Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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to reduce the number of animals, length of time, and

costs involved. At present, none of these tests has

been sufficiently validated to replace the full carcino-

genicity test.

Neonatal Mouse Assay

0036 The exposure of newborn mice to carcinogens has been

found to provide a sensitive assay for tumor produc-

tion, primarily in the liver. Since the newborn liver is

rapidly replicating, tumors develop relatively quickly

compared to adults. Neonatal mice are exposed to two

treatments of test chemical in the first and second

weeks after birth, and allowed to grow for 1 year,

when the study is ended. This assay is sensitive to

genotoxic chemicals which form adducts with DNA.

Transgenic Mouse Carcinogenicity Assays

0037 Several mouse models which display faster tumor

formation compared to parental strains have been

created through genetic engineering. These include

the p53

þ/

heterozygous mouse model which con-

tains one active copy of the wild-type p53 tumor

suppressor gene, the CB6F1-TG-rasH2 hemizygous

mouse model containing five to six copies of the

human c-Ha-ras oncogene, the Tg.AC transgenic

mouse model produced by introducing the v-Ha-ras

oncogene linked to a fetal zeta-globin promoter and

containing 40 copies v-Ha-ras per allele, and the

XPA

/

knockout mouse, which lacks the XPA com-

ponent of the nucleotide excision repair pathway.

Each treatment group can contain 15 animals and

studies are continued for 26 weeks, after which the

background of spontaneously occurring mutations

begins to increase. The p53

þ/

and XPA

/

strains

generally respond to genotoxic carcinogens while the

CB6F1-TG-rasH2 and Tg.AC transgenic strains can

respond to both genotoxic and nongenotoxic carcino-

gens. Bigenic strains combining more than one trans-

genic alteration, such as XPA

/

. p53

þ/

and p53

þ/

Tg.AC, have also been produced to increase the re-

sponsiveness of the assays.

Preneoplastic Foci

0038 Preneoplastic foci are small, contained proliferative

lesions that precede the development of either benign

or malignant neoplastic tumors. They may be

detected as proliferating regions by measuring

uptake of bromodeoxyuridine or tritiated thymidine

into the DNA by immunohistochemistry or autora-

dioraphy, respectively. Preneoplastic foci can also be

identified by the use of immunohistochemistry to

measure expression of proliferating cell nuclear anti-

gen or the placental form of glutathione S-transferase.

Preneoplastic foci can be enumerated in various

tissues, but the liver is examined most frequently.

Tissues can be examined for preneoplastic foci at the

close of the rodent carcinogenicity assay or animal

studies can be carried out specifically for this end-

point. More preneoplastic foci are usually found than

would form eventual tumors, a finding which allows

assessments to be carried out in a shorter time period

with fewer animals compared with the complete

rodent carcinogenicity assay.

Assays for Monitoring Human Population

Exposures

0039Obviously, techniques for monitoring human expos-

ure to carcinogenic compounds must be relatively

noninvasive and a number of assays have been ap-

plied to samples of blood and urine. The presence of

DNA adducts and oxidized DNA bases or nucleosides

such as 8-hydroxyguanine or 8-hydroxyguanosine

can be detected in urine and the DNA of leukocytes

(white blood cells) using specific antibodies or instru-

mental analysis. The comet assay can be used to

detect strand breakage in leukocyte DNA and levels

of repair replication can be measured as well. The

P-postlabeling assay has proven to be very sensitive

to examine adduct formation. Levels of mutagenesis

in lymphocytes can be detected using a variation of

the HGPRT mutagenesis assay and chromosome ab-

errations and sister chromatid exchange can also be

assayed.

See also: Chromatography: High-performance Liquid

Chromatography; Gas Chromatography; Mass

Spectrometry: Principles and Instrumentation;

Applications; Mutagens; Nucleic Acids: Properties and

Determination; Physiology; Pesticides and Herbicides:

Toxicology

Further Reading

Clayson DB (2001) Toxicological Carcinogenesis. Boca

Raton: Lewis.

Douglas JF (1984) Carcinogenesis and Mutagenesis Testing.

Clifton: Human Press.

Franks LM and Teich NM (eds) (1997) Introduction to the

Cellular and Molecular Biology of Cancer, 3rd edn.

Oxford: Oxford University Press.

Friedberg EC, Walker GC and Siede W (1995) DNA Repair

and Mutagenesis. Washington, DC: ASM Press.

Henderson DS (1999) DNA Repair Protocols. Methods in

Molecular Biology, vol. 113. Totowa: Humana Press.

Kilbey BJ, Legator M, Nichols W and Ramel C (1984)

Handbook of Mutagenicity Test Procedures. Amster-

dam: Elsevier Science.

Kitchin KT (ed.) (1999) Carcinogenicity: Testing, Predict-

ing, and Interpreting Chemical Effects. New York:

Marcel Dekker.

