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

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the spectra to be obtained on-line. However, the

widespread use of these two parameters as the only

criteria has led to misidentifications. Thus, it has been

recommended that the following minimum criteria be

fulfilled for identification: (1) the visible (or ultravio-

let for shorter chromophores) spectrum in at least two

different solvents must be in agreement with the chro-

mophore suggested; (2) chromatographic properties

must be identical in two systems, preferably TLC (R

)

and HPLC (t

), and cochromatography with the

authentic carotenoid should be demonstrated; and

(3) a mass spectrum should be obtained, which allows

at least confirmation of the molecular mass. The re-

quirement of a mass spectrum, however, would limit

carotenoid analysis to a very few laboratories around

the world, precluding its execution in areas where it is

very much needed. Moreover, major well-known

carotenoids can be conclusively identified by the

judicious and combined use of chromatographic

data, absorption spectra and specific chemical reac-

tions to confirm the type, location, and number of

functional groups. (See Chromatography: Thin-layer

Chromatography; High-performance Liquid Chro-

matography.)

0043 Mass spectrometry (MS) and nuclear magnetic res-

onance spectroscopy (NMR) are indispensable in the

elucidation of unknown or inconclusive structures of

carotenoids and are increasingly used with food

carotenoids. MS gives the molecular mass, and

typical fragmentations provide information about

the structure. NMR recognizes new end groups and

determines the location of cis double bonds. The

chemical shift data for many end groups and trans/

cis isomers are available from tables for both

H-NMR and

C-NMR. (See Spectroscopy: Nuclear

Magnetic Resonance.)

0044 The quantification step in OCC is straightforward.

The separated carotenoid fractions are collected and

quantified spectrophotometrically through the use of

tabulated absorption coefficients. HPLC quantifica-

tion is carried out by means of internal or external

calibration, for which the concentrations of the

standard solutions are also determined spectrophoto-

metrically. Unfortunately, few carotenoid standards

are available commercially and are of a widely vary-

ing purity. Other carotenoids have to be isolated and

purified from natural sources by OCC or by accumu-

lating separated fractions from several HPLC runs.

Both procedures are time-consuming and require ex-

perience and patience. The instability of the caroten-

oid standards is another serious problem.

0045 Notwithstanding the inherent difficulties and the

many possible sources of errors, reliable data on food

carotenoids can be obtained in the hands of careful

and well-informed analysts.

See also: Analysis of Food; Antioxidants: Natural

Antioxidants; Cancer: Diet in Cancer Prevention;

Chromatography: High-performance Liquid

Chromatography; Colorants (Colourants): Properties

and Determination of Natural Pigments; Guavas;

Mangoes; Mass Spectrometry: Applications; Oxidation

of Food Components; Papayas; Peaches and

Nectarines; Ripening of Fruit; Tomatoes; Vegetables

of Temperate Climates: Carrot, Parsnip, and Beetroot

Further Reading

Britton G, Liaaen-Jensen S and Pfander H (eds) (1995)

Carotenoids, vol 1A. Isolation and analysis. Basel: Bir-

khauser.

Britton G, Liaaen-Jensen S and Pfander H (eds) (1995)

Carotenoids, vol. 1B. Spectroscopy. Basel: Birkhauser.

Goodwin TW and Britton G (1988) Distribution and

analysis of carotenoids. In: Goodwin TW (ed.) Plant

Pigments, pp. 61–132. London: Academic Press.

Gross J (1987) Pigments in Fruits. London: Academic Press.

Gross J (1991) Pigments in Vegetables. Chlorophylls and

Carotenoids. New York: Van Nostrand Reinhold.

Krinsky NI, Mathews-Roth MM and Taylor RF (eds)

(1990) Carotenoids: Chemistry and Biology. New

York: Plenum Press.

Packer L (ed.) (1992) Carotenoids part A. Chemistry,

separation, quantitation, and antioxidation. Methods

in Enzymology 213: 3–538.

Pfander H (ed.) (1987) Key to Carotenoids, 2nd edn. Basel:

Birkhauser.

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.

Rodriguez-Amaya DB (1999a) A Guide to Carotenoid

Analysis in Foods. Washington, DC: International Life

Sciences Institute Press.

Rodriguez-Amaya DB (1999b) Changes in carotenoids

during processing and storage of foods. Archivos Latino-

americanos de Nutricio

n 49: 36S–47S.

Physiology

H C Furr, Craft Technologies, Wilson, NC, USA

R M Clark, University of Connecticut, Storrs, CT, USA

Background

0001The carotenoids are polyisoprenoid compounds (usu-

ally tetraterpenes); most common carotenoids have

40 carbon atoms, although shorter (apocarotenoids)

and longer forms exist. Frequently, carotenoids have

936 CAROTENOIDS/Physiology

six-membered rings at each end of the polyprenoid

chain, although lycopene (with no end rings) is a

prominent exception (Figure 1). b-Carotene (C

MW 536), with two b-ionone rings connected by a

conjugated polyene chain, is the carotenoid proto-

type; formally, it can be considered to be composed

of eight isoprene units joined head-to-tail, except

in the center, so that the molecule is symmetrical about

the center. The large number of conjugated carbon–

carbon double bonds is responsible for carotenoids’

high light-absorbing ability and intense red–yellow

colors (wavelengths of maximum light absorption

are typically 450–500 nm, molar extinction coeffi-

cients typically 132 000–185 000 mol

1

). Effect-

ive photoprotection requires at least nine conjugated

double bonds, and absorption of visible light requires

seven to 13 conjugated double bonds. Higher carote-

noids (C

and C

), of bacterial origin, are products

of addition of C

units to give a ring structure substi-

tuted at the 2-position.

