Phytochem Rev (2008) 7:261–280
DOI 10.1007/s11101-007-9084-y
Betalains in the era of global agri-food science, technology
and nutritional health
Diego A. Moreno Æ Cristina Garcı́a-Viguera Æ
José I. Gil Æ Angel Gil-Izquierdo
Received: 18 October 2007 / Accepted: 23 November 2007 / Published online: 18 January 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Natural pigments from plants are of
growing interest as substitutes for synthetic dyes in
the food and pharmaceutical industry and they
increase their added value if they possess positive
effects on health. These pigments can be added as
such if they are in the legal authorized lists of
additives or can be added as phytochemical-enriched
plant extract achieving the original product, which
has received it, the new nomenclature of functional
food. In this way, we comprise on this review a wide
point of view of a group of natural pigments known
as betalains. From a chemical point of view, betalains
are ammonium conjugates of betalamic acid with
cyclo-DOPA (betacyanins, violet) and aminoacids or
amines (betaxanthins, orange or yellow), which are
compounds present in our diet. Besides and taking
into account that one type of betalain, betanin is
approved as food colorant (E-162) by the European
Union and that enlarges the specific weight of these
compounds in the diet, we have evolved an overview
D. A. Moreno
C. Garcı́a-Viguera

A. Gil-Izquierdo (&)
Department of Food Science and Technology,
CEBAS-CSIC, Apdo Correos 164, Espinardo,
Murcia 30100, Spain
e-mail: angelgil@cebas.csic.es
J. I. Gil
Servicio de Radiodiagnóstico, Hospital Morales
Meseguer, Avda. Marqués de los Vélez s/n, Murcia
30008, Spain
from the biosynthesis, technology and promoting
production, industrial uses as pigments up to phys-
iological and nutritional biovailability or biological
and health-promoting properties of betalains for
accessible information to industrials, researchers
and consumers.
Keywords Amaranthus
Betalains

