isomers exhibit absorption in the near- of carotenoids. There are reports of the detection of resonance UV region, around 320 nm (Rodriguez-Amaya 2001). The visible 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 ferent natural carotenoids (Figure 6). In Flavobacterium R1529, 1970). nicotine blocks zeaxanthin biosynthesis by specifically inhibiting the cyclization reaction (McDermott and others 1974). Lycopene and rubixanthin replace zeaxanthin as the main carotenoid. In Biosynthetic Pathway and Genetic Manipulation the absence of nicotine, lycopene is converted to β-carotene un- of the Pathway for Zeaxanthin Production der anaerobic conditions and into zeaxanthin in the presence of A paramount function of xanthophylls in all photosynthetic or- oxygen. ganisms, including cyanobacteria, is to provide protection against The biosynthesis of IPP and DMAPP from acetyl-CoA via photooxidation. It is proposed that zeaxanthin protects the mem- melavonate has been studied using animal cells and yeasts brane directly against lipid peroxidation by reactive radicals that (Bochar and others 1999). Three acetate units afford the 5 carbon have been created as toxic byproducts during photosynthetic re- atoms of IPP from loss of 1 acetate carboxylic group as CO2. actions. Another mechanism suggests specific xanthophylls to be DMAPP is obtained from IPP by an isomerase. A mevalonate- involved in the de-excitation of singlet chlorophyll (1Chl) that ac- independent 2nd pathway for the biosynthesis of IPP and cumulates in the light-harvesting complexes (LHC) under condi- DMAPP via 1-deoxy-D-xylulose 5-phosphate has been discov- tions of excessive illumination (Demmig-Adams 1990; Demmig- ered in some eubacteria and plants (Rohmer and others 1993). Adams and Adams 1992; Demmig-Adams and others 1996). The nonmevalonate pathway starts with the formation of 1- Carotenoid biosynthesis leads to the all-trans-forms. Hence, all- deoxyxylulose 5-phosphate from pyruvate and glyceraldehyde 3- trans-lycopene, -lutein and -zeaxanthin predominantly occur in phosphate catalyzed by 1-deoxyxylulose 5-phsophate synthase. fresh fruits and vegetables. All-trans-isomers have therefore been The carbohydrate is converted into 2 C-methylerythritol 2, 4- assumed to be the thermodynamically stable form of carotenoids cyclodiphosphate by a series of 4-reaction steps (Eisenreich and (Miebach and Behsnilian 2006). Carotenoids are synthesized others 2002). from the basic C5-terpenoid precursor, isopentenyl diphosphate Genetic studies with Arabidopsis thalina (Rock and Zeevaart (IPP) and dimethylallyl pyrophosphate (DMAPP). IPP is converted 1991; Rock and others 1994), Nicotiana plubaginifolia (Marin into geranylgeranyl phosphate (GGPP) and the dimerization of and others 1996), and the green alga Chlamydomonas reinhardtii     GGPP leads to phytoene (7,8,11,12,7 ,8 ,11 ,12 -octahydro-γ ,γ - (Niyogi and others 1997) revealed the presence of a single-gene carotene) and the stepwise dehydrogenation via phytofluene coding for the zeaxanthin epoxidase enzyme. Thus, a single-gene   (15 Z, 7, 8,11,12, 7 ,8 -hexahydro-γ , γ - carotene), zeta product is apparently responsible for both the biosynthesis of vi-   carotene (7,8,7 ,8 -tetrahydro-γ -γ -carotene), and neurosporene olaxanthin during growth, and development and the epoxidation (7,8-dihydro-γ , γ -carotene) gives lycopene. Subsequent cycliza- reaction leading to the return of zeaxanthin via antheraxanthin tion, dehydrogenation, and oxidation lead to hundreds of dif- to violaxanthin following recovery after irradiance stress. Mutants

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).

Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 31 CRFSFS: Comprehensive Reviews in Food Science and Food Safety with lesions in the zeaxanthin epoxidase gene not only are conse- domonas reinhardtii (Niyogi and others 1997), and Dunaliella quently deficient in antheraxanthin and violaxanthin, but also fail salina (Jin and others 2003). to synthesize neoxanthin (Niyogi and others 1997; Jin and oth- In microalgae, the zeaxanthin content is regulated by light ir- ers 2003). In addition, these mutants accumulate large amounts of radiance. When photosynthetic irradiance is greater than that zeaxanthin that are almost equivalent to the levels of violaxanthin required for the saturation of photosynthesis in the chloro- found in the wild type, even when grown under nonstressed con- plasts of plants and green algae, a reversible violaxanthin de- ditions. Mutations affecting zeaxanthin production exist in green epoxidation reaction occurs to form antheraxanthin and, subse- algae, Scenedemus obliquus (Bishop and others 1995), Chlamy- quently zeaxanthin, resulting in the accumulation of zeaxanthin

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).