0002 The two major carotenoid subgroups are the caro-

tenes (hydrocarbon carotenoids, including lycopene,

a-carotene, and b-carotene) and the oxycarotenoids,

also called xanthophyls (hydoxy-, oxy-, epoxy-, and

furanoxy derivatives, such as lutein, canthaxanthin,

and astaxanthin) (Figure 1). The prefix ‘neo-’ was

previously used to designate carotenoids with at

least one cis configuration in the polyisoprene

chain, and the prefix ‘pro-’ designates some poly-cis-

carotenoids. The prefix ‘apo-’ designates a chain-

shortened structure (e.g., b-apo-8

-carotenal). Be-

cause the basic structures can be further metabolized

by the introduction of hydroxyl, keto, or methoxyl

groups, and by hydrogenation, cyclization, isomeri-

zation, and oxidative degradation, a great variety of

structures exist; it has been estimated that more than

600 carotenoids have already been identified in

nature. Good reviews on the structures and physical

properties of carotenoids have been provided by Isler

et al. and by Bauernfeind, and in a series edited by

Britton, Liaaen-Jensen, and Pfander.

0003Carotenoids are synthesized by higher plants and

by certain fungi, algae, and bacteria; they are not

synthesized by animals but may be biochemically

modified by them. Carotenoids are ubiquitous

in the plant kingdom (no naturally occurring

green plant lacks carotenoids), which suggests that

their two functions are irreplaceable: as protectants

against reactive singlet oxygen and as accessory

photopigments. In photoprotection, chlorophyl and

other cellular components are protected against

irreversible destruction by preferential reaction of

reactive singlet-state oxygen (electronically excited)

with carotenoids. As accessory photopigments,

β-Carotene

β-Cryptoxanthin

β-apo-8⬘-Carotenal

Zeaxanthin

Canthaxanthin Astaxanthin

Lutein

Lycopene

α-Carotene

fig0001 Figure 1 Structures of some representative carotenoids.

CAROTENOIDS/Physiology 937

carotenoids increase the efficiency of photosynthesis

by absorbing light in regions of the visible spectrum

where chlorophyls do not absorb efficiently, then

transferring the captured energy to other photopig-

ments; carotenoids in photosystems are associated

with proteins, and the carotenoid–protein inter-

actions can shift the wavelength of maximum light

absorption. Carotenoids are particularly effective in

both processes, because their singlet electronic states

are higher in energy, and their triplet states are lower

in energy than those of chlorophyls, thus allowing

both photoprotection and light-harvesting to be ener-

getically favorable.

0004 The total carotenoid production in the biosphere

has been estimated at 10

tonnes per year; this is

mostly due to fucosanthanin (characteristic of many

marine algae) and the main carotenoids of green

leaves, namely lutein, violaxanthin, and neoxanthin.

b-Carotene, although quantitatively less important,

occurs widely and is of particular interest as a

major precursor of vitamin A for animal and human

nutrition.

Absorption and Bioavailability of

Carotenoids

0005 There is considerable species variability in the effi-

ciency of absorption and in metabolism of carote-

noids. Humans are apparently unusual in absorbing,

transporting, and storing both carotenes and xantho-

phyls well. Rats do not accumulate b-carotene, but

instead metabolize almost all dietary b-carotene to

vitamin A. In contrast, cattle show serum and milk

accumulation of b-carotene but not of xanthophyls.

Animals with yellow fat absorb and store carote-

noids, whereas animals with white fat do not. Ferrets,

gerbils, preruminant calves, and nonhuman primates

have been used as animal models for human caroten-

oid absorption; rats and mice are also useful for

studies of noncleavable carotenoids and for studies

of pharmacological doses of carotenoids used for

cancer prevention or treatment. Some fish (for

example, salmonids) and birds absorb xanthophyls

particularly well and use them as pigments.

0006 Intestinal absorption of carotenoids in animals re-

quires the presence of bile acids; human subjects with

impaired bile flow (biliary atresia) show low levels of

liver carotenoids compared with normal subjects. The

presence of other dietary lipid promotes expansion of

the mixed bile salt micelle and enhances carotenoid

absorption. Animal and human feeding studies have

shown that the biological matrix of food affects

absorption of dietary carotenoids, and dietary caro-

tenoids in plant tissues have a lower bioavailability

than those in oil solutions. Hence, the plant source

and method of preparation of foods affect bioavail-

ability of b-carotene and other carotenoids: processes

that break down the biological matrix (e.g. grinding

or chopping, mild cooking) improve carotenoid ab-

sorption. Castenmiller and West (1998) have charac-

terized the factors affecting carotenoid absorption as

Species of carotene; Molecular linkage (e.g., esterifi-

cation of xanthophyls); Amount of carotene con-

sumed in a meal; Matrix in which the carotenoid

is incorporated; Absorption modifiers (including

lipid content of the meal); Nutrient status of host (in

particular, vitamin A status); Genetic factors; Host-

related factors; and Interactions (e.g., competition

among carotenoids for absorption).