Betacyanins
Betaxanthins
Colouring agent

Opuntia
Red beet
Introduction
Betalains are water-soluble nitrogen-containing pig-
ments, which comprise the red–violet betacyanins
and the yellow betaxanthins. They are ammonium
conjugates of betalamic acid with cyclo-DOPA and
aminoacids or amines, respectively (Mabry and
Dreiding 1968; Grotewold 2006). One of the best
known controversies in angiosperm systematics in the
1960s and 1970s regarded the taxonomic significance
of the betalains, an unique class of vacuolar pigments
restricted in the angiosperms to thirteen families of
the order Caryophyllales, and the mutual exclusive-
ness of these compounds and the anthocyanins
(Clement and Mabry 1996). The methods recom-
mended for analytical characterization, preparative
isolation, photometric quantification and structure
elucidation of betalains, the latter mainly by MS- and
NMR-techniques, are comprehensively summarized
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in different reviews (Schoefs 2004; Stintzing et al.
2002a, b, 2004a, 2006) and recent reports on isolation
and purification (Degenhardt and Winterhalter 2001;
Gandı́a-Herrero et al. 2006; Liveri et al. 2007),
RP-HPLC and HPLC-DAD analysis (Kujala et al.
2001; Wybraniec and Mizrahi 2004), HPLC and
LC-MS analysis (Fernández-López et al. 2002; Strack
et al. 2003; Kluger et al. 2004), and capillary zone
electrophoresis analysis of betalains (Stuppner and
Egger 1996), as well as the analysis of precursor amino
acids and amines by GC-MS (Kluger et al. 2006).
Extraction of betalains from plant tissues or cell
cultures is commonly performed with aqueous
organic solvent (i.e. methanol); however, the addition
of ascorbic acid (ca. 50 mM) in the extraction
medium is recommended to slightly lower the pH,
which stabilizers betacyanins and inhibits possible
oxidation by polyphenoloxidases (PPOs). When high
tyrosinase activities and certain betaxanthins, e.g.
miraxhanthin V, are present as in case of hairy root
cultures of yellow beets, the addition of ascorbic acid
is absolutely necessary to avoid loss of miraxanthin V
and the appearance of artefacts (Strack et al. 2003).
Within the last 15 years, a series of publications
has described new structures and complete identifica-
tion of some putative ones as well as elucidation of
biosynthetic reactions. Some early enzymatic reac-
tions in betalain biosynthesis were characterized,
polyphenoloxidase (PPO)-type tyrosinase and extra-
diolic DOPA dioxygenase. Furthermore, the decisive
steps in the biosynthesis of both betanidin and
betaxanthins by aldimine formation were recently
identified to proceed non-enzymatically. Finally,
betalain-specific glucosyl- and hydroxycinnamoyl-
transferases were characterized (Strack et al. 2003;
Grotewold 2006).
There is growing interest in the use of natural
pigments for food colouring, since synthetic dyes are
becoming more and more critically assessed by the
consumer. In food processing, betalains are less
commonly used than anthocyanins and carotenoids,
although these water-soluble pigments, stable
between pH 3 and 7, are well suited for colouring
low acid food. The most important source of betanin
as colouring agent is the red beet (Beta vulgaris ssp.
vulgaris) root. The corresponding cell cultures (Tre-
jo-Tapia et al. 1999) and hairy roots (Pavlov et al.
2003, 2005a, b) cannot compete with the plant’s root
(50–60 t/ha with ca. 0.5 g betanin/kg) (Stintizing
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Phytochem Rev (2008) 7:261–280
et al. 2002a, b) with respect to betanin accumulation.
Besides betanin from red beet, also betacyanins from
plants of the Amaranthaceae were tested concerning
colour properties involving betalains as optical attr-
actants for pollination (Borsch 1998), and pigment
stability in model food systems (Cai et al. 2005).
Biosynthesis and occurrence of betalains in plants
Biosynthesis
Betalains are synthesized from tyrosine by the
condensation of betalamic acid (Fig. 1), a central
intermediate in the formation of all betalains, with a
derivative of dihydroxyphenylalanine (DOPA). This
reaction results in the formation of the red to violet
betacyanins, such as those found in red beets or in the
flowers of Portulaca. The condensation of betalamic
acid with an amino acid (e.g., Ser, Val, Leu, Iso, and
Phe) or amino acid derivative (e.g. 3-methoxytyr-
amine) results in the formation of the yellow–orange
betaxanthins. Betacyanins and betaxanthins can be
further classified into several subclasses, based on the
chemical characteristics of the betalamic acid conju-
gate (Strack et al. 2003). Recent advances in the
separation and analysis of betalains, which are
unstable under the acidic conditions normally used
for Nuclear Magnetic Resonance (NMR) spectra
analysis, are likely to shed additional light on the
existence of novel conjugates (Stintzing et al. 2004a).
As in common for many other phytochemicals, light
and hormones have a dramatic effect on the accu-
mulation of betalains (Piatelli 1981).
The complete scheme of biosynthesis of betalains
has been recently reported in the review of Strack
et al. (2003). Briefly, the conversion of tyrosine to
Fig. 1 Betalamic acid
Phytochem Rev (2008) 7:261–280
DOPA is carried out by a tyrosinase-type phenoloxi-
dase (Steiner et al. 1999; Strack et al. 2003), a group
of copper containing bifunctional enzymes involved
in the hydroxylation of phenols to O-diphenols. In
addition to participating in the formation of the
betalamic acid core, the tyrosinase enzyme also
oxidizes DOPA to dopaquinone, contributing to the
biosynthesis of cyclo-DOPA, which conjugates with
betalamic acid to form the chromophore of all
betacyanins, betanidin (Fig. 2). The formation of
betalamic acid from DOPA requires the extradiol
cleavage of the 4,5 bond carried out by a DOPA
dioxygenase, first identified in the basidiomycete fly
agaric (Amanita muscaria) (Hinz et al. 1997). The
plant enzyme was subsequently cloned by a subtrac-
tive cDNA approach using Portulaca grandiflora
isogenic lines with different color phenotypes (Chris-
tinet et al. 2004). The plant enzyme exhibits no
obvious sequence or structural similarity with the
fungal enzymes. Moreover, the plant enzyme displays
regiospecific extradiol 4,5-dioxygenase (Christinet
et al. 2004) in contrast to the 2,3- and 4,5 dioxygen-
ase activity of the Amanita muscaria enzyme (Hinz
et al. 1997). The 4,5-seco-DOPA is subsequently
recyclized, a step likely to occur spontaneously
(Strack et al. 2003). This different activity of the
plant and fungal enzymes permits Amanita muscaria
to accumulate muscaflavin, in addition to betalain, in
the cuticle of the cap. The introduction of the DOPA
dioxygenase from Amanita muscaria into Portulaca
grandiflora petals by particle bombardment resulted
in the accumulation of various betalains, and also of
muscaflavin, a pigment normally not found in plants,
which is synthesized by the extradiol ring cleavage of
Fig. 2 Betanidin: 2,6-Pyridinedicarboxylic acid
263
the 2,3 bond followed by recyclization into the
6-atom, N-containing ring muscaflavin (Mueller et al.
1997a, b).
In light of the fact that the main betaxanthin
(miraxanthin V; Fig. 3) and the major betacyanin
(2-descarboxy-betanidin) in hairy root cultures of
yellow beet (Beta vulgaris L.) are both dopamine-
derived, the occurrence of similar structures for the
minor betacyanins was suggested. The administration
of dopamine to beet seedlings revealed that the
condensation step between 2-descarboxy-cyclo-
DOPA and betalamic acid is the decisive reaction,
followed by glucosylation and acylation (Kobayashi
et al. 2001).
The next step in the biosynthesis of betalains
involves the formation of an aldimine link between
betalamic acid and cyclo-DOPA to produce betani-
din, or an amino acid derivative for betaxanthin. No
enzyme capable of carrying out the aldimine reaction
has yet been identified, opening the possibility that
this step occurs spontaneously in vivo (Strack et al.
2003). It remains unclear how the spontaneous
condensation of betalamic acid with various different
DOPA or amino acid derivatives results in the
specific patterns of betalains consistently obtained
in the same plant (Grotewold 2006).
As in the case with many other plant natural
products (Grotewold 2004, 2006), betalains are stored
in the vacuole as glycosides. Glycosilation of beta-
cyanins occurs both at the level of the cyclo-DOPA
(Wyler et al. 1984; Sasaki et al. 2004) and by the
glucosylation of betanidin (Vogt et al. 1999a; Vogt
2002). The cloned Darotheanthus bellidiformis
5- and 6-O-glucosyltranferases transfer glucose with
Fig. 3 Miraxanthin V
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similar efficiency from UDP-glucose to betanidin, to
form betanin, and to several flavonoids (Vogt et al.
1999a; Vogt 2002), raising the provocative possibil-
ity that there are evolutionary links between these
two pathways (Grotewold 2006). Although yet to be
tested for its ability to glycosylate flavonoids, the
cyclo-DOPA 5-O-glucosyltransferase belongs to a
group of enzymes very distinct from those involved
in the phenylpropanoid pathway (Sasaki et al. 2005).
Tyrosine feeding experiments suggest a strict com-
partmentalization for the betacyanin biosynthetic
pathway, with the possibility of forming multienzyme
complexes (Strafford 1994). However, it remains
unknown whether there is a single or multiple pools
of betalamic acid responsible for the formation of
both betacyanins and betaxanthins, or how these
compounds are transported to the vacuole, their
ultimate site of accumulation (Grotewold 2006).
Occurrence
Betacyanins are a class of water-soluble pigments
that provide the colours in a wide variety of flower
and fruits (Strack et al. 2003; Kluger et al. 2007).
Betanidin and isobetanidin (the corresponding C-15
diasteroisomer) are the simplest betacyanins (Wybra-
niec and Mizrahi 2002). Betalains are of great
taxonomic significance in higher plants. The presence
of betalains in members of the order Caryophyllales
has been an important criterion for their classification
(Table 1). Betalains replace the anthocyanins in
flowers and fruits of plants of most families of the
Caryophyllales (Strack et al. 2003). The presence of
betalains and anthocyanis is mutually exclusive in the
angiosperms, i.e. betalains and anthocyanins have
never been reported in the same plants (Mabry and
Dreiding 1968; Kimler et al. 1970; Lee and Collins
2001; Cai et al. 2005; Grotewold 2006). Within this
order, betalains are absent in a couple of families
including the Caryophyllaceae, which comprises
genera such as Lychnis and Dianthus (e.g., carna-
tions, Dianthus caryophyllus), widely used as
ornamentals and cut flowers for their colourful
anthocyanin pigmentation. While this exclusion
probably makes sense from a functional perspective,
since both types of pigments have overlapping
absorption spectra, and hence colors, the molecular
basis of this exclusion is not clear. Plant-
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Phytochem Rev (2008) 7:261–280
accumulating betalains express at least some of the
flavonoids biosynthetic enzymes (Shimada et al. 2004)
and can accumulate significant quantities of flavonols,
other flavonoids, and in some cases even proanthocy-
anidins, suggesting that it might be the last step in the
flavonoid pathway, the anthocyanidin synthase (ANS),
the only enzyme ‘‘missing’’ in betalain-accumulating
plants (Strafford 1994). The origin of the betalain
biosynthetic pathway in just one order of the angio-
sperms is even more puzzling given that these
pigments are also found in some basidiomycetes.
One possibility is that the anthocyanin and betalain
pigments have coexisted in ancestral plant species and
that one of the two pigments has been selectively lost
because of similar redundant pigmentation functions.
Alternatively, the betalain biosynthetic pathway could
have been acquired independently and more recently
in the fungi and plants. The evolution of this pathway
would have made unnecessary the presence of the
anthocyanins, resulting in the observed exclusion of
both pigments in the Caryophyllales. The paraphyletic
relationship of the betanidin 5- and 6-O-glucos-
yltransferases from Dorotheanthus bellidiformis with
other glucosyltransferases, together with their ability
to utilize both betanidin and flavonoids as substrates
(Vogt et al. 1999a; Vogt 2002), was interpreted to
indicate that these enzymes, originally involved in the
glycosylation of flavonoids, were later recruited to
glycosylate betacyanins. If so, these findings would
suggest that betalains originated later in the evolution
of plants than the anthocyanins. This model raises the
question of how betalains appeared independently in
the fungi, with a fungal betacyanin biosynthetic
enzyme being able to function in the plants (Mueller
et al. 1997a). An alternative model is that anthocya-
nins and betalains coexisted in an ancestral plant, and
that during the evolution of the angiosperms, selective
loss of ANS or of an enzyme necessary for betalain
formation resulted in the current distribution. It
remains to be established what selective advantage,
if any, betalains provide over anthocyanins (Weiss
1995; Grotewold 2006).
Hylocereus polyrhizus and its related species
(Table 1) belong to the vine cacti from the subfamily
Cactoideae of the tribe Cactaceae (Wybraniec and
Mizrahi 2002; Wybraniec et al. 2001, 2007). The
fruits of Hylocereus species, known as red pitaya or
pitahaya, which means ‘‘the scaly fruit’’, in Latin
America, are medium-large beries bearing large
 Table 1 The betalain-producing plant species in the caryophyllalesa
Family Species Common name or Betalains References
representative