926 CARCINOGENS/Carcinogenicity Tests

Klug WS and Cummings MR (2000) Concepts of Genetics,

6th edn. Upper Saddle River: Prentice Hall.

Milman HA and Weisburger EK (eds) (1994) Handbook of

Carcinogen Testing, 2nd edn. Park Ridge: Noyes Publi-

cations.

Pfeifer GP (ed.) (1996) Technologies for Detection of DNA

Damage and Mutations. New York: Plenum Press.

Twyman RM (1998) Advanced Molecular Biology: A

Concise Reference. Oxford: BIOS Scientific.

CAROTENOIDS

Contents

Occurrence, Properties, and Determination

Physiology

Occurrence, Properties, and

Determination

D B Rodriguez-Amaya, Universidade Estadual de

Campinas, Campinas, SP, Brazil

Introduction

0001 Current knowledge on the structures, distribution,

and properties of food carotenoids is presented.

Their role as food colorants and changes during pro-

cessing and storage of foods are discussed. Trends in

the inherently difficult analysis of these compounds

are described, pointing out sources of errors, as well

as means to guarantee the reliability of results.

Structures and Occurrence in Foods

0002 Among the naturally occurring pigments, carotenoids

are notable for their wide distribution, structural

diversity, and varied functions and actions. In nature,

about 100 million tonnes of these compounds are

produced annually. More than 600 carotenoids,

exclusive of cis and trans isomers, have now been

isolated and characterized from natural sources.

This remarkable number includes the enormous var-

iety of carotenoids in algae, bacteria, yeast, and fungi.

In foods, the number is much more restricted, but the

carotenoid composition can still be complex.

0003 Food carotenoids are generally C

tetraterpenoids

formed from eight C

isoprenoid units joined head to

tail, except at the center, where a tail-to-tail linkage

reverses the order and results in a symmetrical

molecule. Lateral methyl groups near the center are

separated by six carbon atoms and the others by five

(Figure 1). An important feature is a centrally located,

extended double-bond system. The p electrons are

highly delocalized over the entire polyene chain,

although electron density appears to be greater at or

towards the end of the chain. The basic skeleton

may be modified in many ways, such as cyclization,

hydrogenation, dehydrogenation, introduction of

oxygen functions, rearrangement, chain shortening,

or combinations thereof, resulting in an immense

array of structures.

0004A semisystematic nomenclature that conveys struc-

tural information has been devised for carotenoids. In

this chapter, the semisystematic names are given in

parentheses in the figures and table; those of other

carotenoids will be cited with the trivial names when

first mentioned in the text. Thereafter, only the better

known trivial names will be used.

0005Hydrocarbon carotenoids (e.g., b-carotene, lyco-

pene) are known as carotenes; oxygenated derivatives

are called xanthophyls. Common oxygen substituents

are: hydroxy (as in b-cryptoxanthin), keto (as in

canthaxanthin), epoxy (as in violaxanthin), and alde-

hyde (as in b-citraurin) groups. Carotenoids may be

acyclic (e.g., z-carotene, lycopene), monocyclic (e.g.,

g-carotene), or bicyclic (e.g., a- and b-carotene).

Cyclization is limited to the formation of a six-

membered (occasionally five-membered) ring at one

or both ends of the molecule.

0006In nature, carotenoids exist primarily in the more

stable all-trans (or all-E) form, but cis (or Z) isomers

do occur. The first C

compound in the biosynthetic

pathway has the 15-cis configuration in most natural

sources. Bixin, the principal pigment of the colorant

annatto,alsooccursnaturallyinthe cisform(Figure2)

Small amounts of cis isomers of other carotenoids

have been increasingly reported. Commonly encoun-

tered cis- b-carotenes are shown in Figure 3.

CAROTENOIDS/Occurrence, Properties, and Determination 927

CHO

15'

14'

13'

20'

12'

11'

10'

19'

18'

17'

16'

15'

14'

12'

11'13'

10'

19'20'

18'

16'

17'

Lycopene (␺,␺-carotene)

␨-Carotene (7,8,7',8'-tetrahydro-␺,␺-carotene)

γ-Carotene (β,␺-carotene)

␤-Carotene (β,β-carotene)

α-Carotene ((6'R)-β,ε-carotene)

β-Cryptoxanthin ((3R)-β,β-carotene-3-ol)

Zeaxanthin ((3R,3'R)-β,β-carotene-3,3'-diol)

Lutein ((3R,3'R,6'R)-β,ε-carotene-3,3'-diol)

Violaxanthin ((3S,5R,6S,3'S,5'R,6'S)-5,6,5'6'-diepoxy-

5,6,5'6'-tetrahydro-β,β-carotene-3,3'-diol

Neoxanthin ((3S,5R,6R,3'S,5'R,6'S)-5',6'-epoxy-6,7-

didehydro-5,6,5',6'-tetrahydro-β,β-carotene-3,5,3'-triol)

β-Citraurin ((3R)-3-hydroxy-8'-apo-β-caroten-8'-al)

fig0001 Figure 1 Structures of common carotenoids in foods of plant origin.