32 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008 The carotenoid pigment zeaxanthin... in the chloroplast thylakoids. The enzyme that catalyzes this re- Localization and Sources of Zeaxanthin action, violaxanthin de-epoxidase, is localized in the lumen of Xanthophylls are relatively hydrophobic molecules. Therefore, the chloroplast thylakoids (Hager and Holocher 1994). When the they are typically associated with membranes and/or noncova- absorbed irradiance is lower than that required for saturation of lently bound to specific proteins. In general, primary carotenoids photosynthesis, zeaxanthin is converted back into violaxanthin are localized in the thylakoid membrane, while secondary by the enzyme zeaxanthin epoxidase with the monoepoxide an- carotenoids are found in lipid vesicles either in the plastid stroma theraxanthin being an intermediate in this reversible oxidation– or the cytosol. Most xanthophylls that are found in cyanobacteria reduction process (Hager 1980). The xanthophyll cycle is thought and oxygenic photosynthetic bacteria are associated with chloro- to be essential for the protection of the photosynthetic appara- phyll (Chl)-binding polypeptides of the photosynthetic apparatus tus from photooxidation, and zeaxanthin, antheraxanthin, and (Grossman and others 1995). Among nonphotosynthetic bacte- violaxanthin are associated with the light-harvesting complexes ria and, to a lesser extent, among photosynthetic bacteria and (Demmig-Adams and Adams 1992). In accordance with the role cyanobacteria, xanthophylls and their glycosides can be found of zeaxanthin and violaxanthin in photosynthesis, ABA2mRNA in cytoplasmic and cell wall membranes where they are thought (abscisic acid 2mRNA) is more abundant in photosynthetic than to influence membrane fluidity (Armstrong 1997). in nonphotosynthetic tissues. ABA, a breakdown product of xanthophyll carotenoids (C40) Plant sources via the C15 intermediate xanthoxin (Walton and Li 1995), mod- Green leafy vegetables are good dietary sources of lutein, but ulates the growth and development of plants, particularly dur- poor sources of zeaxanthin. Dietary sources of zeaxanthin include ing seed formation and also in response to environmental stress yellow corn, orange pepper, orange juice, honeydew, mango, and (Zeevaart and Creelman 1988; Giraudat and others 1994). Mu- chicken egg yolk. Jungalwala and Cama (1962) found zeaxanthin tants blocked in the early steps of carotenoid synthesis, for ex- to comprise about 90% of the total carotenoids in the anthers of ample, some viviparous mutants of maize (vp2, vp5, vp7,or Delonix regia (Gul Mohr) flowers. Zeaxanthin is also the major vp9), lack carotenoids essential for photosystem protection and, carotenoid in cold-pressed marionberry, boysenberry, red rasp- therefore, exhibit photobleaching and ABA deficiency (Neill and berry, and blueberry seed oils, followed by β-carotene, lutein, and others 1986). In contrast, mutants impaired in the downstream cryptoxanthin (Parry and others 2005). Zeaxanthin has also been steps of carotenoid biosynthesis do not show photobleaching. identified in extracts from apricots, peaches, cantaloupe, and The aba1 mutant of Arabidopsis and the aba2 mutant of Nico- a variety of pink grapefruit (Ruby seedless) among carotenoids tiana plumbaginifolia are impaired in the epoxidation of zeaxan- separated and quantified on C18 reversed-phase HPLC columns thin and have been shown to be either slightly or not at all affected with low and high carbon loading (Khachik and others 1989). in PSII photochemical efficiency (Rock and Zeevaart 1991; Rock The major carotenoids of Viburnum tinus leaves are β-carotene and others 1992; Marin and others 1996; Tardy and Havaux 1996; and lutein (38.5% and 47%, respectively) with neoxanthin and Hurry and others 1997). Zeaxanthin is able to replace the miss- zeaxanthin accounting for 7% and 6.8%, respectively (Chiralt ing epoxy-carotenoids, antheraxanthin, violaxanthin, and neo- and others 1990). Carotenoids identified in persimmon fruits are xanthin as a stabilizing component of the light-harvesting com- cis-mutatoxanthin, antheroxanthin, zeaxanthin, neolutein, cryp- plex II in the aba1 mutant of Arabidopsis. toxanthins, α-carotene, and β-carotene, and fatty acid esters of The recent genetic elucidation of bacterial and plant carotenoid cryptoxanthin and zeaxanthin (Daood and others 1992). Zeaxan- biosynthetic pathways leading to the accumulation of zeaxan- thin has also been identified in the skin, flesh, and oil of avocado thin, canthaxanthin, and astaxanthin may offer interesting alter- (Ashton and others 2006). Lucerne contains 2% zeaxanthin and natives for their in vivo production (Misawa and others 1995a, 40% lutein of the total xanthophyll content, while Fremontia (Ster- 1995b; Misawa and Shimada 1998). For instance, blue-green al- culalareaceae) produces as much zeaxanthin as lutein (Goodwin gae can be readily transformed with autonomously replicating 1976). The stereochemical correlation between capsanthin and plasmids, while endogenous genes can be disrupted by homolo- zeaxanthin and the cooccurrence of the 2 pigments in Capsicum gous recombination. A number of commercial possibilities have sp. have suggested a close biogenetic relationship between the been proposed for recombinant blue-green algae (Lagarde and two. Formation of capsanthin from zeaxanthin via antheraxanthin others 2000). Recently, Synecocystis sp. strain PCC 6830 was is indicated (Britton 1976). Of the total pigment content, zeaxan- used as a transformation host to overproduce zeaxanthin in vivo. thin contributes to about 6.5%, 7.3%, and 15.9%, respectively, Moreover, the system developed in the study allowed for gene in the red, orange, and yellow varieties of Capsicum annuum replacement without the introduction of antibiotic resistance cas- (Goodwin 1976). settes in the final overexpressing strains. The absence of cas- In corn, xanthophylls are mostly found in the horny endosperm. settes containing genes that confer antibiotic resistance in such The total xanthophyll content is estimated to be 11 to 30 mg/kg strains is a positive feature highlighting the increasing desire of (Zuber and Darrah 1987). In 1 study, yellow dent corn was found the biotechnology industry to avoid spreading antibiotic-resistant to contain a total xanthophyll content of 21.97 µg/g with lutein cassettes, thereby respecting the concerns of consumers and content of 15.7 µg/g, zeaxanthin content of 5.7 µg/g, and β- environmentalists. cryptoxanthin of 0.57 µg/g (Moros and others 2002). Commercial corn gluten meal has 7 times higher concentration of xanthophylls A genetically engineered zeaxanthin-rich potato (145 µg/g), and deoiled corn contains 18 µg/g, indicating that the In an attempt to provide a good supply of zeaxanthin in staple xanthophylls are probably bound to the zein fraction of corn pro- crops such as potatoes (Solanum tuberosum L.), 2 different potato teins. Wolfberry (Lycium chinese), a small fruit used to improve varieties were genetically modified (Romer¨ and others 2002). vision in traditional Chinese medicine, contains concentrations By transformation with sense and antisense constructs encoding of zeaxanthin dipalmitate that can approach 1 g/kg wet weight zeaxanthin epoxidase, the conversion of zeaxanthin to violaxan- (Zhou and others 1999). The zeaxanthin concentration of fruits thin was inhibited. Both antisense and cosuppression approaches and vegetables is shown in Table 2 (Khachik and others 1989). yielded potato tubers with high levels of zeaxanthin. Depending on the transgenic lines and tuber development, zeaxanthin con- Microbial sources tent was elevated by 4- to 130-fold, reaching values up to 40 µg/g Recently, there has been much interest in the microbial produc- dry weight. tion of zeaxanthin (Ruther and others 1997; Jin and others 2003); Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 33 CRFSFS: Comprehensive Reviews in Food Science and Food Safety however, unlike β-carotene and astaxanthin, very few microbes minerals, and other factors necessary for growth (Goodwin 1971; synthesize zeaxanthin as their predominant carotenoid (Johnson Gierhart 1994). The effect on growth and production of zeaxan- and Schroeder 1996). thin by Flavobacterium sp. was studied using different carbon Flavobacterium sp. Among the sources of microbial xantho- and nitrogen sources in a chemically defined medium by Al- phylls, Flavobacterium sp. is reported to produce zeaxanthin as cantara and Sanchez (1999). The best growth was supported by essentially its only carotenoid. The pigment formed by Flavobac- sucrose, and both asparagine and glutamine were found to stim- terium consists of 95% to 99% zeaxanthin. Flavobacterium- ulate growth and pigment formation. Carotenoid production and produced zeaxanthin is identical to zeaxanthin from Zea mays glucose consumption increased as a function of asparagine con- (Gierhart 1995). Beta-carotene along with β-cryptoxanthin is centration. Flavobacterium sp. was found to utilize asparagine known to act as precursors in the biochemical pathway of zeaxan- primarily as a nitrogen source for growth and production of zeax- thin production, and thus appreciable levels of these carotenoids anthin. In the presence of asparagine, high glucose concentra- (about 5% to 10%) were observed during initial growth phases of tions decreased pigment production without affecting biomass Flavobacterium sp. (Bhosale and others 2004). Hydroxylation of formation. In the absence of glucose, asparagine could not sup- β-carotene and β-cryptoxanthin ultimately leads to accumulation port growth and zeaxanthin production (Table 3). Lactic acid and of zeaxanthin. palmitic acid methyl esters are reportedly pigment promoters of However, for optimal industrial production, it is essential to zeaxanthin. With no special measures taken, fermentation of a continue strain improvement and to select better producers in Flavobacterium sp. in a medium containing glucose and corn order to maximize the yield. Currently, the only effective way to steep liquor generates approximately 10 to 40 mg zeaxanthin/L screen for better producers is to extract carotenoids from microbes (Nelis and DeLeenheer 1989). The yield increased to 335 mg/L and to perform HPLC analysis with ultraviolet/visible or photodi- by supplementation with palmitic esters, methionine, pyridoxine, ode array detectors. Improved yields of zeaxanthin may be ob- ferrous salts by continuous addition of the nutrients, and reduc- tained by culturing a microorganism of the genus Flavobacterium tion of temperature. under conditions whereby the amounts of carbon and nitrogen Another important factor in the production of microbial pig- present in the culture medium are maintained at a substantially ments is the oxygen provided to the culture medium (Goodwin constant ratio (Gierhart 1995). 1971; Britton 1985). In general, an increase in oxygen supply to Cultures of Flavobacterium sp. in a nutrient medium contain- a highly active culture can increase its productivity. The growth ing glucose or sucrose, sulfur-containing amino acids, such as of bacteria and fungi, measured in terms of dry weight, varies methionine, cystine, or cysteine, pyridoxine, and bivalent metal directly with the efficiency of aeration above a predetermined ions selected from the group consisting of Fe2+,Co2+,Mo2+,or level depending on the available substrate. In addition to being Mn2+ were able to produce up to 190 mg zeaxanthin/L, with a required for growth in Flavobacterium, oxygen is also required for specific cell concentration of 16 mg/g dried cellular mass (Shep- desaturation, cyclization, and oxygenation of carotenoids (Smith herd and others 1976). The optimized process claims to provide and Johnson 1958; Edwards 1985; Han and Mudgett 1992; Brit- up to 500 mg of zeaxanthin/L culture at lower costs and more ton 1995). To improve zeaxanthin production by Flavobacterium, rapidly than known methods and microorganisms. a fermentor with a high oxygen transfer rate is preferred. Agita- Several reports have shown different medium constituents tion speed and aeration rate are the 2 factors that strongly affect other than environmental factors to affect zeaxanthin production. the oxygen supply in stirred-tank fermentors. A proper combi- Among these factors, nitrogen and carbon sources play an impor- nation of these factors can regulate the required oxygen supply tant role in zeaxanthin production (Gierhart 1994; Alcantara and to a growing culture. However, any additional supply of oxygen Sanchez 1999). For Flavobacterium, corn steep liquor is benefi- should be matched with the right availability of other nutrients cial for pigment synthesis because of its richness in amino acids, such as corn steep liquor. Some factors that have an unexpectedly

Table 2 --- Zeaxanthin concentration in fruits and vegeta- Table 3 --- Effect of different carbon and nitrogen sources bles. on growth and zeaxanthin production (Alcantara and Sanchez 1999). Zeaxanthin, Food items µg per 100 g Growth Zeaxanthin Conditions (OD at 540 nm) (µg/mL) Beans (drained, green, canned) 44 a Broccoli (drained, cooked, boiled without salt) 23 Carbon sources Carrots (raw) 23 Yeast extract (1%) 2.38 ± 0.070 0.75 ± 0.025 Celery (drained, cooked, boiled, without salt) 8 D-glucose (55 mM) 1.55 ± 0.066 0.15 ± 0.020 Celery (raw) 3 Sucrose (55 mM) 1.82 ± 0.102 0.15 ± 0.027 Collards (drained, cooked, boiled, without salt) 266 Xylose (55 mM) 0.51 ± 0.032 0.07 ± 0.017 Corn (drained, sweet, yellow, canned, whole kernel) 528 No carbohydrate 0.46 ± 0.055 0.07 ± 0.011 b Lettuce (raw) 187 Nitrogen sources (7.5 mM) Lettuce (raw) 70 L-asparagine 3.74 ± 0.050 0.38 ± 0.016 Orange juice (frozen concentrate, unsweetened, diluted) 80 L-glutamine 2.96 ± 0.119 0.36 ± 0.011 Oranges (all commercial varieties) 74 L-cysteine 1.89 ± 0.090 0.07 ± 0.0009 Papaya 20 L-methionine 1.85 ± 0.015 0.08 ± 0.0095 Peaches (raw) 6 Glycine 0.6 ± 0.020 0.03 ± 0.007 Peas (drained, green, canned) 58 L-asparagine without glucose 0.44 ± 0.101 0.08 ± 0.009 ± ± Spinach (drained, cooked, boiled without salt) 179 L-asparagine without MgSO4 0.55 0.030 0.03 0.006 Spinach (raw) 331 L-asparagine + L-cysteine 2.31 ± 0.076 0.28 ± 0.003 Squash, butternut 280 aFermentation was carried out for 48 h, 29 ◦C, and 180 rpm in CD medium supplemented Tangerines, raw (mandarin oranges) 112 with 20 mM NH4Cl. bFermentation was carried in CD medium supplemented with 100 mM glucose.