0007Unequivocal estimation of the efficiency of absorp-

tion of dietary carotenoids is very difficult because of

the ubiquitous presence of carotenoids in the diet

(and thus in plasma and other tissues), slow absorp-

tion kinetics, partial metabolism of carotenoids, and

considerable individual variation in the extent of

absorption and metabolism.

Transport, Distribution, and Storage of

Carotenoids

0008As with other lipids, freshly absorbed carotenoids are

transported in the lymph via chylomicra. It is believed

that the carotenoids, as with other lipid components

of the chylomicron remnants, are taken up by

the liver and then released into the blood stream

as components of very-low-density lipoproteins

(VLDL). The VLDL are taken up by extrahepatic

tissues, their components processed into low-density

lipoproteins (LDL), and eventually into high-density

lipoproteins (HDL). Early studies showed that

in human plasma, the hydrocarbon carotenoids b-

carotene and lycopene are predominantly associated

with LDL (75%) and HDL (25%); the xanthophyl

lutein is approximately equally distributed between

HDL and LDL. In cattle, where the predominant

plasma lipoprotein is HDL, more than 80% of

total plasma b-carotene is transported in this fraction.

The predominant human serum carotenoids in the

USA are b-carotene, a-carotene, lycopene, b-cryptox-

anthin, lutein, and zeaxanthin, and also the colorless

carotenoids phytoene and phytofluene; serum carot-

enoid profiles and concentrations are highly depend-

ent on dietary intake and so depend on food habits. It

is estimated that serum carotenoids represent about

1% of total body carotenoids in the human.

0009Adipose tissue (80–85% of total body carotenoids)

and liver (8–12%) are the major sites of carotenoid

deposition in humans when the total mass of tissue

carotenoid is considered. Typical total human carot-

enoid levels of 100–150 mg are reported from

938 CAROTENOIDS/Physiology

autopsy analyses. Human adipose carotenoid profiles

are similar to those of serum. Reported values for

total human liver carotenoids range between 0 and

97 mg per gram of liver (i.e., 0–0.18 mmol g

1

; total

carotenoids were expressed as b-carotene in older

studies), and include lutein, lycopene, a-carotene,

and b-carotene. Other tissues that are known to

contain high concentrations of carotenoids include

adrenals (20 mgg

1

(37 nmol g

1

) tissue, mostly as b-

carotene) and human macular pigment (containing

predominantly the xanthophyls lutein and zeaxan-

thin), as well as kidney, ovaries, pituitary, placenta,

testes, and thymus in cattle. Chick retinal oil droplets

accumulate astaxanthin, which can be formed from

dietary zeaxanthin or canthaxanthin. It has been sug-

gested that organs with high numbers of LDL recep-

tors and high rates of LDL uptake show higher tissue

levels of carotenoids.

0010 The specific accumulation of certain carotenoids in

particular tissues may furnish clues to distinctive

physiological roles of carotenoids. The macula of

the primate eye accumulates the xanthophyls lutein

and zeaxanthin, with lutein being found predomin-

antly in the center of the macula; loss of these xantho-

phyls is associated with macular degeneration, a

disease characteristic of aging. Bovine corpus luteum

amasses xanthophyls (as much as 60 mg of total car-

otenoids per gram, 105 nmol per gram of tissue), and

it has been suggested that carotenoids play some im-

portant role in bovine reproduction; however, porcine

corpus luteum does not accumulate carotenoids.

Bovine pineal gland has been found to store b-carotene

(as much as 1 mg per gram, 2 nmol per gram of wet

tissue), with perhaps small amounts of xanthophyls

also present; the absence of other carotenoids was

noteworthy. Levels of approximately 1 mg of total

carotenoid per gram of bovine pituitary gland have

been reported. The physiological importance of these

accumulations of carotenoids is, however, not clear.

0011 Carotenoid–protein interactions have been stud-

ied in plants, bacteria, and marine invertebrates.

The strength of specific protein–carotenoid binding

depends, not surprisingly, on carotenoid structure.

Distinctive interactions between a binding protein

and its carotenoid ligand are evident by the spectral

changes of the carotenoid on binding; these spectral

changes may reflect physiological functions as well

as producing color polymorphism among species. A

notable example of these carotenoid–protein inter-

actions is the binding of the xanthophyl astaxanthin

(red-colored in solution) to the protein a-crustacyanin

in the lobster, giving a pigment that is typically blue–

green; on boiling the animal, the carotenoid is

released from its binding protein to yield a bright

red color.

Retinol Equivalents of Different

Carotenoids

0012Of the more than 600 known carotenoids, only about

60 have been reported to be precursors of vitamin A.