Achatocarpaceae Clement and Mabry (1996)

Aizoaceae Lampranthus productus Ice plant Dopaxanthin Kluger et al. (2004), Gandı́a-Herrero
Betanidin et al. (2005b), (2007)
Mesembryanthemum Ice plant Betacyanins and mesembryanthin Vogt et al. 1999b
Phytochem Rev (2008) 7:261–280

crystallinum
Basellaceae Clement and Mabry (1996)
Cactaceae Hylocereus polyrhizus Red-purple Pitaya Betacyanins (10 differents) bougainvillein-r-I, Kluger et al. (2006), Stintzing et al.
betanin, isobetanin, phyllocactin, isophyllocactin, (2002a), (2002b), Wybraniec et al.
hylocerenin (2001)
H. purpusii, H. costaricensis, Red-flesh fruit species Betacyanins hylocerenin and isohylocerenin, Wybraniec and Mizrahi (2002),
H. undatus, H. sp. (hybrids) and other minor apiofuranosyl betacyanins Wybraniec et al. (2007)
Opuntia ficus-indica Cactus pear
Schlumbergera x buckleyi Christmas cactus Phyllocactin, apiofuranosyl betacyanins, Kobayashi et al. (2000)
diacylated betacyanins
Myrtillocactus geometrizans, Garambullo Betaxanthine Reinoso et al. (1997),
M. cochal Barrera et al. (1998)
Selenicereus megalanthus Yellow pitaya Betaxanthins Kluger et al. (2006)
Amaranthaceae Amaranthus spinosus Spiny amaranth Amaranthine, isoamaranthine Stintzing et al. (2004b)
Gomphrena globosa Betaxanthins and several betacyanins Kluger et al. (2007)
Celosia argentea (var. plumosa Feathered amaranth and Amaranthin and betalamic acid, Schliemann et al. (2001)
and var. cristata) common cockscomb dopamine-derived betacyanins
Chenopodiaceae Beta vulgaris Red beetroot Vulgaxanthin (I, II), indicaxanthin, betanin, Kujala et al. (2001), (2002)
prebetanin, isobetanin, neobetanin, betalamic
acid
Swiss chard Betaxanthins (20 different) Kluger et al. (2004)
Betacyanis (9 different)
Suaeda salsa Betacyanins Wang et al. (2007)
Didiereaceae Clement and Mabry (1996)
Halophytaceae
Hectorellaceae
265

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green or red scales. The peel is usually red, and the

pulp varies from red or purple colors of various hues

Chenopodiinae, the betalain-producing taxa; Caryophyllinae, the anthocyanin-producing taxa, includes Caryophyllaceae and Molluginaceae (Clement and Mabry 1996)
to white. The pulp is delicate and juicy and contains
numerous small soft seeds. The plants are grown in

Gandı́a-Herrero et al. (2005b)

the open in tropical areas but must be protected from
Clement and Mabry (1996)

intense solar radiation and subfreezing temperatures

Stintzing et al. (2004b)

when cultivated under subtropical conditions (i.e.

Kluger et al. (2007)

Israel) (Wybranied and Mizrahi 2002). Betacyanins

are present in peel and flesh of fruits of different
References

Hylocereus species and has been identified by GC/

MS, electrospray MS/MS, HPLC as well as 1H and
13
C NMR techniques (Wybraniec et al. 2007).
Betalainic vegetables and fruits (Table 1), such as
Dopaxanthin, Vulgaxanthin I, Portulacaxanthin II,

beetroot and cactus pears, are cultivated for their use

as colouring foodstuffs, whilst other betalain-con-
taining plants are grown for ornamental purposes
(Strack et al. 2003; Gandı́a-Herrero et al. 2005a, c;
Betaxanthins and several betacyanins

Kluger et al. 2007). Betalain-bearing Bougainvillea

Miraxanthin V, Indicaxanthin
Betanin, isobetanin, neobetanin

species (Nyctaginaceae) known for their colourful

bracts as well as Gomphrena globosa L. (Amaranth-
aceae) developing spherical betalainic inflorescences
are widespread ornamental plants and were therefore
subjected to a through investigation of their betalain
compositions. There are remarkable differences in the
Betalains

betacyanin patterns between the purple, red and

orange varieties for both Gomphrena and Bouganvil-
lea inflorescences. Hence, both the betacyanin
profiles and the relative betaxanthin:betacyanin ratios
determine the broad colour palette of Gomphrena
Common name or

petals and Bougainvillea bracts (Kluger et al. 2007).

Erect spiderling
representative

Moss rose

Tyrosinase and betalains

The betalain metabolic pathway is still to be fully

clarified. Tyrosinase or polyphenol oxidase (monophe-
nol, o-diphenol:oxygen oxidoreductase; EC 1.14.18.1.)
Portulaca grandiflora

is a copper enzyme that catalyzes two different

reactions using molecular oxygen: the hydroxylation
Bougainvillea sp.
Boerhavia erecta

of monophenols to o-diphenols (monophenolase activ-

ity) and the oxidation of the o-diphenols to o-quinones
Species

(diphenolase activity; Sánchez-Ferrer et al. 1995;

Gandı́a-Herrero et al. 2005a, b, c). This enzyme is
widely distributed in plants, microorganisms, and
Table 1 continued

Stegnospermaceae

animals where tyrosinase is responsible for melaniza-

Phytolaccaceae
Nyctaginaceae

Portulacaceae

tion (Gandı́a-Herrero et al. 2004).