34 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008 The carotenoid pigment zeaxanthin... strong positive effect on β-carotene production at the expense mal photoautotrophic growth in the absence of the other two of zeaxanthin formation should also be considered. In a study psbA genes (Mohamed and Jansson 1989). Thus, the psbAII lo- on carotenoid production by Flavobacterium multivorum ATCC cus can be used as an integration platform to overexpress genes in 55238, urea and sodium carbonate were found to influence β- Synechocystis. Synechocystis sp. resulted in a 2.5-fold increase carotene production, which represented 70% (w/w) of the total in zeaxanthin accumulation in the mutant strain (Lagarde and carotenoid content (Bhosale and Bernstein 2004). others 2000). The nutrient media that are currently preferred for commercial- Microcystis aeruginosa. The microalgae Microcystis aeruginosa scale fermentation have eliminated corn flour and several other is reported to produce the bioactive carotenoid zeaxanthin (Chen ingredients, and contain either high-maltose corn syrup of sugar and others 2005). beet molasses at concentrations ranging from 1% to 10% (w/v), Spirulina. The blue-green alga Spirulina has zeaxanthin as one along with corn steep liquor at 0.5% to 4% (w/v); ammonium sul- of its carotenoids. A marked enhancement in carapace color, an fate heptahydrate at 0.5% (w/v); sodium chloride at 0.5% (w/v); important quality parameter of cultured prawns, was observed magnesium sulfate heptahydrate at 0.1% (w/v); sodium acetate at when prawns were fed with Spirulina-supplemented diets. Zea- 0.1% (w/v); ferrous sulfate heptahydrate at 0.001% (w/v); yeast xanthin, one of the major carotenoids in Spirulina, was rapidly extract at 0.2% (w/v); thiamine-HCl at 0.01% (w/v); between 1% converted to astaxanthin (Liao and others 1993). Spirulina, also and 6% (w/v) hydrolyzed casein; and vegetable oil at 1% (v/v) increases the yellowness and redness of broiled chicken due to (Garnett and others 1998). Once the ingredients are mixed to- accumulation of zeaxanthin within the flesh (Toyomizu and oth- gether, sufficient NaOH is added to raise the pH to 6.5. The cul- ers 2001). ture medium is sterilized by autoclaving at 121 ◦C for 30 min, ◦ Phaffia rhodozyma. Among yeasts, asporogenous Phaffia cooled to 27 C, and inoculated with 5% to 10% (v/v) of a “liq- rhodozyma, although best known as an astaxanthin producer, uid preculture” containing a strain of F. multivorum which pro- is also reported to be a zeaxanthin producer (Hoshino and others duces the R-R isomer of zeaxanthin without producing S-S or S-R 2004). isomers of zeaxanthin, or any other carotenoids, in significant Zeaxanthinibacter enoshimensis is a zeaxanthin-producing quantities. marine bacterium of the family Flavobacteriaceae, isolated from Several strains of Flavobacterium have been taxonomically the seawater off Enoshima island in Japan (Asker and oth- reevaluated as Paracoccus zeaxanthinifaciens sp. (Berry and oth- ers 2007a). Mesoflavibacter zeaxanthinifaciens is another novel ers 2003). Metabolic engineering can be used to improve zeaxan- zeaxanthin-producing marine bacterium of the family Flavobac- thin production in the bacterium Paracoccus sp. strain PTA-3335, teriaceae (Asker and others 2007b). The carotenoids of the red a mutant derived from a zeaxanthin-producing bacterium origi- algae Corallina officinalis, C. elongate, and Jania sp. are report- nally classified as a species of Flavobacterium (Schocher and Wiss edly composed of β-carotene, zeaxanthin, fucoxanthin, 9-cis- 1975). fucoxanthin, fucoxanthinol, 9-cis-fucoxanthinol, and 2 epimeric Erwinia herbicola. Erwinia herbicola, a nonphotosynthetic mutatoxanthins (Palermo and others 1991). The symbiotic blue- bacterium, is yellow-colored due to accumulation of polar green algae Cyanophora paradoxa and Glaucocystis nostochin- carotenoids, primarily mono- and diglucosides of zeaxanthin earum synthesize only β-carotene and zeaxanthin (Goodwin (Hundle and others 1992). 1976). The thylakoid of the cyanobacteria Anacystis nidulans Neospongiococcum. Among FDA-approved GRAS strains is is reported to have zeaxanthin as one of its major carotenoids Neospongiococcum, the only alga presently designated as GRAS (Murata and others 1981). Dunaliella parva (Andrew and Brit- for feeding poultry to enhance yellow pigmentation (21 C.F.R. ton 1990), Erythrotrichia carnea (Shlomai and others 1992), section 73,275). The green alga Neospongiococcum excentricum Dunaliella bardawil (Ben-Amotz and others 1982), Prochloron is shown to produce up to 0.65% xanthophylls (dry mass basis) sp. (Withers and others 1978), and Pleurochloris commutata (Liao and others 1995). (Goodwin 1976) are some of the other microbial sources of zeax- Dunaliella salina. A zeaxanthin-overproducing mutant strain anthin. 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, Extraction and Isolation of Zeaxanthin the zea1 mutant lacks neoxanthin, violaxanthin, and antheraxan- The advantage of tetrahydrofuran (THF) in comparison to other thin, but constitutively accumulates zeaxanthin in the thylakoid organic solvents of Class 2 as an extraction solvent is that the membrane even under normal growth conditions. Under normal carotenoid is highly soluble in the solvent, which allows for its growth conditions (low light), the mutant strain has 15-fold higher efficient extraction from plant matrices (Khachik 2005). Solvents zeaxanthin content than the wild type. Previous efforts to gener- in Class 2 should be limited in pharmaceutical products because ate zeaxanthin-overproducing E. coli strains using metabolic en- of their inherent toxicity. The choice of extracting solvents is based gineering have resulted in the production of 1.6 mg zeaxanthin/g on a guideline set by the U.S. Dept. of Health and Human Ser- dry weight; however, this value is just one-third of that produced vices, Food and Drug Administration (FDA) in Docket No. 97D- by the zea1 strain of the photosynthetic microalga Dunaliella 0148 published in the Federal Register on May 2, 1997 (Vol 62, salina (6 mg zeaxanthin/g dry weight) (Albrecht and others 1999; Nr 85, p 24301–9). The draft guideline recommends acceptable Jin and others 2003). amounts of residual solvents in pharmaceuticals for the safety of Synechocystis sp. Zeaxanthin is a natural constituet of the outer patients as well as the use of less toxic solvents in the manufacture membrane of Synechocystis sp. PCC6714 (Jurgens and Weckesser of drug substances and dosage forms. 1985). In an attempt to increase zeaxanthin accumulation in a Suitable modes of packaging and delivery for human ingestion photoautotrophic prokaryote, Synechocystis sp. strain PCC 6803, include various forms such as lyophilized coarse-grain powder, a system designed to overexpress genes involved in carotenoid viscous oily liquid, and micelles. An alternate form of packag- synthesis in the organism employs the psbAII gene, which en- ing for human ingestion can utilize tablets, provided that a solid codes the highly expressed D1 protein of photosystem II and has binder material is used which will hold the tablet together after a strong promoter in it. Synechocystis genome contains 3 genes manufacture. Such tablets may be coated with an enteric coat- coding for the D1 protein, psbAI, psbAII, and psbAIII, the latter ing that remains intact until after the tablet passes through the two of which are expressed and can individually support nor- stomach and enters the intestine. Zeaxanthin is believed to be Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 35 CRFSFS: Comprehensive Reviews in Food Science and Food Safety susceptible to degradation at high pressures (Garnett and others a zeaxanthin fluid can be added to various types of foods such 1998). Thus, it is assumed that compressed tablets will be less as margarine, dairy products, syrup, cookie dough, and certain preferred or tablet binders that ensure good cohesion at lower types of meat preparations which are not subjected to harsh cook- pressures could be used. ing. Additional purification steps can be used to produce granular Flavobacterium sp. Flavobacterium multivorum, a nonfastidi- zeaxanthin containing nearly pure zeaxanthin. Such processing ous and nonpathogenic bacterium that rapidly accumulates zeax- methods may be used to create formulations such as ingestible anthin, is considered to be a potential source of the carotenoid tablets to be added to soups, salads, drinks, or other foods. (Dasek and others 1973; Pasamontes and others 1997; Alcan- tara and Sanchez 1999; Masetto and others 2001). Synthesis of Micelles containing zeaxanthin a pure form of the R-R isomer of zeaxanthin involves ferment- Zeaxanthin-containing “micelles,” which are less than 1 µmin ing bacterial cells from a strain of Flavobacterium multivorum diameter, are obtained from either the solvent extract of biomass (ATCC 55238) (Garnett and others 1998). The bacterial strain can or the oily fluid by using certain types of bile salts (Garnett and synthesize zeaxanthin as a sole carotenoid with virtually no sub- others 1998). An oily fluid may be mixed with a suitable bile salt stantial amounts of other carotenoids under proper fermentation such as the phosphate salts of glyco- or taurocholate or by use of conditions. Therefore, the extremely difficult task of purifying zea- gall bladder extracts containing mixtures of bile salts. The bile ma- xanthin by separating it from closely related carotenoids can be terial mixed with either the solvent extract or oily mass and with completely avoided if this bacterial strain or its descendants are salts such as sodium chloride, calcium chloride, or potassium used. chloride is processed in a mechanical homogenizer to produce In the isolation and pure cultivation of the yellow Flavobac- micelles that are dried and diluted to a desired concentration terium microorganisms, material from the natural source is sus- using a carrier or diluent fluid such as vegetable oil. The mix- pended in physiological saline (Gierhart 1994). Streak cultures ture may be enclosed within a capsule or other device that will are applied to Petri dishes and the yellow colonies growing aid in swallowing and help protect the resultant micelles against on the agar are examined for carotenoid content. Colonies of degradation by stomach acid. Flavobacterium are identified by comparing the cells to the tax- Microcystis aeruginosa. High-speed counter-current chro- onomic description provided in Bergey’s Manual (1984 edition). matography has been successfully applied in the isolation and The microorganisms are identified by their zeaxanthin content purification of zeaxanthin from the cyanobacterium Microcystis confirmed by analytical procedures such as high-performance aeruginosa. Preparative high-speed counter-current chromatog- liquid chromatography (HPLC). A method of extracting zeaxan- raphy with a 2-phase solvent system composed of n-hexane-ethyl thin involves carefully drying the cell mass, pulverizing the dried acetate–ethanol–water (8:2:7:3, [v/v/v/v]) resulted in zeaxanthin cell mass, digesting the pulverized material with an inert organic of 96.2% purity in a 1-step separation (Chen and others 2005). solvent, filtering the solution, and isolating the pure zeaxanthin by elution of the filtration residue with an inert organic solvent Chinese wolfberries and marigold flowers such as ethanol, acetone, or chloroform. Briefly, the commercial extraction of xanthophylls (oleoresin) from marigold flowers involves the following stages: ensilage, Lyophilized coarse-grain zeaxanthin powder pressing, drying, hexane extraction, and saponification. The en- In an attempt to isolate zeaxanthin, Flavobacterium multivo- silage is considered critical in determining the efficiency of the rum cells (ATCC 55238) were fermented to produce zeaxanthin overall process. Isolation of lutein and zeaxanthin esters from and separated from the liquid broth by centrifugation. Acetone marigold flowers and Chinese wolfberries may involve the use was added to the cells to extract a majority of the zeaxanthin of hexane as a solvent for extraction at room temperature fol- into the solvent phase. The cells were then placed in a filter press lowed by evaporation of hexane at 60 ◦C. The isolated oleoresin to remove the cell solids and acetone was evaporated from the may be mixed with an alcohol to remove some of the by-product liquid under warm conditions (about 33 to 43 ◦C) and mild vac- impurities. uum (Guerro-Santos and others 2005). The oily residue contained Enzymatic treatment can enhance xanthophyll extraction from zeaxanthin in a large crystalline form and some cell residues. Wa- marigold flowers (Delgado-Vargas and Paredes-Lopez´ 1997; ter was added and the mixture was processed using a standard Barzana´ and others 2002). Petals treated with 0.1% (w/w) mechanical mixer, and forced through a Teflon filter at 15 psig Econase-cep (Enzyme Development Corp., N.Y., U.S.A.) for a pe- pressure. The zeaxanthin and the solids that stayed on the filter riod of 120 h produced a significant increment in xanthophyll were washed with hexane, a mixture of 95% hexane and 5% yield (24.7 g/kg dry weight) that compared favorably with the acetone, and then a mixture of 90% hexane and 5% acetone to yield from the untreated control (11.4 g/kg dry weight) (Delgado- remove impurities. Subsequent washings with pure acetone re- Vargas and Paredes-Lopez´ 1997). Enhanced yields (>85%) of sulting in an acetone/zeaxanthin mixture were passed through recovered carotenoids were also obtained from fresh flowers a silica gel column. Since zeaxanthin would not cling to silica macerated with 0.3% (v/w flower) Viscozyme/Neutrase (Novo- as tightly as various other impurities will, the solvent passing Nordisk, Bagsvaerd, Denmark) and simultaneously treated with through the silica gel eluted the zeaxanthin from the gel. The hexane (Barzana´ and others 2002). However, enzymatic treat- resulting zeaxanthin, after removal of acetone, was 95% to 99% ments present practical limitations owing to the high cost of com- pure. Lyophilization at −70 ◦C and less than 100 mbar resulted mercial enzymes. Supercritical fluid extraction (SFE) is a viable in free-flowing powders at room temperature. alternative for many commercial separation applications to ob- tain carotene, lutein, and oleoresin from marigold flowers (Favati Zeaxanthin in viscous oily liquid and others 1988; Naranjo-Modad and others 2000). The R-R isomer produced by Flavobacterium multivorum Lutein and zeaxanthin may also be isolated by simultaneous (ATCC 55238) can be concentrated in large quantities and at low extraction and saponification at room temperature using tetrahy- cost into a viscous oily fluid containing about 5% to 20% zeaxan- drofuran and alcoholic potassium or sodium hydroxide. To obtain thin by means of a simple solvent extraction process (Garnett and pure zeaxanthin from Chinese wolfberries, zeaxanthin dipalmi- others 1998). The oily fluid may be mixed with a carrier such as tate was saponified by dissolving in THF and treated with 10% vegetable oil and enclosed within a digestible capsule, compara- KOH in methanol or ethanol (Khachik 2005). The mixture was ble to a conventional capsule containing vitamin E. Alternately, stirred at room temperature for 1 h to complete the hydrolysis