In order to serve as a precursor of vitamin A, a carot-

enoid must have at least one unsubstituted ‘b-ionone’

ring (2,6,6-trimethyl-1-cyclohexen-1-yl) with a poly-

ene side chain of at least 11 carbon atoms. Thus,

a-carotene (with one b-ionone ring) has half the

biological activity of b-carotene, and canthaxanthin

(with keto substitutions on both rings; Figure 1) has

no provitamin A activity. Absolute vitamin A activity,

however, depends on a number of other factors, not

all of which are well understood. In general, it seems

that the efficiency of the conversion of carotenoids to

vitamin A depends on the vitamin A status: high

intakes of preformed vitamin A result in poor effi-

ciency of conversion to vitamin A, perhaps due to

metabolic control of the cleavage enzyme(s). Frank

vitamin A deficiency is also associated with impaired

conversion efficiency, perhaps because of damaged

intestinal epithelial function in vitamin A inadequacy.

Because conversion of b-carotene to vitamin A is

impaired in vitamin A deficiency, vitamin A defi-

ciency disease is more rapidly cured by providing

preformed vitamin A than by providing dietary

carotenoids. Excessive vitamin E intake may impair

carotenoid cleavage (or interferes with intestinal ab-

sorption); vitamin E deficiency decreases vitamin A

formation, perhaps because adequate vitamin E is

needed to protect carotenoids and vitamin A from

oxidation. Conversion efficiency of b-carotene and

other carotenoids to vitamin A is also decreased at

high carotenoid intakes, perhaps due to impaired

intestinal absorption or to metabolic control of the

cleavage enzyme(s). Protein deficiency impairs carot-

enoid cleavage, suggesting that protein malnutrition

in humans exacerbates vitamin A deficiency.

0013Common dietary sources of provitamin A carote-

noids are carrots, yellow squash, dark-green leafy

vegetables, yellow maize, tomatoes, papaya, and

oranges. Cereal grains and white maize contain little

or no provitamin A. Red palm oil is one of the richest

sources of provitamin A carotenoids, containing

approximately 0.5 mg of a-carotene þb-carotene per

milliliter; as little as 7 ml of palm oil per day may

provide adequate vitamin A for the preschool child.

On a global basis, b-carotene is the most important

vitamin A precursor, both because of its greater pro-

vitamin A activity and because of its wide distribution

in plant products. The recent incorporation of the

enzymatic pathway of carotenoid biosynthesis into

rice (‘Golden Rice’) promises to help alleviate vitamin

A deficiency in parts of the world where rice is

CAROTENOIDS/Physiology 939

eaten extensively. Other provitamin A carotenoids,

such as a-carotene and b-cryptoxanthin and

b-apo-carotenals, can be nutritionally important

sources of vitamin A from particular foods (Table 1).

Other carotenoids, such as lycopene (which has no b-

ionone rings) and the dihydroxycarotenoids lutein

and zeaxanthin (which are substituted on both ionone

rings), may be major carotenoids in particular foods

and may have other important physiological func-

tions but have no provitamin A activity.

0014 Estimation of provitamin A content of foods from

food composition tables is problematic: carotenoid

composition of raw fruits and vegetables varies with

species, growth conditions, and mode of storage. Fur-

thermore, older tables often express total carotenoids

as ‘b-carotene,’ ignoring the different provitamin A

activity of different carotenoids. Biological activity

of carotenoids in foodstuffs may differ from the

analyzed content because of the mode of preparation,

binding of carotenoids within the foodstuff, and the

nature of the meal (presence or absence of fat), as

discussed above. Conversion factors, recently revised

to account for apparent bioavailability of carotenoids

from foods, are given in Table 2.

Enzymatic Conversion to Vitamin A

0015By comparison of the structure of b-carotene with

that of vitamin A, it would appear that b-carotene

could be cleaved in its center (between the 15 and 15

carbon atoms) to give two molecules of vitamin A per

molecule of carotenoid. Such a mechanism was first

proposed by Karrer. However, in addition to this

central (symmetric) cleavage, a random (asymmetric)

cleavage has been proposed. Glover first suggested

that asymmetric cleavage of the symmetric b-carotene

molecule could be followed by stepwise shortening

to vitamin A. In support of this hypothesis, small

tbl0001 Table 1 Vitamin A activity of common carotenoids relative to b-carotene

Carotenoid Activity (%) Occurrence (partiallist)

b-Carotene 100 Green plants, vegetables, carrots, yellow sweet potatoes,

squash, tomatoes, red and yellow fruits

a-Carotene 50–54 Green plants, carrots, squash, maize, green peppers

g-Carotene 42–50 Carrots, sweet potatoes, maize, tomatoes, algae, some

fruits

b-Carotene 5

-monoepoxide 21 Plants, potatoes, red peppers

b-Carotene 5,6:5

-diepoxide Active? Plants

b-Carotene 5

-monofuranoxide (mutatochrome,

citroxanthin, flavacin)

50 Orange peel, red peppers, tomatoes, sweet potatoes,

cranberries

4-Keto-b-carotene (4-oxo-b-carotene, echinenone,

aphanin, myxoxanthin)