The physiological function of PPO in higher plants
Family

is yet to be fully determined, but it has been implicated

in pigment formation and scavenging molecular
a

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oxygen in the chloroplast, and more recently, it has
been proposed that PPO is involved in the biosynthesis
of betalains of higher plants (Joy et al. 1995; Gandı́a-
Herrero et al. 2004, 2005a). The biosynthetic scheme
of betalains and new branches were proposed based on
the description of new physiological substrates for
tyrosinase. The yellow pigments tyrosine-betaxanthin
(portulacaxanthin II) and dopaxanthin are character-
ized as physiological substrates of the enzyme
(Gandı́a-Herrero et al. 2005a, c). Evidences of the
interconversion between betaxanthins catalyzed by an
enzyme are reported and the transformation of tyro-
sine-betaxanthin to dopaxanthin and its further
oxidation to a series of compounds has been charac-
terized. The identity of the reaction products was
studied by HPLC and ESI-MS and data indicated that
dopaxanthin-quinone was obtained and evolved to
more stable species, related to the violet pigment
betanidin, by intramolecular cyclization. Kinetic
parameters were determined and showed a high
affinity of tyrosinase for these physiological substrates
(Gandı́a-Herrero et al. 2005a, b, c).
The structural unit of the violet betacyanins,
betanidin is reported as a substrate for PPO. The
tyrosinase-mediated oxidation of betanidin from
Lampranthus productus violet flowers was character-
ized by HPLC-MS showing that the addition of
ascorbic acid reversed the reaction product, betani-
din-quinone. The betanidin degradation kinetics was
also studied in the absence of the enzyme and
demonstrated that pH values over 6.0 and high ionic
strength reduce the pigment stability (Gandı́a-Herrero
et al. 2007).
Technology and conditions promoting betalain
production for food and health
Cell suspension culture
The betalain (betacyanin) production from Beta
vulgaris L. in cell suspension cultures indicated that
a medium with reduced total nitrogen concentration,
modified ratio of ammonium to nitrate and supple-
mented iron (Fe) had promoting effects on betacyanin
production, and that omitting zinc (Zn) from the
initial medium resulted in a high betacyanin content
of the cells. The effects of a high iron concentration
and low concentration of zinc on betacyanin
267
production were not cumulative. The modifications
of the growth media enabled the maximum betacy-
anin yield of 550 mg/l to be obtained from a 14-day
culture. This medium promoted a high betacyanin
productivity of 40 mg/l per day (Akita et al. 2002a).
The betalain production in cell suspension cultures of
Beta vulgaris remained unchanged with a modified
media (no ammonium and 50% less nitrate, phos-
phate and calcium compared to standard culture
medium) reducing costs for laboratory tests and also
for the applications for bioreactor production systems
(Trejo-Tapia et al. 1999).
Brief exposure of Beta vulgaris root cultures to
acidic medium (10-min exposure to pH 2 followed by
return to standard growth medium of pH 5.5, and
1.1 mM PO4) resulted in release of betalain pigments
while the capability for regrowth and continued
pigment accumulation, releasing 0.59 mg pigment/g
dry weight over the subsequent 24-h period (Mukun-
dan et al. 1998).
The effects of six microelements (Cu2+, Mn2+,
Fe , Mo2+, Zn2+, Co2+) on the production of
2+
betalains and the growth of suspension cultures of
B. vulgaris were studied and the increase of Co2+
from 1 lM to 5 lM resulted in a 60% increment on
the production of betalains when applied at the
beginning of the culture. Mo2+, Fe2+ and Cu2+
presented a positive but less marked effect, while
the Mn2+ did not show effects on the production of
betalains compared to control (Trejo-Tapia et al.
2001).
Cells of Beta vulgaris have the ability to grow in a
stirred tank under an impeller tip speed as high as
95.3 cm/seg. Comparing the system with cultures in
shake flasks, decreased cell concentration, betalains
production, and growth rate was observed (Rodrı́-
guez-Monroy and Galindo 1999). Cell suspension
cultures of B. vulgaris have been grown in shake
flasks, and in different types of laboratory bioreac-
tors: air lift of 10 dm3, bubble-free Taylor-Couette of
2.5 dm3, fluidized bed of 5 and 50 dm3, and stirred
tank of 2 dm3 (Khlebnikov et al. 1995; Trejo-Tapia
et al. 1999). Cell cultures developed in an air lift
bioreactor of 10 dm3 showed that the non-Newtonian,
shear thinning characteristics of B. vulgaris broths
were determined by the cell concentration as well as
by the extracellular protein secreted by the cells and
the hydrodynamic stress (estimated as the power
input) in the air lift bioreactor was lower than that
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generated in the stirred tank. As a consequence, the
level of protein produced in air lift was considerably
lower than that reported in stirred tanks (Sánchez
et al. 2002).
There is a variety of methods for the release of
secondary metabolites from plant cells including
electropermeabilization, chemical permeabilization,
elicitation, ultrasonic technique, pressure or heat
shock, oxygen or phosphate limitation, and pH
gradient variation. Techniques applicable to most
species and suitable for continuous production of
secondary metabolites from plant cells, as the low-
level electric currents and voltages to extract, trans-
port, and collect betalains from cell-suspension
cultures while retaining higher cell viability (Heuer
et al. 1996; Yang et al. 2003).
Hairy root culture technology
Hairy root cultures are an alternative for the produc-
tion of secondary metabolites, because they are
genetically stable and, in contrast to callus and
suspension cultures, can grow in hormone-free
medium and produce valuable secondary metabolites
at a comparable rate to the original plants (Pavlov
et al. 2005a). One of the most important limitations
for the commercial exploitation of hairy roots is the
development of the bioreactor systems with suitable
design for their cultivation. Beta vulgaris hairy roots
are good model systems for studying processes of
hairy roots cultivation in different bioreactor systems.
Red and yellow B. vulgaris varieties and their hairy
root cultures are potential sources of valuable water-
soluble nitrogenous pigments, betacyanins and
betaxanthins. Many investigations on hairy root
cultures of red beet have been reported, in which
the effects of inoculum, type and age, medium
compositions, amino acid feeding, culture conditions
and elicitations on growth of the hairy root cultures
and betalains biosynthesis were studied (Hempel and
Böhm 1997; Kobayashi et al. 2001; Böhm and Mäck
2004; Pavlov et al. 2003, 2005a; Pavlov and Bley
2006).
It is more appropriate to use an inoculum of
B. vulgaris grown in submerged culture for 14 days,
i.e. when the hairy roots are in the stationary phase of
growth and, at the same time, they have become
adapted to the conditions of the final experiment
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(Pavlov et al. 2003). Hairy root culture of B. vulgaris
cv. Detroit Dark Red (obtained by infection of
4 weeks old leaves of plants with Agrobacterium
rhizogenes ATCC15834, following cultivation under
appropriate conditions for 21 days in 300 ml Erlen-
meyer Flasks). The biosynthesis of betalains occurred
between the 6th and 15th days, being especially
intensive between the 9th and 15th days when
maximum amount of 42.2 mg/flask betalains was
produced (26.2 mg/flask betaxanthins and 16 mg/
flask betacyanins, respectively) (Pavlov et al. 2005a).
The production in temporary immersion RITAÒ
system is suitable for the cultivation of Beta vulgaris L.
and the process of biosynthesis of betalains in hairy
roots grown under submerged conditions. The hairy
root culture biosynthesized 18.8 mg/g dry biomass
betalains (9.6 mg/g betacyanins and 9.2 mg/g betax-
anthins) at immersion frequency with 15 min flooding
and 60 min stand-by periods (Pavlov and Bley 2006).
The temporary immersion systems are based on a
principle similar to that of mist bioreactors, preferring
temporary contact between the plant in vitro cultures
and the liquid medium rather than permanent contact.
Once the betalains are effluxed from hairy roots,
developing processes for in situ and ex situ recovery
of the betalains with the help of adsorbents (i.e.
alumina, silica, Amberlite,...) becomes necessary
(Mukundan et al. 2001; Rudrappa et al. 2004).
The hairy roots of Beta vulgaris grow on a simple
medium producing good levels (1.2% or 88.4 mg/l)
of betalains were screened for the use of elicitors for
scaled-up production of betalains. In an attempt to
enhance betalain productivity, the hairy roots were
contacted with several biotic elicitors (purified gly-
cans of microbial origin (200–500 mg/l), extracts of