36 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008 The carotenoid pigment zeaxanthin... of zeaxanthin dipalmitate to zeaxanthin. The solvents were co- 20 ◦C to prevent decomposition of the carotenoids during the distilled under reduced pressure and zeaxanthin crystallized as analysis for better reproducibility of the results. A normal phase a dark yellow-orange solid. The crystals were centrifuged and LC-atmospheric pressure chemical ionization (LC-APCI) MS-MS vacuum-dried at 60 ◦C. The yield of zeaxanthin was 16 mg from technique can simultaneously quantify β-carotene, α-tocopherol, 36 mg of zeaxanthin dipalmitate (Figure 7). β-cryptoxanthin, zeaxanthin, and lutein in biological materials (Hao and others 2005). In reversed-phase HPLC, where partition Corn is the major chromatographic mode, the order is more or less the Supercritical fluid extraction (SFE) can extract yellow pigments reverse of that encountered in normal-phase adsorption open- from corn gluten meal, a by-product of corn starch processing ◦ column chromatography. Polymeric C18 phases have excellent (Jing 2004); optimum conditions are a temperature of 40 C, a selectivity for structurally similar carotenoids such as the geo- pressure of 20 MPa, and a time of 120 min, with 20% absolute metric isomers of β-carotene (Bushway 1985; Quackenbush and ethyl alcohol as a cosolvent. Yields of corn pigment extracted by Smallidge 1986; Lesellier and others 1989; Craft and others 1990) SFE were 2.2 times that obtained by solvent extraction. However, and of lutein and zeaxanthin (Epler and others 1992). However, the pigment was found to be sensitive to sunlight and, therefore, the total carbon load is lower in the wide-pore polymeric phases, should be stored in the dark at low temperatures. resulting in weak retention of the carotenoids (Craft 1992). The Lipophilic carotenoids, notably (all-E)-lutein and (all-E)- peaks also tend to be broader and columns from different produc- zeaxanthin, and also neoxanthin, violaxanthin, β-carotene, and tion lots are more variable than with monomeric columns. The chlorophylls a and b from spinach and sweet corn were iso- C30 column provides an excellent resolution of photoisomerized lated and analyzed by high-speed counter-current chromatog- standards of lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β- raphy (HSCCC) (Aman and others 2005a). Pretreatment with pro- carotene, and lycopene (Emenhiser and others 1995, 1996). teinase can increase the extraction efficiency of lutein, zeaxan- Temperature regulation is recommended to maintain day-to- thin, and total carotenoids from corn gluten meal (Lu and others day reproducibility. Variations in column temperature result in 2005). substantial fluctuation of the carotenoids’ retention times. With a monomeric C18 column and acetonitrile–dichloromethane– Orange juice methanol (70:20:10) as mobile phase, no separation of lutein and Carotenoid pigments from Brazilian Valencia orange juice zeaxanthin, and 9-cis- and trans-β-carotene occurred at ambient were isolated by open-column chromatography (OCC) after (30 ◦C) temperature (Sander and Craft 1990). At subambient tem- extraction using acetone and saponification with 10% methano- perature (−13 ◦C), good separation of lutein and zeaxanthin and lic KOH (Gama and Sylos 2005). The pigments were identi- baseline separation of 9-cis- and trans-β-carotene were achieved. fied as α-carotene, zeta-carotene, β-carotene, α-cryptoxanthin, In a Vydac 201TP54 (polymeric) column with acetonitrile– β-cryptoxanthin, lutein-5,6-epoxide, violaxanthin, lutein, anther- methanol–dichloromethane (75:20:5) as mobile phase, optimum axanthin, zeaxanthin, luteoxanthin A, luteoxanthin B, mutatox- resolution of lutein, zeaxanthin, β-cryptoxanthin, lycopene, α- anthin A, mutatoxanthin B, auroxanthin B, and trollichrome B. 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- Leaves of Physalis cups drofuran in methanol as mobile phase, resolution of lutein and In the leaves of Physalis, zeaxanthin occurs in the form of the zeaxanthin and of β-carotene and lycopene was better at lower palmitic acid ester physalien, from which the pigment is obtained temperature (Craft and others 1992). by saponification. Physalien may also be isolated from Physalis Cereal and cereal products. A rapid procedure for the extrac- berries (Kuhn and Wiegund 1929). tion and determination of carotenoids from cereals and cereal by-products involves sample saponification and extraction fol- Determination of Zeaxanthin lowed by normal-phase HPLC, allowing separation of the major  carotenoids of cereals, particularly lutein and zeaxanthin (Panfili Standards for all trans-zeaxanthin, 12 apo-zeaxanthinal, and and others 2004). Among the cereals analyzed, the highest lev- parasiloxanthin are available from DSM (DSM Nutritional Prod- els of carotenoids were found in corn (11.14 mg/kg dry weight), ucts Ltd., Kaiseraugst, Switzerland). All-trans-zeaxanthin is also which contained β-cryptoxanthin (2.40 mg/kg dry weight), zeax- available from Fluka (Buchs, St Gallen, Switzerland). anthin (6.43 mg/kg dry weight), and α + β-carotenes (1.44 mg/kg dry weight). HPLC, MS Plant pigments. A procedure for the simultaneous determina- The most common method for analysis of carotenoids is tion of lutein and zeaxanthin stereoisomers in thermally pro- HPLC employing various detection techniques. Both normal- cessed vegetables by HPLC with diode array detection was and reversed-phase systems, either in isocratic or gradient elu- developed by Aman and others (2005b). (Z)-isomers of lutein tion modes, are employed with reversed-phase systems being and zeaxanthin were prepared by iodine-catalyzed photoiso- more preferred. Antioxidants are added to the mobile phase, merization. Their structures were analyzed by 1D- and 2D-LC- and the column temperature is usually maintained at around NMR spectroscopy, APCI-MS in the positive mode, and UV/Vis

Figure 7 --- Chemical structure of zeaxanthin dipalmitate (Peng and others 2005).

Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 37 CRFSFS: Comprehensive Reviews in Food Science and Food Safety spectroscopy. Near-baseline separation was observed for (13-Z)- dichloromethane/methanol/water/propionic acid, 71:22:4:2:1 lutein, (13-Z)-lutein, (all-E)-lutein, (9-Z)-lutein, (9-Z)-lutein, (13- [v/v/v/v/v]). Prior to HPLC analysis, fatty tissues were defatted Z)-zeaxanthin, (all-E)-zeaxanthin, and (9-Z)-zeaxanthin. with acetone/methanol (1:1 [v/v]) at −80 ◦C or on a silica gel A reversed phase-HPLC method may be used to analyze column. The method gave satisfactory resolution of polar com- the full complement of higher plant photosynthetic pigments pounds (retinol and retinal forms, astaxanthin and phoenicox- (cis-neoxanthin, neoxanthin, violaxanthin, anteraxanthin, lutein, anthin or zeaxanthin) and an acceptable elution time for less zeaxanthin, cis-lutein, chlorophyll b, chlorophyll a, and α- and polar molecules (retinyl palmitate, α- and β-carotene). Levels of β-carotene) (Rivas and others 1989). The separation on a C18 retinoid forms and carotenoids were determined in eyes, blood, column takes about 10 min, using a single high-pressure pump and eggs of mature rainbow trout. and 3 different mobile phases in 3 isocratic steps. The method introduces a major improvement in higher plant photosynthetic TLC pigment analysis, resolving in all photosynthetic pigments while Carotenoids are so strongly colored that use of special reagents achieving good separation of lutein from its isomer zeaxanthin. for the detection is normally unnecessary (Bolliger and Konig Positive-ion fast-atom bombardment tandem MS (FAB MS-MS) 1969). A number of precautions must be taken to prevent loss using a double-focusing mass spectrometer with linked scanning of pigments. These include applying the sample quickly and run- at constant B/E and high-energy collisionally activated dissocia- ning the chromatogram without delay, applying the sample under tion (CAD) was used to differentiate 17 different carotenoids, in- subdued illumination and running the chromatogram in the dark, cluding zeaxanthin (Breemen and others 1995). Carotenoids were and using an atmosphere of nitrogen if possible. The rapid fad- either synthetic or isolated from plant tissues, including carrots ing of carotenoids on developed thin layers may be delayed by and tomatoes. Both polar xanthophylls and nonpolar carotenes spraying the chromatogram with a solution of liquid paraffin in formed molecular ions during FAB ionization. Following colli- light petroleum (Bolliger and Konig 1969). sionally activated dissociation, fragment ions of selected molec- Zeaxanthin can be separated from other carotenoids using a ular ion precursors showed structural features indicative of the silica gel G (activated) plate and a solvent system consisting of presence of hydroxyl groups, ring systems, ester groups, and alde- dichloromethane:ethyl acetate (4:1 [v/v]) where lutein and zeax- hydes groups, and the extent of aliphatic polyene conjugation. It anthin separate with Rf values of 0.35 and 0.24, respectively is suggested that the fragmentation patterns observed in the mass (Bolliger and Konig 1969). On silica gel G (activated) plate de- spectra may be used as a reference for structural determination veloped with benzene:ethyl acetate:methanol (75: 20: 5, by vol.), of carotenoids isolated from plant and animal tissues. the Rf values of lutein and zeaxanthin are reportedly 0.57 and Fruits, fruit juices. An HPLC-diode array detector (HPLC-DAD) 0.53, respectively (Davies and others 1970a, 1970b). method was used to quantify the content of zeaxanthin dipalmi- Kieselguhr paper can separate carotenoids of many types and tate, a major carotenoid in Fructus lycii, on a C18 column with is particularly recommended as being superior to other micro- the mobile phase consisting of acetonitrile and dichloromethane scale methods for the resolution of cis-trans isomeric xanthophylls (42:58). Zeaxanthin dipalmitate was the predominant carotenoid, (Liaaen-Jensen 1971). The Rf values of lutein and zeaxanthin are comprising 31% to 56% of the total carotenoids (Peng and others 0.72 and 0.59, respectively, when developed in 10% acetone in 2005). petroleum ether, and 0.91 and 0.87, respectively, in 20% acetone An isocratic RP-HPLC method was developed for routine anal- in petroleum ether. ysis of the main carotenoids related to the color of orange juice, using a more selective wavelength (486 nm) in which NIR reflectance spectroscopy the absorption in the red-orange region of the visible spec- NIR reflectance spectroscopy may be used for screening large trum is maximum. Separation was carried out using a mixture numbers of corn samples for breeding programs aimed at produc- of methanol:acetonitrile:methylene chloride:water (50:30:15:5 ing corn hybrids with increased carotenoid concentration (Be- [v/v/v/v]) to which small amounts of BHT and triethylamine were rardo and others 2004). Genotypic variation in corn carotenoid added (0.1%) as the mobile phase. Application of the method to concentration was investigated in 64 genotypes, including vari- Valencia ultrafrozen orange juices showed the major carotenoids eties of different geographical origin, commercial hybrids, lines in to be lutein + zeaxanthin (36%), lutein 5, 6-epoxide (16%), an- selection, cornflakes, popcorn, and sweet corn. Carotenoid con- theraxanthin (14%), and β-cryptoxanthin (12%). centration in meals from each genotype was determined using An HPLC-MS technique was developed for identification and NIR reflectance spectroscopy and HPLC. Comparison of the 2 an- determination of carotenoids and their fatty acid esters in citrus alytical techniques revealed similar results for each; correlations fruit juices (orange and tangerine juice concentrates) (Wingerath of between 0.84 (lutein) and 0.94 (zeaxanthin) were identified. 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- Isomerization and Oxidation of Zeaxanthin assisted laser desorption ionization (MALDI) post-source-decay The highly unsaturated carotenoid is prone to isomeriza- (PSD) MS, and comparison with synthetic reference compounds. tion and oxidation (www.hni.ilsi.org). Oxidative degradation, the The xanthophylls lutein, zeaxanthin, α-cryptoxanthin, and β- principal cause of extensive losses of carotenoids, depends on cryptoxanthin were detected in extracts of saponified tangerine the availability of oxygen and is stimulated by light, enzymes, concentrate. metals, and co-oxidation with lipid hydroperoxides. Carotenoids Poultry products. An HPLC-DAD with a C30 phase was used appear to have different susceptibilities to oxidation, with ζ- for the simultaneous separation of xanthophylls in egg yolks. Peak carotene, lutein, and violaxanthin being cited as more labile identification was carried out by LC-(APCI) MS (Schlatterer and (Rodriguez-Amaya 2001). Formation of epoxides and apoc- Breithaupt 2006). arotenoids (carotenoids with shortened carbon skeleton) appears Fish. An HPLC procedure was developed to separate 2 retinol to be the initial step (Figure 8). Subsequent fragmentations yield a forms (retinol1 and retinol2 or dehydroretinol), their correspond- series of low-molecular-weight compounds similar to those pro- ing retinal forms, and carotenoid pigments commonly found in duced in fatty acid oxidation. Thus, total loss of color and bi- fish tissues (Guillou and others 1993). A reversed-phase C18 ologic activities are the final consequences. Heat, light, acids, column was used with an isocratic solvent system (acetonitrile/ and adsorption on an active surface (such as alumina) promote

38 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008 The carotenoid pigment zeaxanthin... isomerization of trans-carotenoids, their usual configuration, to lead to bleaching and the formation of artifacts such as epoxy the cis forms (Figure 9). carotenoids and apocarotenals (Britton 1991). Oxygen can be ex- Oxygen, especially in combination with light and heat, is cluded at several steps during analysis and storage with the use of highly destructive. The presence of even traces of oxygen in stored vacuum and a nitrogen or argon atmosphere. Antioxidants such samples (even at deep-freeze temperatures) and of peroxides in as butylated hydroxytoluene, pyrogallol, and ascorbyl palmitate solvents (for example, diethyl ether and tetrahydrofuran) or of any may also be used, especially when the analysis is prolonged. They oxidizing agent, even in crude extracts of carotenoids, can rapidly 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 Figure 8 --- Possible scheme for the degradation of 2005). In 1 study, all-trans zeaxanthin was incubated at 75 ◦C for carotenoids (www.hni.ilsi.org). 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 ◦Cin24h;5dat44◦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 Figure 9 --- Chemical structure of zeaxanthin in the confor- or the specific area of the material (powder) yielded significant mation: all-trans,9-cis, 13-cis, and 15-cis. zeaxanthin losses (Kim and others 2004). Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 39 CRFSFS: Comprehensive Reviews in Food Science and Food Safety

Food products such as fruit juices, soy drinks, yoghurt, ice and slightly soluble in chloroform giving a clear intensive orange- cream, biscuits, cereals and cereal bars, margarine, and soft can- red solution. It is composed of trans-zeaxanthin and minor quan- dies with added zeaxanthin (and lutein) remain stable for periods tities of cis-zeaxanthin, 12-apo-zeaxanthinal, diatoxanthin, and of up to 12 mo. “Jelly bears” and extruded cereals showed losses parasiloxanthin. Nonfermentation processes suffer from several of zeaxanthin of 25% and 78% after 9 and 12 mo, respectively disadvantages; they typically require numerous reaction steps, (Table 4) (Koenig-Grillo 2002). Encapsulation of carotenoids with and each step has a less than 100% yield, so that the final yield lipids, dextrins, and polyvinylpyrrolidone is 1 approach being of zeaxanthin at the end of the multistep processing tends to be evaluated in order to reduce carotenoid degradation in foods. relatively poor. In addition, chemical synthesis tends to yield un- Decrease in degradation during storage is reported for encapsu- desirable S-S and S-R stereoisomers of zeaxanthin, as well as lated carotenoids (Selim and others 2000; Basu and Del Vecchio various conversion products such as oxidized zeaxanthin, and 2001; Rodriguez-Huezo and others 2004). zeaxanthin molecules that have lost one or more of the double The small red berry, wolfberry (Fructus barbarum L.), is one of bonds in the straight portion or end rings. the richest natural sources of zeaxanthin. Enhanced bioavailabil- Zeaxanthin synthesis follows a sequence of reactions in which ity of zeaxanthin in a milk-based formulation of wolfberries was the final step is a double Wittig condensation of a symmetri- obtained when homogenization of the berries was performed in cal C10-dialdehyde as the central building block with 2 equiva- hot-skimmed milk (80 ◦C) compared with the hot water (80 ◦C) lents of the appropriate C15-phosphonium salt (Figure 10). The and warm-skimmed milk treatment of the berries (40 ◦C) (Benzie 1st step in the process is the production of an enantiopure C9- and others 2006). 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 Chemical Synthesis of Zeaxanthin into the C15-phosphonium salt employed in the Wittig condensa- Synthetic zeaxanthin is an orange-red crystalline powder with tion. The C10-dialdehyde is commercially available. A similar ap- little or no odor. It is practically insoluble in water and ethanol, proach is used for the synthesis of other symmetrical carotenoids,

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).