44–54 Algae, sea urchins, Daphnia, Hydra, red sponges, brine

shrimp, crustaceans

3-Keto-b-carotene (3-oxo-b-carotene) 52

3-Hydroxy-b-carotene (b-cryptoxanthin) 50–60 Yellow maize, green peppers, lichens, persimmons,

papayas, lemons, oranges

4-Hydroxy-b-carotene (isocryptoxanthin) 48 Brine shrimp

3,4-Dehydro-3

-hydroxy-b-carotene (anhydrolutein,

deoxylutein)

Active, c. 10 Alfalfa meal, acidulated soybean soapstock

3-Hydroxy-4-keto-b-carotene (hydroxyechinenone) Probably active Algae, bacteria, flowers

b-Apo-2

-carotenal Active Citrus fruit

b-Apo-8

-carotenal 36–72 Citrus fruit, green plants

b-Apo-10

-carotenal Active Citrus fruit, green plants, alfalfa meal

b-Apo-12

-carotenal 120 Alfalfa meal

b-Apo-8

-carotenoic acid Active Maize, animal tissue

b-Apo-8

-carotenoic acid ethyl ester 25–78

Lycopene Inactive Tomatoes, carrots, green peppers, pink citrus fruit

3,3

-Dihydroxy-b-carotene (zeaxanthin) Inactive Spinach, paprika, yellow maize, green peppers, fruits

3,3

-Dihydroxy-a-carotene (lutein) Inactive Green leaves, yellow maize, potatoes, spinach, green

peppers, carrots, tomatoes, fruits

3,3

-Dihydroxy-4,4

-diketo-b-carotene (astaxanthin) Inactive Oranges, crustaceans, lobster, fish, algae, Daphnia, trout,

salmon

4,4

-Diketo-b-carotene (canthaxanthin, aphanicin) Inactive Mushrooms, trout, Daphnia, Hydra, microorganisms,

algae, crustaceans, brine shrimp

Capsanthin Inactive Red peppers, paprika

Capsorubrin Inactive Red peppers, paprika

Bixin Inactive Annatto seeds

Adapted and shortened from Bauernfeind JC, Adams CR and Marusich WL (1981) Carotenes and vitamin A precursors in animal feed. In: Bauernfeind JC

(ed.) Carotenoids as Colorants and Vitamin A Precursors, pp. 563–743. New York: Academic Press.

940 CAROTENOIDS/Physiology

amounts of labeled b-apo-8

-, b-apo-10

-, and b-apo-

-carotenal (and their alcohols and acids) have been

found in rat tissues after feeding radioactive b-carot-

ene. These apo-carotenals can be formed by chemical

reaction of oxidizing agents (hydrogen peroxide, po-

tassium permanganate) with b-carotene in vitro, and

they are found in small amounts in plants and in

animal-feeding experiments. Enzymatic asymmetric

cleavage activity has been demonstrated in vitro, but

its relative importance has not been established.

However, in vitro experiments (initially carried out

by Olson and Hayaishi and by Goodman and Huang)

using partially purified intestinal homogenates (from

rat, rabbit, or guinea-pig) have demonstrated sym-

metric cleavage of b-carotene, with retinal (vitamin

A aldehyde) as the only detectable product. This en-

zymatic activity requires the presence of O

, and has

been named ‘15,15

- b-carotenoid dioxygenase’ (EC

1.13.11.21). The enzyme has recently been cloned

from mammalian sources, and its further character-

ization promises to explain much about the factors

affecting provitamin A activity of b-carotene and re-

lated carotenoids. This enzymatic activity has been

demonstrated in intestine, liver, kidney, and several

other tissues in vitro. b-Carotene is not a source of

vitamin A in the cat, and it has been reported that

15,15

-dioxygenase activity is not present in cat intes-

tine, consistent with the ‘central cleavage’ hypothesis.

The possiblity remains that excentric cleavage is a

source of retinoic acid, which is an important modu-

lator of gene expression.

001 6 Some fish and birds can also convert the xantho-

phyls astaxanthin, canthaxanthin, and isozeaxanthin

to vitamin A; some freshwater fish can convert

lutein to 3,4-didehydroretinol (vitamin A

). Keto

carotenoids can be reduced to the corresponding alco-

hols and esterified with long-chain fatty acids.

Toxicity of Carotenoids

0017There is virtually no toxicity associated with high

intakes of carotenoids, in marked contrast to vita-

min A. Excessive human ingestion of b-carotene can

cause high serum carotenoid levels and yellowing of

light-colored skin, but such over-intake is very

unusual, and no harmful physiological effects from

even such high intakes of b-carotene have been

reported. The effects disappear when high carotenoid

intakes cease. Ingestion of large amounts of carote-

noids does not give rise to hypervitaminosis A, prob-

ably because of the decreased efficiency of conversion

of carotenoids to vitamin A at high intakes. Terato-

genicity due to carotenoids has never been suggested.

b-Carotene supplementation in humans does not

produce elevated serum triacylglycerols or choles-

terol. However, prolonged intake of high doses

(20 mg per day) of b-carotene has been associated in

epidemiologic studies with increased risk of lung

cancer in cigarette smokers, perhaps because of the

prooxidant effects of b-carotene at relatively higher

oxygen concentrations in the lungs. Although inges-

tion of 3–10mg of carotenoid per day from foods and

dietary supplements appears to be completely safe

and advisable, there are concerns that higher doses

of b-carotene (20–30 mg per day) may not be safe in

smokers.