whole microbial cultures (0.25–1.25%) and the
respective culture filtrates (5–25% v/v). Similarly,
abiotic elicitors, particularly metal ions, upto 10-folds
of that present in the nutrient (Murashige and Skoog)
medium, were tested. It was observed that though
there was a significant suppression of biomass in
almost all the treatments, a significantly high pro-
ductivity of betalain was observed in Penicillium
notatum DCP-treated cultures (158 mg/l on 7th day)
among biotic elicitors, pullulan-treated cultures
(202 mg/l on 10th day) among purified glycans and
calcium treated cultures (127 mg/l on 7th day) among
abiotic elicitors, whereas control cultures showed
productivities of only 43.3 mg/l on 7th day and
Phytochem Rev (2008) 7:261–280
88.4 mg/l on 10th day. Since most of the elicitors
caused early elicitation (on 7th day) and suppressed
biomass resulting in reduced overall productivity, a
strategy of using elicitor at late exponential growth
phase was considered and such a strategy was
adoptable to scaled up process using a bubble-column
bioreactor, where too the addition of elicitor at late
exponential phase resulted in about 47% higher
productivity of betalains (Savitha et al. 2006).
Ecophysiological factors
Half-sib recurrent selection programs at the Univer-
sity of Wisconsin (1978 and 1995) to increase
betalain (betacyanin and betaxanthin) concentration
in red and yellow table beets (Beta vulgaris L. ssp.
vulgaris), respectivelty. Cycles of selection from both
the red and yellow table beet breeding programs were
evaluated for pigment and total dissolved solids
distribution in five tissue sections (outer, middle and
center zones of the root; leaf and petiole) in two
environments (early and late planting) during 2002.
Betaxanthin concentration increased with the later
planting date in the majority of the tissue zones and
the absolute pigment concentration of the outer root
zone increased the most over cycles of selection
(46.6 mg/100 g fresh weight betaxanthin and
201 mg/100 g fresh weight betacyanin for yellow
and red populations, respectively). However, the
greatest rate of gain was in the center and middle
tissue zones. Selection based on the outer 2 cm of
root tissue has effectively increased pigmentation of
the entire beet plant (Gaertner and Goldman 2005).
Crops that have been cultivated in the Andean
region for thousands of years have a high level of
resistance to drought, frost, salinity, pests, and
diseases, and have only been little improved over
the years. The Andean crops, which include grains,
tubers, roots, fruit trees, aromatic, and medicinal
plants, have a great potential for increased use and for
transformation into a range of processed products.
There are differences in quality and quantity of
primary constituents and secondary metabolites,
including anthocyanins and betalains. Agroindustrial
research should search for genotypes for each specific
use (Jacobsen et al. 2003).
Seeds, 5-day and 10-day-old seedlings of C3
halophyte Suaeda salsa were watered, sprayed and
269
infiltrated with 0, 0.10%, 0.33%, and 1.00% hydrogen
peroxide (H2O2) solution to examine whether H2O2 is
involved in the betacyanin accumulation. The H2O2
treatments led to the most significant betacyanin
accumulation in the shoots when seeds were watered
with H2O2 solution (Wang et al. 2007).
Although betalains synthesis is modulated by light,
the effect of the diverse wavelengths on betalains
synthesis is poorly known. Using quinoa seedlings
(Chenopodium quinoa Willd.) grown in darkness
during 7 days and then kept under continual illumi-
nation (using red, green, UV-A and incandescent
lamps) during 5 days, the synthesis of pigments was
demonstrated to be light dependent, being the coty-
ledons its preferential place of synthesis. The
maximum yield in betalains production in cotyledons
and hypocotile was achieved in presence of UV-A
light in both distilled water and L-DOPA, the natural
precursor of betalains, followed by the values
obtained with red and incandescent lamps (Gallardo
et al. 1999).
Mesembryanthemum crystallinum L. (ice plant)
(Aizoaceae) grown frorm seeds in the greenhouse and
salt-stressed (1 M NaCl in Hoagland’s solution) were
induced with high intensities of white light resulting
in a rapid cell-specific accumulation of flavonols, and
betacyanins within bladder cells of the leaf epidermis
was first detected 18 h after the initiation of light
treatment in bladder cells located at the tip of young
leaves followed by the bladder cells located on the
epidermis of fully expanded leaves. UV-A light
apparently is sufficient to induce accumulation of
betacyanins and flavonoids (Vogt et al. 1999b; Ibdah
et al. 2002).
The crop management strategies based on geno-
typic and ecophysiological effects for the production
of vegetables enriched with phytochemicals, which
can be served as fresh market products or be used as
raw material for functional foods and supplements are
active research lines nowadays. The case of betalains
has to be included in the trend.
Industrial uses of betalain pigment from different
sources
Natural red pigments from plants are of growing
interest as substitutes for synthetic dyes in the food
and pharmaceutical industry. In most countries the
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use of food additives (including colourants) in
governed by strict regulation. The legislation speci-
fies which colourant may be used, the source, the
purity, to which food may be added and at what level
may be added to a specific food. Beetroot is the only
allowed source of betalain approved additives for use
in foods in the United States (Title 1 of the Code of
Federal Regulations, 21 CFR 73, 40) and in the
European Union (E-162), and commercially, they are
exempt from batch certification (Castellar et al.
2003). However, unfortunately earth like flavour
characteristics caused by geosmin and high nitrate
concentrations associated with the formation of
carcinogenic nitrosamines may affect commercial
use (Strack et al. 2003; Stintzing and Carle 2004).
With a focus on food, the scare attention towards
betalains may be due to the fact that red beet has long
been considered the only edible betalainic source.
However, other sources of betalains are species of the
genus Amaranthus, which are used as food colourants
in China. Other Cactaceae such as cactus fruits from
Opuntia sp., Hylocereus polyrhizus or Myrtillocactus
geometrizans, and also Swiss chard and yellow beet
have stimulated scientists to study other sources of
betalains and their possibility of use as food colou-
rants from the nutritional and technologically related
analytical issues (Stintzing and Carle 2007a; Herbach
et al. 2006). In addition, a good review, including
selected features of betalains from the stability and
degradation-structural and chromatic aspects point of
view, has recently been published (Stintzing and
Carle 2007b).
In general, the stability of betalains in manufac-
tured products is affected by numerous pigment-
specific and external factors such as: pigment content,
degree of glucosylation or acylation, matrix constit-
uent, chelating agents, antioxidants, temperature, pH,
oxygen, light, water activity and nitrogen atmosphere.
Besides pigment concentration and the particular
betalain structures, pH and water activity will have
considerable impact on pigment stability. In order to
ensure optimum pigment and colour retention, the
particular time-temperature conditions during manu-
facture must be carefully controlled. In addition
external factors during storage such as temperature,
light and oxygen exposure need to be considered
(Herbach et al. 2006; Stintzing and Carle 2007a).
The most interesting applicable feature is the
stability in the pH range from 3 to 7, which makes
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Phytochem Rev (2008) 7:261–280
betalains suitable for their application in a broad
range of low acid and neutral foods, and used as
anthocyanins substitutes (Stintzing and Carle 2004).
In the present work the most interesting betalain
sources with possible use in the industrial area, as
food colourants, would be reviewed.
Table beet and beetroot
Beet (Beta vulgaris subsp. vulgaris) has been bred to
produce a number of varieties, being beetroot (var
rubra) a variety with a strongly coloured root due to
its high betalain concentration. The red-purple beta-
cyanin comprise the major part of pigments in
beetroot, and of these betanin comprises 75%–95%
(Mortensen 2006). The pigments are not extracted,
instead of the beetroots are pressed and the water
partially removed to give a product containing 0.5%
pigments. Colour hue does not change with pH in the
range 4–7, but presents low heat stability (Mortensen
2006). Adaptation to pH about 4 has turned out to be
recommendable during processing, as well as a time