928 CAROTENOIDS/Occurrence, Properties, and Determination

0007 The major structural differences that account for

variations in properties and biological activities of

carotenoids, therefore, are: length and rigidity of the

molecule, length of the conjugated double-bond

system, cyclized or acyclic nature of the end groups,

and presence of polar substituents in the predomin-

antly hydrocarbon molecule.

0008 Although masked by the green chlorophyls, carote-

noids are found universally in the chloroplasts of

photosynthetic tissues. Leaves of all species studied

revealed the presence of the same major carotenoids:

lutein, b-carotene, violaxanthin, and neoxanthin.

Small amounts of a-carotene, a-cryptoxanthin

((3

R,6

R)-b,e-caroten-3

-ol) or b-cryptoxanthin, zeax-

anthin, antheraxanthin ((3S,5R,6S,3

R)-5,6-epoxy-

5,6-dihydro-b,b-carotene-3,3

-diol), and lutein-5,6-ep-

oxide ((3S,5R,6S,3

R,6

R)-5,6-epoxy-5, 6-dihydro-

b,e-carotene-3,3

-diol) may also be found. The xantho-

phyls are unesterified, and the relative ratios are

fairly constant, but the absolute concentrations

vary considerably. Lettuce also has lactucaxanthin

((3R,6R,3

R,6

R)-e,e-carotene-3,3

-diol) at levels com-

parable with, or slightly higher than, neoxanthin.

0009In ripe fruits carotenoids are located in chromo-

plasts and the hydroxycarotenoids are mostly esteri-

fied with fatty acids. The composition varies

dramatically from fruit to fruit, but eight major pat-

terns can be discerned: (1) insignificant levels of car-

otenoids; (2) small amounts generally of chloroplast

carotenoids (e.g., grape); (3) considerable amounts

of lycopene (e.g., tomato, watermelon, red-fleshed

guava and papaya); (4) predominance of b-carotene

and/or b-cryptoxanthin (e.g., apricot, peach, loquat);

(5) large amounts of epoxides (e.g., mango,

HOOC

COOCH

Bixin (methyl hydrogen 9'-cis-6,6'-diapocarotene-6,6'-dioate)

Capsanthin ((3R,3'S,5'R )-3,3'-dihydroxy-β,κ-caroten-6'-one)

Capsorubin ((3S,5R,3'S,5'R )-3,3'-dihydroxy-κ,κ-carotene-6,6'-dione)

HOOC

COOH

Crocetin (8,8'-diapocarotene-8,8'-dioic acid)

fig0002 Figure 2 Principal carotenoids of natural extracts used as food colorants.

CAROTENOIDS/Occurrence, Properties, and Determination 929

carambola); (6) preponderance of unusual or species-

specific carotenoids (e.g., red pepper); (7) substantial

amounts of poly-cis-carotenoids (e.g., tangerine

tomato); (8) significant levels of apocarotenoids (car-

otenoids with shortened carbon skeleton) (e.g., citrus

species). Some merging of these patterns can be seen

in some fruits.

0010 In the few carotenogenic roots (e.g., carrot, sweet

potato), the carotenes are preponderant. In corn

(seed), the xanthophyls predominate.

0011 Since plants are able to synthesize carotenoids de

novo, the composition of plant foods is enriched by

the presence of trace amounts of biosynthetic precur-

sors along with derivatives of the main components

(Figure 4, Table 1). In a given food, compositional

variations occur as a consequence of varietal differ-

ences, climatic conditions, agricultural variables,

stage of maturity, postharvest handling, conditions

during storage, and transportation. Ripening of fruits

is generally accompanied by enhanced carotenogenesis,

the carotenoids increasing in number and quantity.

0012 Carotenoids are not as widely distributed in animal

foods, and the total content is much less. Incapable of

carotenoid biosynthesis, animals depend on dietary

carotenoids, which are selectively or unselectively

absorbed, converted to vitamin A, deposited as such

or slightly altered to form carotenoids typical of

animal species (Figure 5). Astaxanthin is the major

carotenoid in most crustaceans, either free, esterified,

or as carotenoprotein complexes. b-Carotene, echine-

none and canthaxanthin are the other pigments usu-

ally encountered. When found in fish, carotenoids are

located in the skin and flesh. The xanthophyls

predominate over the carotenes; astaxanthin is the

most common, followed by lutein (dominant in fresh-

water fishes) and tunaxanthin (e,e-carotene-3,3

-diol)

(characteristic of marine fishes).