40 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008 The carotenoid pigment zeaxanthin... such as lycopene, β-carotene, and astaxanthin, also commer- Acceptable Daily Intake of Zeaxanthin cially available (Soukup and others 1996; Ernst 2002). The safety of zeaxanthin in supplements has been confirmed In the method described by Loeber and others (1971), a mix- by the New Dietary Ingredient Notification for dietary 3R, 3R ture of Wittig salt (146 mg, 0.26 mmol), 2, 7-dimethylocta-2,4,6- zeaxanthin. The notification was filed by Roche Vitamins and trienedial (16 mg, 0.1 mmol), and 1.2-epoxybutane (0.5 g) was ◦ was reviewed by FDA without objection in 2001 (Docket Nr heated for 1 h at 100 C under nitrogen in a sealed tube, and 95S-0316: 75-Day Premarket Notifications for New Dietary In- cooled. The crude product was chromatographed on alumina gredients, Report Nr 96). In addition, the Joint Food and Agricul- (gradient elution with light petroleum, benzene, and ethyl ac- ture Organization/World Health Organization (FAO/WHO) Ex- etate); the main red band yielded an orange solid (35 mg, 62%). ◦ pert Committee on Food Additives (JECFA) recently completed a Crystallization from methanol gave zeaxanthin, m.p, 211 C; λmax −l toxicological evaluation of zeaxanthin. JECFA established a group (C6H6) 589, 462, and 439; V max, 3610, 1035, and 965 cm . The Acceptable Daily Intake (ADI) for lutein and zeaxanthin of 0 to product did not separate from natural zeaxanthin on mixed TLC. 2 mg/kg body weight for zeaxanthin (JECFA, Summary and Con- [Kieselgel HF 254; 30% acetone in light petroleum (b.p. 60 to ◦ clusions, 63rd meeting, Geneva, June 2006). There are also di- 80 C)]. rect animal data demonstrating retinal protective effects of dietary Zeaxanthin and its dipalmitate and physalien have been syn- zeaxanthin (Thomson and others 2002). thesized by Isler and others (1956a, 1956b). Starting from the optically pure hydroxyketone 1, an efficient synthesis of (3R, 3R)- zeaxanthin is described by Widmer and others (1990) (Figure 11). Functional Properties of Zeaxanthin Creemers and others (2002) synthesized pure all-E zeaxanthin The intake of a carotenoid-rich diet is epidemiologically related as follows: TiCl3 (0.22 g, 1.4 mmol) was added to dry THF un- to a lower risk of some types of cancer and AMD (Perez-Galvez der argon; LiAlH4 (0.04 g, 1.1 mmol) was added to the suspen- and others 2003). Zeaxanthin is used in the prevention of human sion and the mixture was stirred for2hatRT.3-Hydroxyretinal macular degeneration and also for poultry pigmentation (Berry (0.21 g, 0.7 mmol) in THF was slowly added and the mixture and others 2003). For infants, human milk is the main source was stirred overnight at RT. HCl (1 M) was added and the mix- of lutein and zeaxanthin until weaning occurs. The carotenoids ture was extracted twice with diethyl ether. The combined or- appear to be important as protective factors in the retinal pigment ganic layers were washed with NaCl, dried with MgSO4, and epithelium of the newborn infants (Jewell and others 2001, 2004). concentrated in vacuo. After purification on a silica-gel column The evaluation of zeaxanthin in the cosmetic area, especially in (50% diethyl ether/petroleum ether), and subsequent recrystal- solar protection, is under way owing to its natural function as a ◦ lization (CH2Cl2/EtOH, −20 C), pure all-E zeaxanthin (0.02 g, protectant of cells from photosensitization. 0.04 mmol, 10%) was obtained. In vitro, the conversion of lutein to meso-zeaxanthin is readily Nutraceutical value of zeaxanthin achieved in a base-catalyzed reaction (Bone and others 1993), Age-related macular degeneration (AMD). AMD, the leading and this is the basis of an industrial process for its synthesis and cause of irreversible blindness in adults, is the result of degen- use in poultry feeds. The conversion hypothesis is supported by erative changes that occur in the central region of the retina and the distribution of the individual carotenoids in the retina (Bone the macula, eventually leading to loss of vision. “Dry” macu- and others 2007). lar degeneration is caused by a thinning of the macula’s layers,

Figure 11 --- Synthesis of (3R, 3R)-zeaxanthin as described by Widmer and others (1990).

Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 41 CRFSFS: Comprehensive Reviews in Food Science and Food Safety and vision loss typically is gradual. However, tiny, fragile blood produces reactive oxygen species (ROS). Zeaxanthin, which is vessels can develop underneath the macula. “Wet” macular de- fully conjugated, may offer better protection than lutein against generation can result when these blood vessels hemorrhage, and oxidative damage of the lipid matrix caused by the blue and blood and other fluid can further destroy macular tissue, even near-ultraviolet light radiation in macular membranes. A pro- causing scarring. In this case, vision loss can be rapid, over tective effect of lutein and zeaxanthin against the oxidative months or even weeks. Lutein and zeaxanthin may play a ma- damage of egg yolk lecithin liposomal membranes induced by jor role in the prevention of AMD. They are not metabolized to exposure to UV radiation and incubation with 2,2-azobis(2- vitamin A, and they accumulate in the macular region of the hu- methypropionamidine)dihydrochloride, a water-soluble peroxi- man retina, where they prevent damage by absorbing high-energy dation initiator, is shown by Sujak and others (1999). How- blue light through their antioxidant properties (Handelman and ever, both lutein and zeaxanthin were found to protect lipid others 1988; Britton 1995; Snodderly 1995). It is suggested that membranes against free radical attack with almost the same people supplement their diets with 4 mg of lutein/zeaxanthin efficacy. As a free-radical scavenger, β-carotene and zeaxan- daily. Dietary studies have confirmed the association between thin, with the same chromophore, are less effective than ly- frequent consumption of spinach or collard greens, particularly copene. Lycopene is considered to be the most effective free good sources of lutein and zeaxanthin, and lower AMD risk radical scavenger owing to its 11 conjugated coplanar double (www.aoa.org). Foods provide other phytochemicals that might bonds. act in a synergistic way to help the absorption and utilization Cancer. Zeaxanthin has cancer-preventive properties. Sponta- of the nutrient or to aid in the way it protects the body from neous liver carcinogenesis in C3H/He male mice was suppressed damage. when fed for 40 wk with zeaxanthin at a concentration of 0.005% Zeaxanthin and lutein are specifically accumulated in the mac- and mixed as an emulsion in drinking water (Nishino and others ular region of the retina where they bind to the retinal pro- 1999). tein, tubulin (Handelman and others 1988, 1991). Zeaxanthin Inhibition of LDL oxidation. The carotenoid plays an impor- is specifically concentrated at the macula, whereas lutein is dis- tant role in the inhibition of macrophage-mediated LDL ox- tributed throughout the retina. Within the central macula, zea- idation (Keri and others 1997). Zeaxanthin when incubated xanthin is the dominant component, up to 75% of the total, with human monocyte-macrophages for 24 h with human low- whereas in the peripheral retina, lutein predominates, usually density lipoprotein (LDL) was found to inhibit LDL oxidation in being 67% or greater. Khachik and others (2002) found the ra-  a concentration-dependent manner, suggesting that zeaxanthin tio of lutein to dietary 3R, 3R –zeaxanthin in the lens to be ap- might help in slowing atherosclerosis progression. proximately 1.5:1, which suggests a preferential uptake for di-  etary 3R, 3R –zeaxanthin from the diet and plasma to the lens. Pigmentation of poultry and fish The human retina accumulates lutein and zeaxanthin together Zeaxanthin is preferred over other carotenoids for enhancing with nondietary metabolites of lutein and zeaxanthin includ-    pigmentation in poultry and fish due to its potency to provide a ing meso-zeaxanthin [(3R, 3 S)-β, β-carotene-3, 3 diol] and 3 -  β ε true color and ability to deposit evenly in the flesh and eggs (Orn- oxolutein [(3-hydroxy-3 -oxo- - -carotene)] (Bernstein and oth- dorff and others 1994). Zeaxanthin is at least 1.5 times as potent ers 2001). A significant fraction of lutein in the central retina is as lutein. When administered to poultry in high doses, carotenoid converted into meso-zeaxanthin. In the center of the retina, the or carotenoid-containing compounds such as canthaxanthin, al- ratio of meso-zeaxanthin to lutein is the highest and approaches + falfa, and cayenne pepper cause abnormal red or purple colors in 1:1. Data from autopsy eyes indicate that the (lutein meso- the flesh and color striations in yolks. High doses of lutein in the zeaxanthin):zeaxanthin ratio varies throughout the retina from feed have been shown to impart a greenish hue to poultry flesh 2:1 in the center, where most of the conversion occurs, to 3:1 and egg yolks. Beta-carotene is not deposited well in the flesh in the periphery (Bone and others 1997). Thus there may be of poultry, and canthaxanthin apparently deposits in the iris of an advantage in providing meso-zeaxanthin in supplements at the eye. Zeaxanthin, in contrast, imparts a yellow-red color even the expense of lutein if the goal is to raise the overall zeaxan- at high doses and deposits well in poultry flesh, based on high thin (Bone and others 2007). Lycium Chinese Mill, also known zeaxanthin-containing corn feed studies. Labeled zeaxanthin fed as Chinese boxthorn used in traditional Chinese medicine for to laying hens undergoes conversion to (6S,6S)-ε, ε-carotene-3,3 the treatment of a number of disorders, including visual prob- dione and the intermediate (3R,3S)-3-hydroxy-β, ε-carotene-3- lems, has zeaxanthin in its fatty acid ester form as the principal one, both of which appear in the egg yolks (Schiedt and others carotenoid. 1981). Some fish and crustaceans, such as shrimp, goldfish, and Cataract. Zeaxanthin and lutein may also be protective against carp, apparently can convert zeaxanthin into the red-colored pig- age-related increase in lens density and cataract formation. ment astaxanthin, suggesting that feeding of zeaxanthin to such Cataracts are the leading cause of impaired vision, with a large fish and crustaceans will enhance desirable red coloration. The percentage of the geriatric population exhibiting signs of the le- pigment-containing biomass can be used to color foodstuffs with- sion. Cataracts are developmental or degenerative opacities of out the necessity for isolating pure zeaxanthin (Gierhart 1994). the lens that result in a gradual, painless loss of vision. Studies For poultry products, there are problems with stability and bio- examining lutein and zeaxanthin levels in extracted cataractous logical availability, particularly with xanthophylls from marigold lenses have found up to 3-fold higher levels in the newer ep- and alfalfa. Most marigold products must be solvent-extracted ithelial tissue of the lens than in the older inner cortex portion. and saponified, and may require the inclusion of antioxidants in The epithelial cortex layer comprises 50% of the tissue, yet it has the extraction process (Gierhart 1994). 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 Absorption of Zeaxanthin and others 1999). Humans cannot synthesize carotenoids and, therefore, must Conjugated double bonds are particularly effective at absorb- rely on dietary sources to provide sufficient levels. The human ing harmful UV rays and also quenching singlet oxygen that body absorbs zeaxanthin in the same way as other carotenoids