of cool storage to allow regeneration of betacyanin
colour following thermal exposure bellow 100°C
(Stintzing and Carle 2007b).
The process of spray drying of betalain dye from
beetroot using different carries (malto dextrine, gum
acacia and soluble starch) has been previously inves-
tigated (Koul et al. 2002). These studies showed that
with decrease in percentage of carries in the juice, the
percent yield of betalains increased. Even more, the
shelf life studies of the spray dried betalain dry power,
over 180 days, showed that the dye is quite stable at
temperatures between 4°C and 20°C. Results obtained
by Kujala et al. (2000) also corroborate that the
content of betacyanins in beetroot is higher under cold
storage conditions (5°C—196 days).
Other authors (Vitti et al. 2005) determined the
effect of different storage temperature on the quality
of beetroot minimally processed. A reduction on
colour intensity was observed, due to the alterations
in the amount of pigments, higher at 10–15°C storage
temperature than at 0°C. Based on the obtained
results, these authors, concluded that it was possible
to keep the product for 10 days at 0°C, preserving the
quality of minimally processed beetroots.
Processing technologies for red beet, the improve-
ment of betalain stability during processing and
Phytochem Rev (2008) 7:261–280
storage and the quality control of betalains in red
beet, and the possible uses as plant origen food
colourants or dairy products has been recently
reviewed (Stintzing and Carle 2007b), so it would
not be further discussed here. As well, kinetic studies
evaluating the degradation of betalains in beet root
have been carried out in the University of Venezuela,
concluding that reactions followed a first order
kinetic model when pigments, previously purified,
were exposed to white light, at pH 6.1 and storage at
25°C (Moreno-Alvarez et al. 2002).
The impact of heating at 85°C during 8 h on
overall colour and betalain pattern of red beet was
investigated, resulting in a yellow–orange solution,
due to 2 novel yellow and 2 orange-red betanin
degradation products. On the basis of these results, a
scheme of the thermal degradation of betanin is
proposed (Herbach et al. 2004a). Also, mixtures of
mono- bi- and tri-decarboxylated betacyanins
together with their corresponding neobetacyanins
were identified as degradation products, by LC-
DAD-MS/MS of betacyanins from beetroot (Wybra-
niec 2005).
On the other hand, Akita et al. (2002b) investi-
gated the application of the red pigment from
cultured cells from table beet, indicating that heat
and light stability were similar to those of the
pigment from beetroots. They have also proved the
potential use of these pigments for colouring fish and
pork meat sausages and this cell culture system can
be available for the stable pigment production on a
practical level.
Regarding the possible induction of the pigments
by irradiation, Shin et al. (2003/2004) explored
whether betalains biosynthesis in hairy root culture
can be induced under different radiation sources (blue,
red, red plus blue, blue plus far red and red plus far
red). The hairy roots were induced by leaf disc
method with Agrobacterium rhizogenes, and results
showed that the growth depended on radiation quality.
The highest biomass accumulation was under the blue
plus far red treatment, which efficiently induced
betalain pigmentation in the cultured hairy roots.
Opuntia and Hylocereus fruits
The betalains in these plants cover a broader colour
spectrum from yellow-orange (Opuntia sp.) to
271
red-violet (Hylocereus sp.) compared to red beet. In
addition, in contrast to red beetroot, cactus fruits do
not contain geosmin and pyrazines that are respon-
sible for the unpleasant pettiness of the former. Hence
cactus fruit juice may be applied without negative
sensorial impacts (Moßhammer et al. 2005a; Stint-
zing et al. 2003). Cactus fruits show no toxicity, their
pigments do not provoke any allergic reactions and
can be used as a certification- free food colouring. In
addition, these plants have minimal soil and water
requirements and may be regarded as an alternative
for the agricultural economy of arid and semiarid
regions (Castellar et al. 2006).
In general the colorant properties and stability of
pigments from Opuntia fruits were studied by
Castellar et al. (2003), concluding that the thermal
stability of the pigments extracts was dependent on
the pH, with maximum stability at pH 5, and storage
temperature of 4°C, where deactivation half- life time
is over 1 year.
Tuna pulp (Opuntia boldinghii) is a species
without commercial use. However, recent research
has shown its potential as a colourant for citric
beverages (Moreno Alvarez et al. 2003). In this study
a citric beverage (15% orange juice + 15% grape-
fruit + 65% water + 5% tuna pulp) was formulated
with different concentrations of ascorbic acid, pas-
teurised and stored at 7°C. Results pointed out that
the betalains presented a chemical stability until the
end of the experiment presenting antibacterial and
antiviral properties, extending the shelf life of all the
beverages up to 21 days.
Cactus pear (Opuntia ficus-indica) is the most
extensively studied Opuntia species. A development
of a process for the production of betalain based
colouring foodstuff from this specie was proposed by
Moßahmmer et al. (2005b). Unexpectedly, even after
repeated thermal treatment neither non-enzymatic
browning nor HMF formation was observed in the
clarified juices at pH 4, after filtration and pasteur-
ization. Therefore, juice concentrates from cactus
pear are expected to be a suitable colouring foodstuff
for low acid products such as ice-creams or yoghurt.
In a further study the same authors Moßahmmer et al.
(2006a), evaluated different methods for the produc-
tion of juice concentrates and fruit powders. First of
all the crushing process was simplified, also they
observed that the total yield was increased by 10%
through processing whole instead of peeled fruit and
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by further optimisation of pulp enzymation. Further-
more, the replacement of pasteurisation by cold-
sterile microfiltration, applied for non-thermal juice
preservation, resulted in identical betalain retentions.
Acceptable overall pigment retentions of 71%–83%
combined with the retention of the initial colour
properties after reconstitution of semi-concentrated
and concentrated preparations, proved the viability
for industrial processing. Finally, the use of fruit
cactus pear fruits powders for colouring dessert
preparations, fruit or cereal bars, instant dishes and
chocolates was proposed. In another work (Moßahm-
mer et al. 2006b) the impact of thermal treatment and
storage on colour of cactus pear juice was measured.
In this case the authors heated the juice at different
high temperatures (75, 85 and 95°C) during 60 min
and the impact on overall colour and betalain
retention was evaluated, after a 24 h colour regener-
ation period. Addition of isoascorbic acid and storage
under dark or light conditions were also evaluated.
The results showed that colour changes and betalains
losses were higher with increased temperatures and
heating times. Samples kept in the dark exhibited
better colour and pigment retentions, as compared to
illuminated ones. The addition of 0.1% isoascorbic
acid to heat-treated yellow-orange cactus pear juice
was shown to significantly delay both betacyanin and
betaxanthin degradation upon heating resulting in an
improved colour stability. These results indicate that
betaxanthin from yellow-orange cactus pear appear to
be a promising alternative to synthetic dyes, partic-
ularly when stabilised with isoascorbic acid.
Other source of red-purple betacyanin could be
obtained from the fruits of Opuntia stricta, due to its
high betalain content (800 mg kg-1 f. w.), five times
higher than those found in red fruits of O. ficus-indica,
and even higher than those shown by commercial red
beets used for their purple colour. Since the betacyanins
are the same as those in beetroot, their acceptance as
food additives would not need any new classification.
Taking this into account, Castellar et al. (2006)
obtained a concentrated extract of betacyanins from
O. stricta fruits to be used as an alternative red-purple
food colourant. A processing scheme was proposed
paying special attention to the extraction procedure.
Concentrated extract was characterised and its storage
stability studied. Furthermore, colour parameters were
determined and compared with other commercial
natural red food colourants (red beet concentrated
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Phytochem Rev (2008) 7:261–280
juice, red carrot concentrate, red grape skin extract,
elderberry juice powder, hibiscus extract powder and
red cabbage extract). Results showed that the 40-fold
concentrated extract had a high colour strength (3.9,