0013Avian species preferentially accumulate certain

xanthophyls, which provide the color of eggs, body,

skin, and fat. Cattle absorb b-carotene efficiently,

but not xanthophyls; thus, diet-derived b-carotene

predominates in milk.

Physical and Chemical Properties

0014The conjugated double-bond system constitutes the

light-absorbing chromophore that gives carotenoids

their attractive color and provides the visible absorp-

tion spectrum, which serves as the basis for their

identification and quantification. A few examples of

the structure–spectrum relationship will be cited,

referring to carotenoids in petroleum ether. Most

carotenoids absorb maximally (l

max

) at three wave-

lengths. The greater the number of conjugated double

bonds, the higher the l

max

values. The acyclic lyco-

pene, with 11 conjugated double bonds, is red and

absorbs at the longest wavelengths (l

max

at 444, 470,

and 502 nm). At least seven such bonds are needed

for a carotenoid to have a perceptible color. Thus,

z-carotene is light yellow, and, being also acyclic, its

spectrum has three well-defined peaks but at much

shorter wavelengths (378, 400, and 425 nm). The two

carotenoids that precede z-carotene in the desatura-

tion biosynthetic pathway, phytoene (three conju-

gated double bonds) and phytofluene (five

conjugated double bonds), are colorless and absorb

maximally at 276, 286, and 297 nm and 331, 348,

and 367 nm, respectively. Cyclization results in steric

All-trans-β-carotene

15-cis-β-carotene 13-cis-β-carotene 9-cis-β-carotene

fig0003 Figure 3 Common geometrical isomers of b-carotene.

930 CAROTENOIDS/Occurrence, Properties, and Determination

hindrance between the ring methyl group at C-5 and

the hydrogen at C-8 of the polyene chain, taking the p

electrons of the ring double bond out of plane with

those of the chain. Consequently, a hypsochromic

shift, hypochromic effect, and loss of spectral fine

structure are observed. Thus, bicyclic b-carotene,

although possessing the same number of bonds as

lycopene, is yellow–orange and has a l

max

at 450

and 477 nm and a mere inflection at 425 nm. Mono-

cyclic g-carotene is red–orange and exhibits a spec-

trum intermediate between those of lycopene and

b-carotene. The double bond in the e-ring of

a-carotene is out of conjugation, so it is light yellow,

and the absorption peaks are slightly more defined

and at slightly shorter wavelengths (422, 445, and

473 nm) compared to b-carotene. Conjugated

carbonyl groups cause a bathochromic shift and loss

of fine structure to the extent that the three-maxima

spectrum is replaced by a broad curve, asymmetrical

with a maximum at 458 nm and a shoulder at 482 nm

for echinenone (orange) and symmetrical with the

maximum at 466 nm for canthaxanthin (red–orange).

Hydroxy substituents result in virtually no change in

color and absorption spectrum. Thus, lutein resem-

bles a-carotene, and b-cryptoxanthin and zeaxanthin

are similar to b-carotene in spectral properties. Cis

isomerization of one of the chromophore’s bond

causes slight loss in color, small hypsochromic shift

and hypochromic effect, accompanied by the appear-

ance of a cis peak in the ultraviolet region, the intensity

of which is greater as the cis double bond approaches

the center of the chromophore. The 5,6-mono- and

Phytoene

Phytofluene

␨-carotene

␤-zeacarotene neurosporene

α-zeacarotene

rubixanthin

γ-carotene

lycopene

δ-carotene

β-carotene-5,6-

epoxide

β-carotene-5,6,

5',6'-diepoxide

β-carotene

luteochrome

aurochrome

β-cryptoxanthin

zeaxanthin

antheraxanthin

violaxanthin

neoxanthin

neochrome

lycoxanthin

lycophyl

β-cryptoxanthin-5,6-

epoxide

cryptoflavin

mutatoxanthin

auroxanthin

α-carotene

α-cryptoxanthin or

zeinoxanthin

lutein

lutein-5,6-epoxide

(taraxanthin)

flavoxanthin or

crysanthemaxanthin

(1)

(2)

(3)

(4)

(5)

(4)

(5)

(2) (2)

(3)

(4)

(1)

(3)

(5)

(1)

(2)

(3)

(4)

(5)

luteoxanthin

fig0004 Figure 4 Later stages of carotenoid biosynthesis and possible transformations of carotenoids. Reactions: (1) desaturation, (2) cycli-

zation, (3) hydroxylation, (4) epoxidation, and (5) epoxide–furanoxide rearrangement. From Rodriguez-Amaya DB (1993) Nature and

distribution of carotenoids in foods. In: Charalambous G (ed.) Shelf Life Studies of Foods and Beverages. Chemical, Biological, Physical

and Nutritional Aspects, pp. 547–587. Amsterdam: Elsevier Science, with permission.