42 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008 The carotenoid pigment zeaxanthin... and fat-soluble vitamins. There is no difference between syn- intestine, whereas 50% or more of the carotenoid is absorbed thetic and natural zeaxanthin in terms of the way they are pro- from micellar solutions. Thus, the physical form in which the cessed by the human system. The average concentration (ng/g) carotenoid is presented to intestinal mucosal cells is of crucial of zeaxanthin in human tissues and skin is reported to be liver, importance (Olson 1994). 591; lung, 90; breast, 14; prostate, 35; colon, 32; and skin, The 1st step in carotenoid bioavailability is release from 6 (Khachik and others 1998; Scholz and others 2000). Aver- the food matrix. Food processing activities such as ther- age carotenoid concentration (ng/tissue) in human ciliary and mal processing, mincing, or liquefying result in changes to RPE/choroid is 2.54 and 4.85, respectively (Bernstein and others carotenoid chemistry, probably through isomerization or oxi- 2001). dation reactions (Kopsell and Kopsell 2006). However, freez- The biological availability of zeaxanthin depends not only on ing or low-temperature storage generally preserves carotenoid the composition of the matrix containing it, but also on other concentration by reducing potential enzymatic oxidation. Pro- foods consumed at around the same time. The major factors lim- cessing activities usually increase bioavailability through in- iting the bioavailability include molecular structure, interactions creased release of bound carotenoids from the food ma- of xanthophylls with other nutrients (mainly dietary fat), the man- trix; however, thermal degradation in carotenoid chem- ner in which the food is processed, the physical form, speciation, istry might adversely affect bioavailability in some food molecular linkage of the carotenoid in the plant tissue, the nutri- crops. tional status and physiological state of the subject, and genetic Carotenoids are lipid-soluble molecules that follow the absorp- background and the physical disposition of xanthophylls in the tion pathway of dietary fat (Figure 12). Early in the digestive pro- food matrix (Yeum and Russell 2002). Carotenoids are best ab- cess, carotenoids are partially released from the food matrix by sorbed in the presence of fat, but as little as 3 to5ginameal mastication, gastric action, and digestive enzymes (Deming and appear to ensure carotenoid absorption (van Het Hof and oth- Erdman 1999). Carotenoids released from the food matrix mi- ers 2000a). Zeaxanthin in egg yolks might be highly bioavailable grate and solubilize into lipid globules of varying sizes in the because of its association with the lipid matrix of the egg yolk stomach, where they are eventually transformed into smaller (Handelman and others 1999). The carotenoids in egg yolk are in lipid emulsion particles by normal digestive motility. Solubiliza- a digestible lipid matrix consisting of cholesterol (200 mg/yolk), tion of individual carotenoid molecules into lipid emulsions is phospholipids (1 g/yolk), and triacylglycerols (4 g/yolk) (Human thought to be a selective process related to the specific polar- Nutrition Information Service 1989). Such a lipid matrix may be ity of each molecule. Nonpolar carotenes most likely migrate to optimal for carotenoid absorption from the diet. Only about 5% the triacylglycerol-rich core of the particle, while the more polar of the carotenoids in whole, raw vegetables are absorbed by the xanthophylls orient at the surface monolayer along with proteins,

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.

Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 43 CRFSFS: Comprehensive Reviews in Food Science and Food Safety phospholipids, and partially ionized fatty acids. Borel and oth- tion into nascent chylomicrons synthesized in the absorptive in- ers (1996) reported this selective orientation of carotenoids in testinal epithelial cells. phospholipid-triacylglycerol droplets using an in vitro biological Chylomicrons are secreted from the enterocyte into the lym- emulsion model. Beta-carotene, a nonpolar carotene, was shown phatic circulation for transport to the liver. Prior to hepatic up- to solubilize in the core of the droplets, while zeaxanthin, a more take, chylomicrons in the bloodstream are rapidly degraded by polar xanthophyll, accumulated at the surface. Selective orienta- lipoprotein lipase associated with tissue endothelium and trans- tion of carotenoids in membranes, micelles, and lipoproteins is formed into chlylomicron remnants. During this process, some most likely similar to that of other lipid molecules and is based carotenoids may be taken up by extrahepatic tissues (Deming upon the polarity, length, and structure of the molecule (Dem- and Erdman 1999). However, most chylomicron remnants deliver ing and Erdman 1999). Carotenoids solubilized in lipid emulsion carotenoids to the liver where they are stored or resecreted into particles are transported from the stomach to the duodenum of the bloodstream in very low density lipoproteins (VLDL) (John- the small intestine (Deming and Erdman 1999). Dietary fat in the son and Russell 1992). Circulating VLDLs are subsequently delipi- duodenum triggers the release of bile acids from the gall blad- dated to low-density lipoproteins (LDL). Similar to other nonpolar der and regulates levels of pancreatic lipase. Bile acids aid in lipids, carotenes such as β-carotene and lycopene are thought to the reduction of lipid particle size and stabilization into mixed migrate to the hydrophobic core of lipoproteins, while the more micelles. Dietary fat increases the synthesis and secretion of pan- polar xanthophylls reside closer to the surface where the likeli- creatic cholesteryl esterase (CEL) and chylomicrons. CEL is ca- hood of exchange among lipoproteins is enhanced (Romanchik pable of hydrolyzing triacylglycerols, phospholipids, cholesteryl and others 1995). In the fasting state, LDLs are the main carriers of esters, and vitamin A and E esters in the presence of bile salts. nonpolar carotenoids, β-carotene, α-carotene, and lycopene, in Carotenoids are solubilized into mixed micelles along with di- human serum (Pateau and others 1998). The more polar xantho- etary triacylglycerols, their hydrolysis products, phospholipids, phylls are evenly distributed between high-density lipoproteins cholesterol esters, and bile acids (Deming and Erdman 1999). (HDL) and LDL, and to a lesser extent VLDL. Carotenoids released Studies on the capacity of CEL to hydrolyze zeaxanthin esters from lipoproteins, especially LDL, are taken up by extrahepatic incorporated into micelles showed mixed micelles containing tissues. zeaxanthin esters extracted from wolfberry and incubated with Red pepper (Capsicum annuum L.) and its dietary prod- CEL (1000 unit/L) decreased by 84% after 4 h (Chitchumroon- ucts containing a variety of carotenoids may contribute to the chokchai and Failla 2006). The decrease in zeaxanthin diesters carotenoid pattern of human blood and tissues. The avail- was majorly associated with the appearance of free zeaxanthin ability of carotenoids from paprika oleoresin, including zeax- in the micelles. In contrast, free zeaxanthin was not detected in anthin, β-cryptoxanthin, β-carotene, and the paprika-specific the medium without CEL. oxocarotenoids, capsanthin, and capsorubin, was assessed in Uptake of carotenoids into the intestinal mucosa occurs by human volunteers (Perez-Galvez and others 2003). At different passive diffusion (Deming and Erdman 1999). The process re- time points, the carotenoid pattern in the chylomicron fraction quires solubilization of the mixed micelle in the unstirred water was analyzed to evaluate carotenoid absorption. Of the ma- layer surrounding the microvillus cell membrane of the entero- jor carotenoids present in paprika oleoresin, only zeaxanthin, cyte. The mixed micelles collide and diffuse into the membrane, β-cryptoxanthin, and β-carotene were detectable in consider- releasing carotenoids and other lipid components into the cytosol able amounts. Although the xanthophylls were mainly present of the cell. Parker (1996) suggested a concentration gradient be- as mono- or diesters, only free zeaxanthin and β-cryptoxanthin tween the micelle and the cell membrane to most likely deter- were found in the human samples. Although the bioavailability of mine the rate of diffusion. High doses of carotenoid may saturate the pepper-specific carotenoids, capsanthin, and capsorubin from uptake leading to insufficient removal from the plasma mem- paprika oleoresin is very low, the oleoresin is a suitable source of brane, thereby reducing this concentration gradient. Carotenoid the provitamin A-carotenoids, β-carotene, and β-cryptoxanthin, uptake into the enterocyte does not ensure its metabolism or ab- and the macular pigment zeaxanthin. 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). Assessment of Bioavailability of Zeaxanthin Selective surface orientation of more polar xanthophylls into mi- celles suggests that their uptake into the enterocyte and incor- In vivo methods poration into chylomicrons may occur before that of nonpolar The in vivo bioavailability studies imply the consumption of a carotenes. Gartner and others (1996) reported the preferential certain dose of a nutrient and changes of its concentration (mea- uptake of lutein and zeaxanthin from the intestinal lumen into sured by standard analytical procedures) in the blood plasma chylomicrons in humans, even in the presence of high amounts compared with time (such as postprandial period, or the time of β-carotene. Interestingly, while most xanthophylls are present after the meal) (Parada and Aguilera 2007). Three parameters in fruits as esters (Khachik and others 1991), only free xantho- are derived from the kinetics: the area under the curve (AUC), phylls have been found in the chylomicrons and serum of humans the maximal plasma concentration (C max), and the time to reach (Wingerath and others 1995), suggesting a requirement for hydrol- C max, t max. AUC is a measure of the absorption intensity, whereas ysis of carotenoid esters prior to uptake and incorporation into C max and t max give an idea of the rate of absorption (Heacock chylomicrons. Several human studies show that free xanthophylls and others 2004; Manach and others 2005). Another method to are present in the triglyceride-rich fraction of plasma after inges- assess bioavailability is to measure the plasma concentration of tion of a meal enriched with esterified zeaxanthin (Wingerath a nutrient through an extended period (days, weeks) of constant and others 1995; Breithaupt and others 2004) and cryptoxanthin consumption of a specific food (van het Hof and others 1999; (Breithaupt and others 2003). Wingerath and others (1995) also Richelle and others 2002). Relevant parameters in this case are reported an increase in the concentration of free cryptoxanthin, C sat (value at which the concentration remains constant in the zeaxanthin, and lutein in chylomicrons and sera of human sub- time) and t sat (time at which C sat is attained). jects after they ingested tangerine juice concentrate that was rich A single-blinded, placebo-controlled, human intervention trial in the esterified forms of the xanthophylls. These observations was used to provide data on the effect of dietary supplemen- suggest that carotenoid esters are hydrolyzed before incorpora- tation with whole wolfberries on fasting plasma zeaxanthin