OD 535 nm, 1% v/v sol), a high betanin concentration
(4.7 g l-1) and low viscosity (59.0 cP). It also showed
high stability (237 days at 4°C), mainly due to its low
pH (3.4). All these parameters were in the same range
as those obtained for the commercial liquid concen-
trated studied (red beet, red carrot and grape skin). Also
it presented a vivid red–purple colour, which differs
from the other studied natural red food colourants.
Therefore, concentrated betacyanin extract from the
fruits of O. stricta, with its pleasant taste and flavour,
could offer new opportunities for the use of betacyanin
as a food colourant.
Purple pitaya (Hylocereus polyrhizus) fruits have
been proposed as a promising source of betalains for
red- violet colouring foodstuff. Herbach et al. (2007)
have recently studied the effect of processing and
storage on juice colour and betacyanin stability of
this fruit juice. In this study, juice processing at pilot-
plant scale is reported for the first time. Different
pasteurisation systems and storage experiments using
mucilage-free juice, light exposition and ascorbic
acid addition were also quantified. Authors concluded
that by applying minimal heat-load, two-thirds of the
betacyanin content was retained after pasteurisation.
However, the high mucilage content of pitaya was
disadvantageous for juice clarification by filtration.
On the other hand, the mucilage may contribute to
minimise betalain degradation upon heating and
storage. Moreover, the genuine hydrocolloids may
be advantageous for the application of purple pitaya
juice as a colouring foodstuff for dairy products,
where unclarified juices containing the total mucilage
of the fruit could be advantageous. Regarding
pasteurisation, betacyanin loss and colour alteration
were minimal upon pasteurisation in an HTST system
and a standard tubular heat exchanger. Storage
stability of betacyanins was shown to be superior to
the respective pigments in red beet and significantly
enhanced by the addition of 1% ascorbic acid.
Also, the same authors studied the changes in
betacyanin content and colour shade of juices from
purple pitaya as affected by thermal processing and
consecutive cold storage to allow pigment reconsti-
tutions (Herbach et al. 2004b). In contrast to isolated
betanin from red beet juice, purple pitaya juice was
Phytochem Rev (2008) 7:261–280
shown to display high pigment stability and nearly
unaffected colour shade upon moderate thermal
treatment. Also the identification of the heat induced
degradation products from this fruit betalains have
been reported elsewhere (Herbach et al. 2005;
Wybraniec and Mizrahi 2005).
The colour of fruit juice blends from Opuntia and
Hylocereus cacti were also investigated (Moßhammer
et al. 2005a), in a pH ranging form 3 to 7. All
samples were found to be stable as indicated by
betalain contents as well as colour parameters (hue
and chroma) over the complete pH range monitored.
Garambullo (Myrtillocactus geometrizans)
The stability of garambullo was evaluated in the
presence of stabilizing agents. The results showed
that the pigment is stable at pH 4.8–5.2, at low
temperatures. Also, in the presence of 0.1% ascorbic
acid the half-life was doubled and the energy of
activation was increased (Barrera et al. 1998).
Similar results were obtained by Reynoso et al.
(1997) that described the protecting effect of ascorbic
acid on the red colour, even when the extract was
exposed to drastic treatments such as sterilization.
Nevertheless, the loss of colour was greater when the
temperature increased to 100°C. Same authors also
described that when stability studies were performed
with pure pigments from garambullo (low concen-
tration of labile betaxanthine, 2.92%) and red beet
(19% betaxanthine), the Cactaceae pigment was 15%
more stable than the red beet. However, the red beet
was more stable in the crude extract.
Then again, it was also proved that iron had a
greater degradation effect (52%) than chromium
(32%), after 4 days storage, but ascorbic acid could
protect the pigment when these metals are present.
Regarding extraction procedures, Reynoso et al.
(1997) obtained a higher betalain concentration when
the juice was fermented, in order to reduce the total
solid content that contributed to the purification of the
pigments. However, when pigment extract was
fermented and spray-dried the degradation was too
fast to accurate measurements, reason why a concen-
trated sample was used for the stability studies above
mentioned. On the other hand, Barrera et al. (1998),
indicated that the major concentration of betalains
was extracted with methanol-HCl (99:1), however,
273
these pigments were more unstable than those
obtained by aqueous extraction.
It could be concluded that garambullo pigment has
a potential as a source of natural pigment for the food
industry, especially for those that do not require high
temperature processing, such as dairy products.
Amaranthaceae plants (Amaranthus and Celosia)
Red–violet betacyanin pigments from the genus
Amaranthus plants have provoked interest as a
potential alternative source of betalains since some
genotypes produce particularly high biomass and
contain higher levels of pigment than beetroot. Cai
et al. (2005) described the pigment stability and
colour properties, the feasibility of spray-dried pro-
duction and the possible utilisation of Amaranthus
betacyanins in model food systems (jellies, ice-
creams and beverages). These authors concluded that
betalains from this source exhibited brighter colour
characteristics than red radish anthocyanins, with
similar colour stability during 20-week storage
(B14°C) or during the initial 4-week storage
(B25°C), but less stable at 37°C. Also these pigments
were feasible as natural colourants for higher pH
beverages and ice-creams conditions. Also they
demonstrated that ascorbic acid at 0.1%–0.5% had a
high protective effect in betacyanin stability in jelly.
The genus Celosia consisting of about 60 species
is native from subtropical and temperate zones and
are wide spread ornamental plants, but seedlings,
dried young leaves and inflorescences are used
in traditional Chinese medicine. Cai et al. (2001)
evaluated the colorant properties and stability of
betaxanthins from Celosia argentea. Their results
demonstrated that the yellow pigments had potential
new source of water-soluble food colourant under
selected conditions. The lyophilised betaxanthin
powders from yellow inflorescences exhibited a
bright yellow colour and high colour purity with
strong hydrophobicity. The aqueous solutions were
bright yellow in the pH range 2.2–7, presenting the
highest stability at pH 5.5, and susceptible to heat at
temperatures lower than 40°C, with the exclusion of
light and air and stored at low temperature (4°C). The
lyophilised betaxhantins presented much better
storage stability than the corresponding aqueous
solution.
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In conclusion, the most interesting betalains
applicable feature is the stability in the pH range
from 3 to 7, when processed at low pasteurization, in
the presence of ascorbic acid, and stored at low
temperature which makes these pigments suitable for
their application in a wide range of low acid and
neutral foods.
Bioavailability and metabolism of betalains
From a nutritional point of view, betalains represents
a group of phytochemicals with a restricted occur-
rence in the diet since the plant food sources of them
are quite reduced. Only red beet, swiss chard,
amaranthus, cactus pear, pitaya, and some tubers
and their derived products provide betalains to our
diet (Frank et al. 2005; Stintzing and Carle 2004;
Campos et al. 2006; Kluger et al. 2006). However,
the use of betanin as food colorant and the plant-
betalain enriched extract in functional foods increase
the consumption of this type of phytochemicals
(Gliszczyńska-Świgło et al. 2006; Piga 2004).
Scarce studies have been reported on bioavailibility
and metabolism of betalains. However, their quality
are off of any doubt since the design of the
experiments have been well developed on human
volunteers. Major of them have been carried out
previous oral administration of red beet juice or cactus
pear. To our knowledge, studies implying the human
intake of swiss chard or amaranthus have not been
reported. The intake of red beet juice or cactus pear
revealed that betalains are bioavailable (Tesoriere
et al. 2004a; Frank et al. 2005; Sembries et al. 2006;
Netzel et al. 2005; Kanner et al. 2001). They are
absorbed from the gut into the systemic circulation in
Fig. 4 pH-dependent
Glc O H
structure of betalains.
Example of betanin/
isobetanin
HO
-OOC
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Phytochem Rev (2008) 7:261–280
their intact forms, which indicated that, as shown with
some glycosylated flavonoids (Manach et al. 2005),