CAROTENOIDS/Occurrence, Properties, and Determination 931

5,6,5

-diepoxide, having lost one and two ring

double bonds, respectively, absorb maximally at

wavelengths some 5 and 10 nm shorter and are lighter

colored than the parent compounds. On transform-

ation to the 5,8-furanoid oxide, two double bonds

(one ring, one chain) are lost. Thus, the 5,8-mono-

and 5,8,5

-diepoxide are 20–25 and 50 nm lower,

respectively, than those of the parent compounds.

Solvent effects can be pronounced; the l

max

values

are higher relative to petroleum ether by 2–6nm in

acetone, 10–20 in chloroform and dichloromethane,

and 18–24 nm in toluene.

0015The intensity and hues of plant foods depend on

which carotenoids are present, their concentrations,

Astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione)

Canthaxanthin (β,β-carotene-4,4'-dione)

Echinenone (β,β-caroten-4-one)

fig0005 Figure 5 Some carotenoids typical of foods of animal origin. Astaxanthin occurs as a mixture of the (3S,3

S), (3R,3

R) and (3R,3

forms.

tbl0001 Table 1 Carotenoid distribution in some foods

Food Major carotenoid

concentration (mg/g)

Other carotenoids

Carrot b-Carotene (40–120) z-Carotene, g-carotene, d-carotene, lycopene, lutein, neurosporene, b-zeacarotene

a-Carotene (20–50)

Egg yolk Lutein (6) Zeaxanthin, isolutein, b-cryptoxanthin, b-carotene, a-carotene, neoxanthin,

z-carotene

Guava (pink-fleshed) Lycopene (47–60) b-Carotene, z-carotene, g-carotene, zeinoxanthin, b-cryptoxanthin,

b-carotene-5,6,5

-diepoxide, phytofluene, rubixanthin, cryptoflavin, lutein,

neochrome

Loquat b-Carotene (6–8) g-Carotene, neurosporene, lutein, violaxanthin, neoxanthin,

b-Cryptoxanthin (5–7) b-cryptoxanthin-5,6-epoxide, phytofluene, z-carotene, luteoxanthin, neochrome

Mango b-Carotene (6–75) Luteoxanthin, neoxanthin, zeaxanthin, auroxanthin, a-cryptoxanthin, b-cryptoxanthin,

z-carotene, phytoene, phytofluene, antheraxanthinViolaxanthin (18–22)

Papaya (red-fleshed) Lycopene (21–40) b-Carotene, z-carotene, b-cryptoxanthin-5,6-epoxide, g-carotene, b-zeacarotene,

cryptoflavin, antheraxanthinb-Cryptoxanthin (7–10)

Peach b-Cryptoxanthin (4–6) b-Carotene, z-carotene, violaxanthin, luteoxanthin, auroxanthin, lutein, zeaxanthin

Tomato Lycopene (30–50) Phytoene, phytofluene, b-carotene, z-carotene, g-carotene, neurosporene

Tree tomato b-Cryptoxanthin (14) z-Carotene, b-carotene-5,6-epoxide, lutein, zeaxanthin

b-Carotene (8)

Semisystematic names: neurosporene (7,8-dihydro-c, c-carotene); d-carotene ((6R)-e ,c-carotene); b-zeacarotene (7

-dihydro-b, c-carotene); isolutein

((3S,5R,6S,3

S,6

R)-5,6-epoxy-5,6-dihydro-b,e-carotene-3,3

-diol); zeinoxanthin ((3R,6

R)-b,e-caroten-3-ol; b-Carotene-5,6,5

-diepoxide (5,6,5

diepoxy-5,6,5

-tetrahydro-b,b-carotene); rubixanthin ((3R)-b,c-caroten-3-ol); b-cryptoxanthin-5,6-epoxide (5,6-epoxy-5,6-dihydro-b,b-caroten-3-ol);

cryptoflavin (5,8-epoxy-5,8-dihydro-b,b -caroten-3-ol); neochrome (5

-epoxy-6,7-didehydro-5,6,5

-tetrahydro-b,b-caroten-3,5,3

-triol); auroxanthin

((3S,5R,8RS,3

S,5

R,8

RS)-5,8,5

diepoxy-5,8,5

-tetrahydro-b,b-caroten-3,3

-diol); luteoxanthin (5,6,5

-diepoxy-5,6,5

-tetrahydro-b,b-carotene-

3,3

-diol); phytoene (7,8,11,12,7

,11

,12

-octahydro-c,c-carotene); phytofluene (7,8,11,12,7

-hexahydro-c,c-carotene).