44 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY—Vol. 7, 2008 The carotenoid pigment zeaxanthin... concentration (Cheng and others 2005). A total of 14 human process is simulated under controlled conditions using commer- subjects took 15 g/d wolfberry (estimated to contain almost 3 mg cial digestive enzymes (for example, pepsin, pancreatin, and so zeaxanthin) for 28 d. Age- and sex-matched controls (n = 13) took on) while the final absorption process is commonly assessed us- no wolfberry in their diet. On supplementation, plasma zeaxan- ing “Caco-2 cells” cultures. “Caco-2 cells” is the short name for thin concentration increased 2.5-fold, indicating zeaxanthin in polarized human colon carcinoma cells line (Verwei and oth- whole wolfberries to be bioavailable and that intake of a modest ers 2005). Methods that simulate under laboratory conditions the daily amount could markedly increase fasting plasma zeaxanthin gastrointestinal digestion process are known as gastrointestinal levels. models (GIMs) (Parada and Aguilera 2007). The influence of saponification on the deposition in egg yolks The in vitro digestion/Caco-2 cell culture procedure is a rapid of carotenoids from the oleoresin of red pepper (Capsicum an- and cost-effective model for screening the bioavailability of nuum) was studied by Hamilton and others (1990). Four repli- carotenoids from plant foods (fresh spinach, fresh carrots, and cates of 10 hens depleted of carotenoid stores were fed for 3 tomato paste) (Figure 13). Chitchumroonchokchai and others wk with a basal diet of white corn and soybean meal, amended (2004) studied lutein bioavailability by simulating the gastric and with (1) saponified or (2) nonsaponified oleoresin. Trans-lutein, small intestinal phases of digestion, followed by a Caco-2 cell trans-zeaxanthin, and trans-capsanthin accounted for >85% of assay. According to Chitchumroonchokchai and Failla (2006), the total carotenoids deposited in the yolks. From the ratios of ingested xanthophyll esters are processed in a manner similar total carotenoid concentration in the yolks to those in the feed, to dietary cholesteryl esters in the lumen of the small intestine. the carotenoids from (1) were deposited twice as efficiently as With differentiated cultures of Caco-2 intestinal cells and CEL-null those from (2). mice, CEL enhanced intestinal uptake and transport of cholesterol The main drawbacks of in vivo data are the variability in phys- by cleaving cholesteryl esters both before and after their partition- iological state of individuals and the possible interaction of the ing in micelles to increase the extracellular concentration of free nutrient with other components in the diet (Boileau and others cholesterol. In contrast, CEL does not affect apical uptake of either 1999). Whole plasma pharmacokinetics may not be the most free cholesterol or free zeaxanthin. Once taken up by the entero- practical way to measure carotenoid status as plasma concentra- cyte, the metabolism of cholesterol and the xanthophylls differs. tions are not only a measure of the absorption of carotenoids Cholesterol is esterified by acyl-CoA:cholesterol acyltransferase from the diet, but also a measure of exchange from tissue and incorporated into nascent chylomicrons for secretion into storage, bioconversion, and excretion (Castenmiller and West lymph. In contrast, the concentration of free zeaxanthin remain- 1998). Also, ethical restrictions to abide by severe protocols ing relatively constant in Caco-2 cells suggests that enterocytes when humans and/or animals are used in biological research do not esterify xanthophylls before their transfer to nascent chy- severely limit these types of studies (van het Hof and others lomicrons. The cells preferentially took up free zeaxanthin from 2000b). micelles and the extent of transport (4.5%) in chylomicrons was similar to that reported for lutein in Caco-2 cells (Chitchumroon- In vitro methods/gastrointestinal models (GIMs) chokchai and others 2004). This low amount is consistent with In vitro methods are being extensively used since they are rapid, the estimated absorption of 3.3% of 5 mg zeaxanthin adminis- safe, and do not have the ethical restrictions of in vivo methods. tered to human subjects. Studies are now required to determine They are used as a suitable alternative to in vivo assays to deter- whether, as expected, zeaxanthin uptake occurs in a manner sim- mine bioavailability. In vitro methods simulate either the diges- ilar to that of lutein and to examine possible interactions between tion and absorption processes (for bioavailability) or only the di- zeaxanthin and the other dietary carotenoids that affect their gestion process (for bioaccessibility), and the response measured absorption. is the concentration of a nutrient in the final extract. The digestion 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 Figure 13 --- In vitro method to determine bioavailability in- more lutein than zeaxanthin, new products are being developed volving a digestion absorption step using a Caco-2 cell with higher amounts of zeaxanthin. A new dietary supplement, culture (Parada and Aguilera 2007). with zeaxanthin and lutein in the same 2:1 ratio as found in a Vol. 7, 2008—COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 45 CRFSFS: Comprehensive Reviews in Food Science and Food Safety healthy macula, is now available as “EyePromise Restore” (ZeaV- Bishop NI., Urbig Y, Senger H. 1995. Complete separation of carotenoid biosynthesis by a ision, LLC, Saint Louis, Mo., U.S.A.). The supplement contains a unique mutation of the lycopene cyclase in the green alga, Senedesmus obliquus. FEBS Lett 367:158–62. patented dose of zeaxanthin and lutein, the carotenoids that make Block G, Patterson B, Subar A. 1992. Fruit, vegetables, and cancer prevention: a review of up the pigment in the macula (www.eyepromise.com). epidemiological evidence. Nutr Cancer 18:1–29. Bochar DA, Friesen JA, Stauffacher CV, Rodwell VW. 1999. Biosynthesis of mevalonic acid from acetyl-CoA. In: Cane D, editor. The comprehensive natural product chemistry. Isoprenoids, including carotenoids and steroids. Vol. 2. Oxford, U.K.: Pergamon Press. Conclusions p15–44. Zeaxanthin is effective against AMD, cataract, and LDL oxida- Boileau TWM, Moore AC, Erdman JW Jr. 1999. Carotenoids and vitamin A. In: Pappas AM, editor. Antioxidant status, diet, nutrition, and health. New York: CRC Press. p 133– tion, thus emphasizing its use in nutraceutical formulations. Bile 58. acids may play a role in the cellular uptake of the carotenoid. Bolliger HR, Konig A. 1969. In: Stahl E, editor. Thin-layer chromatography. New York: The xanthophyll ester is hydrolyzed before absorption, and di- Springer-Verlag. p 259–311. Bone RA, Landrum JT, Hime GW, Cains A, Zamor J. 1993. Stereochemistry of the human etary fat greatly stimulates the absorptive process. Zeaxanthin is macular carotenoids. Invest Ophthal Vis Sci 34:2033–40. preferred over other carotenoids for enhancing pigmentation in Bone RA, Landrum JT, Friedes LM, Gomez CM, Kilburn MD, Menendez E, Vidal I, Wang W. poultry and fish due to its ability to deposit evenly in the flesh 1997. Distribution of lutein and zeaxanthin stereoisomers in the human retina. Exp Eye Res 64:211–18. and eggs. Flavobacterium sp., which synthesizes zeaxanthin as Bone RA, Landrum JT, Cao Y, Howard AN, Alvarez-Calderon F. 2007. Macular pigment the major pigment, has been widely studied for the production response to a supplement containing meso-zeaxanthin, lutein and zeaxanthin. Nut Met of the carotenoid. In the aerobic fermentation for the produc- 4:12. 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