0018The only carotenoid that has been clearly associated

with adverse effects is canthaxanthin (4,4

-diketo-b-

carotene), a carotenoid that has been used in treat-

ment of patients with erythropoietic protoporphyria

tbl00 02 Table 2 ConversionfactorsforestimatingvitaminAvaluefromcarotenoidcomposition

Factors

1retinolactivityequivalent(RAE)

¼1 mgall-transretinol

¼12 mgall-trans b-carotene

¼24 mgotherprovitaminAcarotenoids

¼3.33IUvitaminA

(Notethat1RAE¼1.15 mgofretinylacetate(MW328)¼1.83 mgofretinylpalmitate(MW524),i.e.,molarequivalence)

1InternationalUnitofvitaminA(IU

)

¼0.3 mgall-transretinol

¼0.3RAE

¼3.6 mgall-trans b-carotene

¼7.2 mgotherprovitaminAcarotenoids

Formulas

(1)Retinoland b-carotenegivenin mg,RAE¼ mgretinolþ(mg b-carotene/12)

(2) b-CaroteneandotherprovitaminAcarotenoidsgivenin mg,RAE¼(mg b-carotene/12)þ(mgotherprovitaminAcarotenoids/24)

AdaptedfromOlsonJA(1987)Recommendeddietaryintakes(RDI)ofvitaminAinhumans:Appendix.AmericanJournalofClinicalNutrition45:704–716

and from Panel on Micronutrients, Subcommittees on Upper Reference Levels of Nutrients and of Interpretation and Use of Dietary Reference Intakes,

and the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes (2001) Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic,

Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon,Vanadium, and Zinc. Washington, DC: National Academy Press. Available

onlineathttp://books.nap.edu/.

CAROTENOIDS/Physiology 941

and related skin disorders. High daily doses

(50–100 mg) have resulted in canthaxanthin retino-

pathy (crystalline deposits in the retina leading to

impaired night vision); the effect is slowly reversible

on cessation of intake. Pharmacological doses of

canthaxanthin have depressed liver accumulation of

dietary vitamin A in rats. Human ingestion of large

amounts of canthaxanthin gives a yellowish color-

ation to light-colored skin; this is the basis of some

‘skin tanning’ pills.

Therapeutic Role of Carotenoids

0019 In addition to the important role of some carotenoids

as vitamin A precursors, there is evidence that carote-

noids can quench singlet oxygen and can serve as rad-

ical-trapping antioxidants; it has also been shown that

carotenoids may have a role (apart from being precur-

sors of retinoids) in enhancing the immune function in

animals. Singlet-oxygen trapping and antioxidant

capabilities are shown by carotenoids that have no

provitamin A activity (such as lycopene and astax-

anthin) as well as by provitamin A carotenoids.

0020 Both b-carotene and canthaxanthin have been used

in large dose regimens to ameliorate photosensitivity

associated with erythropoietic protoporphyria (a

genetic disease of porphyrin metabolism). b-Carotene

also prevents photosensitivity resulting from quini-

dine ingestion, but not photosensitivity to ultraviolet

light, although it has been reported that human serum

carotenoid levels decrease after repeated exposure to

ultraviolet light.

0021 Epidemiological evidence strongly suggests a

role for carotenoids in human cancer prevention,

although such studies may be confounded by other

factors. The strongest evidence is for a protective

effect of carotenoids against lung cancer; tomato

products (which provide lycopene) may be more

effective than carrots and squash (sources of

b-carotene). In particular, consumption of tomato

products (high in lycopene) has been associated epi-

demiologically with a reduced risk of prostate neo-

plasms. Protective effects of carotenoids against

laryngeal cancer, gastric cancer, invasive bladder

cancer, and cervical dysplasia and cervical cancer

have been suggested also; these effects are not

shown by vitamin A, suggesting a direct role of car-

otenoids. Neither carotenoids nor vitamin A protect

against cancers of the esophagous or gastrointestinal

tract, breast, head, or neck. A variety of cancers in

animal models are prevented or delayed by carote-

noids, including skin tumors and mammary tumors.

In studies in cells in culture, carotenoids can prevent

malignant transformation, sister chromatid ex-

change, and mutagenic effects in bacterial systems.

0022A high consumption of carotenoid-containing

foods is associated with a reduced risk of coronary

heart disease, although there is a lack of consistency

in identifying the responsible compounds.

0023Carotenoids have been found to enhance both

specific and nonspecific immune functions, including

proliferation of T- and B-lymphocytes, induction of

specific effector cells capable of killing tumor cells,

and increased production of tumor necrosis factor,

and secretion of factors required for communication

between cells (prostaglandins and leukotrienes). It

has been suggested that the mechanism(s) may

involve quenching singlet oxygen and trapping free

radicals (generated by neutrophils to kill invading

cells), or maintaining cell-membrane fluidity (import-

ant for the function of membrane receptors and for

the release of immunomodulating factors). Again,

some of the immunologic functions of carotenoids

seem to be independent of any provitamin A activity,

although retinoids are also important in immune

function.