932 CAROTENOIDS/Occurrence, Properties, and Determination

and physical state. In animals, complexation of

carotenoids with proteins extends the color to green,

purple, blue, or black. A well-known example is the

blue carotenoprotein crustacyanin of the lobster cara-

pace, an astaxanthin complex. On denaturation of

the protein (e.g., heating), astaxanthin is released,

and its vivid red color ensues.

0016 Carotenoids are lipophilic, generally insoluble in

water but soluble in organic solvents such as acetone,

tetrahydrofuran, ethyl ether, and chloroform. Caro-

tenes dissolve well in hexane and petroleum ether,

whereas the more polar xanthophylls dissolve more

readily in methanol and ethanol. In plants and

animals, carotenoids occur as crystals, in solutions

in fat depots, in colloidal dispersions, or combined

with proteins in the aqueous phase.

0017 Because of the many double bonds, carotenoids are

prone to trans–cis isomerization. A large number of

cis isomers are theoretically possible for each carot-

enoid. Only a few are actually formed because the cis

configuration in some double bonds creates steric

hindrance between nearby hydrogen atoms and

methyl groups, making the isomers unstable. The

steric hindrance is small when it occurs between

hydrogen atoms, so isomers with cis double bonds

in this situation are relatively stable and are easily

formed (e.g., 9-cis, 13-cis, 15-cis)(Figure 3).

0018 The highly reactive, electron-rich polyene chain is

also subject to oxidative degradation. Carotenoids

apparently have different susceptibilities, z-carotene,

lutein, and violaxanthin being cited as more labile.

In contrast to the wealth of information on the

oxidation of lipids, carotenoid oxidation is not well

understood. It involves initially epoxidation and the

formation of apocarotenoids. The preferred site of

epoxidation is the terminal double bond; oxidative

chain cleavage appears to commence at the C-7,8

position in b-carotene. Subsequent fragmentations

presumably yield low-mass compounds similar to

those obtained in fatty acid oxidation.

0019 Also related to the conjugated double-bond system

is the antioxidant property attributed to carotenoids

more recently. The primary mode of action is quench-

ing of singlet oxygen and interaction with free rad-

icals. The oxygen quenching ability is maximal, with

carotenoids having nine or more double bonds. The

acyclic lycopene was found to be more effective than

the bicyclic b-carotene. In a free radical-initiated

system, canthaxanthin and astaxanthin, both with

conjugated keto groups, were also shown to be better

antioxidants than b-carotene and zeaxanthin. At

elevated oxygen pressures, however, carotenoids are

reported to act as prooxidants.

0020 Xanthophyls undergo specific group reactions

which serve as simple chemical tests in the

determination of the structure. For example, primary

and secondary hydroxy groups are acetylated by

acetic anhydride in pyridine. Allylic hydroxyls, isol-

ated or allylic to the chromophore, are methylated

with acidified methanol. Epoxy groups in the 5,6-

or 5,6,5

-positions are easily detected by acid-

catalyzed conversion to the furanoid derivatives.

Aldehyde and keto carotenoids undergo reduction

with LiAlH

or NaBH

Use as Food Colorants

0021Carotenoids as food colorants find their way into

food products by direct addition or indirectly through

an animal’s feed. Commercial formulations are of

two types: natural extracts and synthetic nature-

identical carotenoids.

0022Annatto, paprika, and saffron as dry powders or

extracts have been used for years. Annatto is a series

of red coloring preparations all based on the extracts

of Bixa orellana seeds, where the pigments are con-

centrated in the thin seed coat. The apocarotenoid

bixin (Figure 2) is the main component of oil-soluble

formulations, and its saponification product, nor-

bixin, the major coloring matter of water-soluble

preparations. Oleoresin of paprika is the oil extract

of Capsicum annuum, which imparts a pinkish

yellow to crimson red color to foods, the predomin-

ating pigments being capsanthin and capsorubin. Saf-

fron consists of the dried stigma of Croccus sativus

and is used as a spice and yellow coloring agent. It

contains mainly crocin, the digentiobioside of croce-

tin, a diapocarotenedioic acid. Other commercial

sources of carotenoids are lutein-rich marigold petals

for poultry feed and b-carotene-rich microalgae.

Industrial production of natural carotenoids by

biotechnology is gaining more interest. (See Color-

ants (Colourants): Properties and Determination of

Natural Pigments.)

0023The first carotenoid prepared by chemical synthe-

sis, b-carotene, was introduced commercially by

Roche in 1954. It was followed by b-apo-8

-carotenal

-apo-b-caroten-8

-al) in 1960, b-apo-8

-carotenoic

acid ethyl ester (ethyl 8

-apo-b-caroten-8

-oate) in

1962, and canthaxanthin in 1964. In 1968, BASF

introduced citranaxanthin (5

-dihydro-5

-apo-18

nor-b-caroten-6

-one), and in 1984, Roche launched

(3RS,3

RS)-astaxanthin as feed additives. Crystalline

carotenoids suffer from problems that render their

commercialization in this form impractical: instabil-

ity, insolubility in water, and limited solubility in fats

and oils. To satisfy the needs of the food industry,

special application forms have been developed

through sophisticated physicochemical operations.