hydrolysis is not a prerequisite for absorption. In
addition, other unknown metabolites have been also
detected in urine that may fit with conjugates or
microbial metabolites after suffering similar metabol-
ical pathways than flavonoids (Manach et al. 2005).
The maximum concentration in plasma was achieved
after 3 h of the intake at 0.2 lM and 6.9 lM of betanin
and indicaxanthin, respectively (Tesoriere et al.
2004a; Frank et al. 2005). The bioavailability ranged
0.28%–0.9 % for betanin and isobetanin after red beet
juice administration (Netzel et al. 2005; Kanner et al.
2001).
Betalains are cationized compounds with a posi-
tive nitrogen in a polyene system. This nature may
help at their absorption site thinking on a similar
absorption mechanism of the anthocyanins, flavo-
noids with cationic structure (Tesoriere et al. 2004a;
Pérez-Vicente et al. 2002; Talavera et al. 2005;
Felgines et al. 2005). Nevertheless, the distribution
of the charges on the betalain structure shows a pH-
dependent structure, presenting a bis-anion and tris-
anion forms at pH equal or higher than 7 (Frank et al.
2005) (Fig. 4). This behaviour has not been demon-
strated at in vivo physiological level yet.
To the best of our knowledge, only one study has
been designed to describe the fate of these phyto-
chemicals in target tissues. Betalains are able to go
across the red blood cell membranes. The concentra-
tion inside the cells was 30 nM and 1 lM betanin and
indicaxanthin, respectively (Tesoriere et al. 2005).
A fact which may suppose an interference for the
bioavailability of betalains is their capacity to interact
with biological macromolecules. Dialysis experiments
provided evidence that betanin and indicaxanthin can
Glc O H
+ COO-
+ COO-
N -O N
-OOC N COO-
N COO-
H H
3.5<pH<7 7<pH<9.5
Bis-anion Tris-anion
Phytochem Rev (2008) 7:261–280
bind to biological membranes and to large unilamellar
L-a-dipalmitoyl-phosphatidylcholine (DPPC) or soy-
bean-phosphatidylcholine liposomes (Tesoriere et al.
2007). Following studies carried out for the same
research team has confirmed that betanin is located at
the hydrophobic interior of the DPPC bilayer (Liveri
et al. 2007). Betalains, despite their high solubility in
water may be less bioavailable at the absorption site
due to these associations and may associate to
macromolecules at systemic level in the human body
at the postabsortive stage. However, clinical trials
must be carried out to support these hypothesis.
As conclusion, betalains are bioavailable mole-
cules and are present as intact forms at systemic
level. However, bioavailability studies are starting
and new studies should be carried out in order to
establish the real extension of the association of
betalains with macromolecules and the possible
occurrence of them at systemic level. This fact may
change the bioavailability of these phytochemicals.
Moreover, the fate of these molecules in vivo repre-
sents a prerequisite to planify biological assays on
health protection with the right physiological con-
centrations and nature of the circulating metabolites.
Biological and health-promoting properties
of betalains
Antioxidant-related actions
Betalains shows a cationized structure with a positive
nitrogen in a polyene system. Their cyclic amine,
which is similar to that of the antioxidant ethoxyq-
uine (Belitz and Grosch 1987; Lin and Olcott 1995),
has reasonably been considered to be the reactive
group conferring to this class of molecules reducing
properties. Therefore and by theory, these phyto-
chemicals (betacyanins and betaxanthins) carries a
phenolic and an acyclic amine group, which are
excellent electron donors and then are able to
stabilise radicals (Kanner and Harel 1985; Kanner
et al. 1996; Kanner et al. 2001).
On base of this knowledge and their redox
properties, several in vitro experiments assayed with
radicals generated in chemical systems were assayed.
The radical 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical stable in alcoholic media was highly scaven-
gered by different types of betalains or plant extract
275
containing these phytochemicals (Cai et al. 2003;
Siriwardhana and Jeon 2004; Pavlov et al. 2005b).
The destruction of the 2,20 -azino-bis(3-ethylbenzthi-
zanoline-6-sulphonic acid) (ABTS) generated in
aqueous media by horse-radish peroxidase/hydrogen
peroxide-mediated oxidation of ABTS was success-
fully achieved by betalains but in greater extension
by betacyanins than betaxanthins (Escribano et al.
1998; Butera et al. 2002; Gliszczyńska-Świgło et al.
2006; Stintzing et al. 2005). Other chemicals model
like inhibition of the lipid peroxidation or the oxygen
radical absorbance capacity (ORAC) showed the
same positive results on the antiradical actions of
betalains (Stintzing et al. 2005; Zakharova and
Petrova 1998; Butera et al. 2002; Siriwardhana and
Jeon 2004; Kanner et al. 2001).
The second step was to prove if betalains prevent
active oxygen-induced and free radical-mediated
oxidation of biological molecules. Following exper-
iments have demonstrated that betalains have
received attention for their antioxidant activity in a
number of biological lipid environments in vitro,
from human low density lipoprotein to cell mem-
branes (Kanner et al. 2001; Tesoriere et al. 2003,
2005). Particularly, they have exerted in vitro pro-
tection against different oxidative stresses caused on
LDL and membrane lipids and myeloperoxidase
acting as reductant of its redox intermediates in
neutrophils (Kanner et al. 2001; Tesoriere et al.
2003; Allegra et al. 2005). Moreover, these phyto-
chemicals exert phase II enzyme-inducing activities,
overall, quinone reductase in hepatoma cells (Wetta-
singhe et al. 2002; Lee et al. 2005) and inhibit
ICAM-1 expression on endothelial cells (Gentile
et al. 2004). The betaxanthin indicaxanthin showed
in vitro positive actions against b-thalassemia by the
incorporation into the redox machinery of b-thalas-
semic red blood cells to defend them from oxidation
(Tesoriere et al. 2006).
The third issue is to know if these phytochemical
are able to exert any action as antioxidant in vivo.
None of the experiments described above would have
sense if they had not the following action on in vivo
systems. Recent studies have demonstrated that
betalains provide protection against oxidative stress-
related disorders. Particularly, two studies published
in the same year provided new data on antioxidant
protection exerted by betalains in humans. An exper-
iment carried out with 8 volunteers who consumed
123
276
cactus pear fruit pulp detected the incorporation of
betalains in LDL and suggested that betanin and
indicanxanthin may be involved in the protection of
LDL against oxidative modifications (Tesoriere et al.
2004b). Moreover, the following clinical trials
showed that consumption of cactus pear fruit posi-
tively affected the body’s redox balance and
decreased the oxidative damage of lipids and the
intake of red beet juice delayed the LDL oxidation.
These effects were linked to betalains (Tesoriere et al.
2004b; Sembries et al. 2006).
Other actions
Currently, the biological properties non-related with
antiradical mechanism have not been afforded on
their real extension yet and therefore, this data
continues lacking. To our knowledge, only one study
described the antimalarial activities of Amaranthus
spinosus L. and Boerhaavia erecta L. containing
betalains. This action was linked to the betacyanin
level thanks to the power of this phytochemical to
chelate the parasites indispensable inner cations Ca2+,
Fe2+, and Mg2+ for different biological synthesis of
Plasmodium berghei berghei like Mg and Fe ions as
cofactors of Plasmodium enzyme ribonucleotide
reductase (Hilou et al. 2006). Other effect shared
with flavonoids has been attributed to betalains
thanks to the hepatoprotection provided by cactus
pear fruit against induced toxicity by CCl4 in rats
(Galati et al. 2005).
As conclusion, the starting experiments on bio-
logical and health-promoted properties of betalains
are directly related to their antioxidant properties
since the chemical and in vitro antiradical properties
have been positively corroborated by very scarce
clinical trials in humans by the moment. Moreover,
these compounds are circulating metabolites as native
compound found in the original foods. However, new
studies are lacking about the action of other unknown
metabolites from betalains detected in urine, which
may open new ways of biological actions of these
phytochemical against human disorders.
Acknowledgements Authors are grateful to the Spanish
CSIC and MEC for the project Intramural Especial (Ref.
200770I003). Diego A. Moreno thanks the European Social
Fund (ESF) and the Spanish Ministerio de Educación y Ciencia
123
Phytochem Rev (2008) 7:261–280
and CSIC for funding through the ‘‘Ramón y Cajal’’ S&T
Programme.
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