0024The xanthophyls lutein and zeaxanthin are accu-

mulated specifically in the macula of the primate eye,

and high dietary intakes of these two carotenoids (and

elevated plasma concentrations) are associated with a

reduced risk of age-related macular degeneration.

0025Elucidation of these biochemical effects of carote-

noids and further explanation of the metabolism

of conversion of carotenoids to vitamin A remain

central issues in the study of carotenoid function.

See also: Carotenoids: Occurrence, Properties, and

Determination; Protein: Deficiency; Retinol: Properties

and Determination; Tocopherols: Properties and

Determination

Further Reading

Bauernfeind JC (ed.) (1981) Carotenoids as Colorants and

Vitamin A Precursors. New York: Academic Press.

Beyer P, Al-Babili S, Ye X et al. (2002) Golden Rice: Intro-

ducing the beta-carotene biosynthesis pathway into rice

endosperm by genetic engineering to defeat vitamin A

deficiency. Journal of Nutrition 132: 506S–510S.

Britton G, Liaaen-Jensen S and Pfander H (eds) (1995a)

Carotenoids. 1A. Isolation and Analysis. Basel: Birk-

hauser.

Britton G, Liaaen-Jensen S and Pfander H (eds) (1995b)

Carotenoids. 1B: Spectroscopy. Basel: Birkhauser.

Castenmiller JJ and West CE (1998) Bioavailability and

bioconversion of carotenoids. Annual Review of Nutri-

tion 18: 19–38.

Isler O, Gutmann H and Solms U (eds) (1971) Carotenoids.

Basel: Birkhauser Verlag.

Lee CM, Boileau AC, Boileau TW et al. (1999) Review of

animal models in carotenoid research. Journal of Nutri-

tion 129: 2271–2277.

942 CAROTENOIDS/Physiology

Straub O (1987) In: Pfander H (ed.) Key to Carotenoids.

Basel: Birkhauser.

Subcommittees on Upper Reference Levels of Nutrients and

Interpretation and Uses of Dietary Reference Intakes,

and the Standing Committee on Scientific Evaluation

of Dietary Reference Intakes (2000) Dietary Reference

Intakes for Vitamin C, Vitamin E, Selenium and

Carotenoids: A Report of the Panel on Dietary Antioxi-

dants and Related Compounds. National Academy

Press, Washington, D.C.

Von Lintig J and Wyss A (2001) Molecular analysis of

vitamin A formation: cloning and characterization

of beta-carotene 15,15

-dioxygenase. Archives of Bio-

chemistry and Biophysics 385: 47–52.

CASEIN AND CASEINATES

Contents

Methods of Manufacture

Uses in the Food Industry

Methods of Manufacture

C R Southward, Fonterra Research Centre,

Palmerston North, New Zealand

This article is reproduced from Encyclopedia of Dairy Sciences,

Introduction

0001 The commercial production of casein, the principal

protein in cows’ milk, has occurred for most of the

20th century. During the period to 1960, the major

proportion of casein was used in technical (or non-

food) applications. More recently, however, there has

been a significant change from technical to edible uses

for casein products and this has been reflected in the

introduction of requirements for pasteurization of

milk intended for casein manufacture (c. 1970) and

the greater number of specifications for microbial

quality and freedom from impurities. At the present

time, the major producers of casein include the Euro-

pean Community (in particular, Denmark, France,

Germany, the Irish Republic, and The Netherlands)

and New Zealand, with Poland, Australia, and India

producing smaller quantities. World production of

casein products is currently estimated at 220 000–

250 000 tonnes. This article describes the manufac-

ture of the main casein products.

General

0002 The proteins that exist in milk can be broadly divided

into two groups – casein and whey proteins. Caseins

may be considered as those proteins that are precipi-

tated when unheated (raw) milk is acidified to pH 4.6

(the isoelectric point of casein) whereas whey proteins

remain in solution. Commercial casein is a mixture of

four different caseins – a

-, a

-, b- and k-casein, and

may sometimes be referred to as whole casein. As a

phosphoprotein, casein belongs to a relatively rare

class of proteins. It contains 0.7–0.9% phosphorus,

covalently bound to the casein by a serine ester

linkage. Casein exists in milk in combination with

calcium, inorganic phosphate, and citrate as a col-

loidal suspension of complex micelles and accounts

for 2.6–2.9% by weight of whole milk.

Manufacture

0003Casein may be precipitated from skimmed milk to

produce several products such as acid casein, rennet

casein, or coprecipitate. All these products are

insoluble in water after precipitation. However,

addition of alkali to acid casein yields water-soluble

caseinate.

Acid Casein

Acidification

0004When milk (pH 6.6) is acidified, the calcium and

inorganic phosphate are removed from the casein

micelles, the net charge on the micelles decreases,

and the micelles become less and less stable until the

casein precipitates. Complete precipitation of the

casein occurs at the isoelectric point, pH 4.6. Acidifi-

cation of the milk may be carried out by one of the

following processes:

0005Inoculation of milk with lactic acid-producing

bacteria such as Lactococcus lactis subsp. lactis

CASEIN AND CASEINATES/Methods of Manufacture 943

or cremoris. These bacteria, commonly known as

‘starters,’ convert some of the lactose in the milk

to lactic acid during the period of incubation

(about 16–18 h). This is the most commonly

employed method of manufacture in New Zealand

(Figure 1).