Micronized oil suspensions are the major marketable

CAROTENOIDS/Occurrence, Properties, and Determination 933

forms for coloring fat-based foods. For water-based

foods, water-dispersible emulsions or colloidal prep-

arations are available.

0024 Advantages cited for carotenoids as food colorants

are: natural connotation, high tinctural potency, un-

affected by reducing conditions, noncorrosive, good

stability in the pH range of most food products, pro-

vitamin A activity, and other beneficial effects on

human health. Their disadvantages are: limited

color range, higher price compared with synthetic

dyes, sensitivity to oxidative degradation, solubility

problems. The last two disadvantages have been over-

come in the application forms mentioned above.

Stability on Processing and Storage

0025 Naturally present or added carotenoids are subject to

isomerization and oxidation during food processing

and storage. Isomerization to cis isomers, provoked

by the release of constituent acids during slicing and

pureeing of foods, heat treatment and exposure to

light, results in some loss of color and vitamin A

activity. Oxidation depends on: availability of oxygen;

the carotenoids present and their physical state; water

activity; presence of antioxidants (e.g., tocopherols

and ascorbic acid); exposure to light; presence of

metals, enzymes and peroxides; severity of the pro-

cessing treatment (i.e., destruction of the cellular

structure that protects the carotenoids, increase of

surface area, duration and temperature of heat treat-

ment); packaging material; and storage conditions.

Completely losing their color and biological activ-

ities, the carotenoids give rise to volatile compounds

that contribute to the aroma/flavor, desirable in tea

and wine and undesirable in dehydrated carrot. (See

Retinol: Properties and Determination.)

0026 Reports on carotenoid retention during processing

and storage of foods appear conflicting, some

claiming no loss or increase in carotenoid content

and others showing considerable reductions. Claimed

increases are likely to be artifacts of the analytical

process due to loss of carotenoids in fresh samples

because of enzymatic activity, greater extractability of

carotenoids from processed samples, unaccounted

loss of water, and leaching of soluble solids. On the

other hand, care must be taken so as not to attribute

carotenoid losses during analysis to processing and

storage effects.

0027 Notwithstanding the diverging results, processing

under good manufacturing practices should have a

small effect on carotenoids; stability is good to excel-

lent in frozen and heat-sterilized foods throughout the

normal shelf-life. Oxygen content is minimized by

hot packing, vacuum packing and oxygen scavenging

with ascorbic acid. Stability in dehydrated and

powdered fruits and vegetables is generally poor,

unless the product has been carefully processed and

stored in inert atmosphere in packaging impermeable

to oxygen and light. A considerable portion of the

carotenoids may be physically removed on peeling

and juicing of fruits and by milling of seeds and

grains. The time lag between peeling, cutting or

pure

eing and processing should be kept to a minimum

so as not to allow enzymatic oxidation of carote-

noids, which can be a more serious problem than

thermal decomposition. Crude red palm oil contains

a substantial amount of carotenoids, which are de-

graded on refining.

Analysis

0028Although more reliable data on food carotenoids

are being acquired, erroneous data persist in the

international literature. This reflects the inherent

difficulty in carrying out this type of analysis. The

principal confounding factors are: (1) the existence

of a large number of naturally occurring carotenoids,

(2) qualitative and quantitative variation in the

carotenoid composition among foods, (3) a wide

range of carotenoid concentrations in a given food,

and (4) the instability of carotenoids.

0029A major problem in carotenoid analysis arises from

their instability. Thus, whatever the method chosen,

precautionary measures to avoid artifact formation

and quantitative losses must be taken, such as com-

pletion of the analysis within the shortest possible

time, exclusion of oxygen, protection from light,

avoidance of high temperature and contact with

acids, and use of high purity solvents, free from

damaging impurities (e.g., peroxides in ether and

tetrahydrofuran).

0030The general procedure for carotenoid analysis con-

sists of sampling and sample preparation, extraction,

partition, or transfer to a solvent compatible with the

subsequent chromatographic step, saponification and

washing (for some samples), chromatographic separ-

ation, identification, and quantification. Common

errors are: samples not representive of the food lots

under investigation; inefficient extraction; physical

losses during the different steps; incomplete chro-

matographic separation; erroneous identification,

quantification, and calculation; and isomerization

and oxidation (enzymatic or nonenzymatic) during

analysis.