0006 Direct addition of dilute acid to skimmed milk.

Hydrochloric acid, sulfuric acid, phosphoric acid,

or lactic acid (or, occasionally, other organic acids)

may be used for this purpose. The most common

method of producing acid casein in countries other

than New Zealand is by means of hydrochloric

acid.

0007 Indirect acidification of skim milk. A number of

alternative processes have been patented in which

skimmed milk is acidified by one or more of the

following:

0008 contact by means of ion exchange resins in the

acid (cation) form

0009 addition of acidified whey

0010 electrodialysis

It is believed that these processes have relatively

minor commercial significance.

Cooking/Acidulation

0011 In the manufacture of acid casein, acidification of the

milk is followed, or occasionally preceded, by heating

of the mixture. Heating promotes agglomeration of

the casein curd particles which subsequently shrink

(syneresis) to expel whey. At the same time, the curds

become firmer and are able to withstand the mechan-

ical processing that follows. Heating of the acidified

milk is sometimes termed ‘cooking’ (as used in cheese

manufacture) and may be carried out, usually in the

temperature range 45–55



C, by:

0012 injection of steam into the pipeline carrying the

acidified milk

0013 indirect heating by means of a heat exchanger

0014 a combination of both – preheating through a heat

exchanger with steam injection to complete the

heating process (Figure 1)

In each case, the cooked curds and whey are held in a

‘cooking pipe’ for a period of about 10–20 s before

they emerge into an acidulation vat. The curds and

whey may remain here for a period varying from 30 s

to about 15 min, during which time the curds are

agitated gently in the whey until equilibrium between

the calcium in the curds and that in the whey is

attained.

0015 Alternative processes may use a syneresis tube; the

cooked curds and whey are held in a large-diameter

tube for a period of several minutes (also undergoing

acidulation or equilibration).

Dewheying and Washing

0016Following acidulation, the curds and whey are

discharged by gravity to a dewheying screen above

the first washing vat. The whey is removed and the

curds fall into the vat. Alternatively, the curds and

whey may be separated more completely using a hori-

zontal solid-bowl centrifuge (decanter) or a casein-

dewatering press before the curds are transferred to

the first wash. A combination of screening and decanter

dewheying can be used to reduce the hydraulic load on

the decanter. The purpose of washing is to remove

whey (containing mainly lactose) from the curds so

that the casein produced is relatively pure. The tem-

perature of the wash water may be varied, depending

upon particular requirements. Casein is usually sub-

jected to multiple washes and these are operated in a

counterflow to the direction of the curds, with the

purest curd meeting the cleanest water.

Dewatering

0017After washing, the curds are mechanically dewatered

to remove excess water before drying. As drying is a

relatively expensive operation, it is worthwhile re-

moving as much water as possible from the curd

before transferring the casein to the drier. The texture

of the curd is affected by temperature; as the tempera-

ture of the wash water is increased, the curd releases

more water during dewatering but becomes firm and

more plastic and is consequently harder to break up

and dry. Therefore, it is necessary to regulate carefully

the temperature of the last wash to optimize the con-

flicting requirements of minimum water content and

maximum friability of the curd.

0018Equipment for dewatering casein curd consists of

roller or belt presses, decanters, and screen-bowl cen-

trifuges. The roller press, used for many years, is

designed to reduce the moisture content of curd to

about 55%. The belt press will do a similar duty.

Solid-bowl decanters or screen-bowl centrifuges are

capable of reducing the moisture content of acid

casein curd to about 52%.

Drying

0019Drying of casein curd is most commonly carried out

using horizontal vibrating fluid-bed driers. These

driers have two or more perforated stainless-steel

decks (Figure 1). The combined effect of vibration

of the decks and the flow of hot air (typical tempera-

ture range 75–115



C) up through the holes in the

decks causes the casein curd to become fluidized and

materially helps in the removal of moisture from the

particles. Most of the water is removed during

the early stages of drying of the casein as it is evapor-

ated from the surface of the particles. The later stages

944 CASEIN AND CASEINATES/Methods of Manufacture

Tanker

reception

Whole milk storage

Unloading Pasteurizer

Preheater

Steam injector

Cas

Screen

Acidulation

Washing and

separation

by screening

Cas

Press

Casein drier

Fines recovery

cyclone

Whey

Exhaust

Tempering

Roller mill

Oversize

particles

Cas

Blending

80 mesh

60 mesh

30 mesh

Sifter

Metal

removal

Storage silos

Bag-filling

hopper

Bag

sewing

Metal

detector

Palletized bags

to store

Bag

flattening

Weighing

Coagulation silos

Separator

Flow

control

Starter

vessels

Cream

Holding

tubes

fig0001 Figure 1 Outline of the manufacturing steps involved in producing lactic acid casein from skim milk. Wm, whole milk; Sm, skimmed

milk; Co, coagulum; Cas, casein.

CASEIN AND CASEINATES/Methods of Manufacture 945