0031Carotenoid analysis has been carried out to differ-

ent extent, depending on the information sought. For

a long time, only the major provitamin A carotenoids

were determined. With the mounting evidence on the

importance of carotenoids in reducing the risk of

degenerative diseases and the recognition that this

934 CAROTENOIDS/Occurrence, Properties, and Determination

role is not related to the provitamin A activity, deter-

mination of nonprovitamin A carotenoids has also

been pursued. The complete carotenoid composition

is the ultimate aim of carotenoid analysis. However,

considering that the carotenoid composition of foods

typically consists of one to four principal compon-

ents, with a series of carotenoids in trace amounts, it

is doubtful that the added information is worth the

greater complexity, higher cost, and longer analysis

time. The determination of major carotenoids

appears adequate for the generation of data for food

databases.

0032 Because of the various factors that affect the carot-

enoid composition of foods, proper sampling and

sample preparation to obtain representative and

homogenous samples for analysis are of paramount

importance. Errors in these initial steps can easily

surpass those of the analysis per se. In addition,

results should be accompanied by pertinent informa-

tion, such as the variety, stage of maturity, part of the

plant analyzed, season, and geographic origin.

0033 The classical technique for separating carotenoids

is open-column chromatography (OCC). MgO:

Hyflosupercel and neutral alumina are the preferred

adsorbents. Fractions are eluted successively with

solvents of increasing polarity (e.g., increasing

proportion of ethyl ether and acetone in hexane or

petroleum ether), the separation being monitored

visually. Reproducibility and efficiency of separation

depend heavily on the analyst’s skill and experience.

0034 Reversed-phase high-performance liquid chroma-

tography (HPLC) on a C

column is currently the

method of choice. Reasons for the popularity of the

column are: its weak hydrophobic interaction

with the carotenoids (expected to be less destructive

than the polar forces in normal-phase OCC), com-

patibility with most carotenoid solvents, and the

polarity range of carotenoids, and wide commercial

availability.

0035 Most carotenoid separations have been carried out

with 5-mmC

spherical particles packed in a 250 

4.6 mm column. Some laboratories are already using

shorter and narrower (narrow bore) columns, smaller

particles (3 mm) and C

stationary phase (especially

for separating geometric isomers).

0036 Monomeric phases are simpler to use and are more

reproducible. Polymeric C

phases, however, have

an excellent selectivity for structurally similar

carotenoids (e.g., geometric isomers, lutein and zea-

xanthin). However, the peaks tend to be broader, and

columns from different production lots are more

variable than the monomeric columns

0037 The most important properties to be considered in

selecting the mobile phase are polarity, viscosity, vola-

tility, and toxicity. In addition, it must be inert with

respect to the carotenoids. Many solvent systems have

been suggested as mobile phases for carotenoids, but

the primary solvents are acetonitrile and methanol,

and most systems are actually slight modifications

of some basic combinations. Acetonitrile has been

widely used because of its lower viscosity and slightly

better selectivity for xanthophyls when a monomeric

column is used. However, higher recoveries of

carotenoids were reported with methanol-based

solvents. Carotenoid recovery with acetonitrile-

based solvents can be improved with the addition of

ammonium acetate and triethylamine. Methanol is

also more available, less expensive, and less toxic

than acetonitrile. Small amounts of other solvents

(e.g., tetrahydrofuran, ethyl acetate) are added to

obtain the desired retention, increase solubility, and

improve resolution.

0038Gradient elution should only be used when the

analysis cannot be done isocratically. Isocratic separ-

ation is rapid, can be performed with simple equip-

ment, and results in a stable baseline and more

reproducible retention times. It is usually sufficient

for the determination of provitamin A carotenoids or

the principal carotenoids.

0039Gradient elution offers a greater resolving power,

improved sensitivity, and elution of strongly retained

compounds. It is more likely to resolve the whole

range of carotenoids found in a given food. However,

it has several disadvantages: an increased complexity,

requirement for more sophisticated and expensive

equipment, a need for column reequilibration be-

tween runs, a greater differential detector response,

and often a poor reproducibility.

0040Because of the qualitative and quantitative vari-

ation of the carotenoid composition of foods, it is

doubtful that a single chromatographic condition

can be established for the different foods. At least

some modification of the mobile phase is needed

when changing from one food to another. Analysts

should also guard against undue confidence that

modern instruments inadvertently give. It is very

easy to make errors in HPLC. Unsatisfactory and

variable recoveries from the HPLC column, especially

of lycopene, have been reported.

0041The injection solvent must be compatible with the

HPLC mobile phase. A chromatogram with peak

tailing and broad, doubled peaks is obtained when

the carotenoids are much more soluble in the injec-

tion solvent than in the mobile phase. However, the

sample will not dissolve completely if the injection

solvent is too weak.

0042The retention times and the ultraviolet–visible ab-

sorption spectra (l

max

and fine structure) provide the

first clues for the identification of carotenoids. The

availability of the photodiode array detector allows

CAROTENOIDS/Occurrence, Properties, and Determination 935