Original article
Betalains: properties, sources, applications, and stability – a review
Henriette M.C.Azeredo*
Embrapa Agroindu´ stria Tropical, CEP: 60511-110, Fortaleza, Ceara, Brazil
(Received 26 June 2006; Accepted in revised form 7 September 2006)
Summary Consumers are increasingly avoiding foods containing synthetic colourants, which lead food industries to
replace them by natural pigments, such as carotenoids, betalains, anthocyanins and carminic acid. Betalains
are water-soluble nitrogen-containing pigments, composed of two structural groups: the red-violet
betacyanins and the yellow-orange betaxanthins. This review synthesises the published literature on basic
chemistry of betalains, their sources and chemical stability. Moreover, several works are mentioned which
have demonstrated the potent antioxidant activity of betalains, which has been associated with protection
against degenerative diseases.
Keywords Antioxidants, beetroots, betalains, natural colourants, pigments.
Introduction
Colour is one of the most important attributes of foods,
being considered as a quality indicator and determ ining
frequently their acceptance. Many naturally coloured
foods, such as fruit products, are submitted to colour
losses during processing, requiring the use of colourants
to restore their colour. Natural colourants have many
disadvantages when compared to synthetic ones, includ-
ing higher cost in-use and lower stability. However,
people have increasingly avoided synthetic colourants,
preferring natural pigments, which are considered to be
harmless or even healthy. These requirements compelled
numerous regulation changes worldwide. For instance,
the USA permitted list of synthetic colourants was
reduced from 700 to only seven until the beginni ng of
the XXI Century (Downham & Collins, 2000). The
current market for all food colourants is estimated at
US$ 1 billion, with natural pigments responding for
only one fourth of the total. However, the market for
synthetic colourants has tended to decline in favour of
natural ones (Fletcher, 2006).
Nature produces a variety of compounds adequate for
food colouri ng, such as the water-soluble anthocyanins,
betalains, and carminic acid, as well as the oil soluble
carotenoids and chlorophylls. The health-benefit prop-
erties of natural pigments have been focussed by many
works, especially those of carotenoids and antho-
cyanins, whose antioxidant properties have been
extensively studied. Betalains, because of their relative
scarceness in nature, have not been much explored as
bioactive compounds, but some studies have indicated
their potential as antioxidant pigments. These findings
have helped to motivate utilisation of betalains as food
colourants.
Betalains as colourants
Betalains are water-soluble nitrogen-containing pig-
ments, which are synthesised from the amino acid
tyrosine into two structural groups: the red-violet
betacyanins and the yellow-orange betaxanthins. Beta-
lamic acid, whose structure is presented in Fig. 1a, is the
chromophore common to all betalain pigments (Strack
et al., 2003). The nature of the betalamic acid addition
residue determines the pigment classification as beta-
cyanin or betaxanthin (Fig. 1b and c, respectively).
The structural differences reflect in varying appear-
ance of the betalain subgroups. Betacyanins contain a
cyclo-3,4-dihydroxyphenylalanine (cyclo-Dopa) residue.
The con densation with the closed structure of cyclo-
Dopa extends the electronic resonance to the diphenolic
aromatic ring. This extra conjugation shifts the absorp-
tion maximum from 480 nm (yellow, betaxanthins) to
about 540 nm (violet, betacyanins) (Jackman & Smith,
1996; Strack et al., 2003).
Betanidin is the aglycone of most betacyanins; differ-
ent substitution (glycosylation and acylation) patterns
of one or both hydroxyl groups located at position 5 or
6 of betanidin result in the formation of the various
betacyanins. Most of these are 5-O-glucosides, but 6-O-
glucosides have also been detected. No betacyanin is
*Correspondent: E-mail: ette@mpc.com.br
R. Dra. Sara Mesquita, CEP: 60511-110, 2270 Fortaleza, Ceara,
Brazil.
International Journal of Food Science and Technology 2009, 44, 2365–2376
2365
doi:10.1111/j.1365-2621.2007.01668.x
2008 Institute of Food Science and Technology
known to have both posit ions substituted with sugar
residues (Delgado-Vargas et al., 2000). Further glyco-
sylation of the 5-O-glucoside is very common and so is
esterification with hydroxycinnamic acids (Strack et al.,
2003). The most common betacyanin is betanidin-5-O-
b-glycoside (betanin), the major pigment in red beets
(Stintzing & Carle, 2004). Betacyanins display two
absorption maxima – one in the UV-range (270–
280 nm) because of cyclo-Dopa and a second one in
the visible range (535–540 nm, depending on the sol-
vent). The red and violet co lours result from different
substitution patterns of betacyanins. Glycosylation of
betanidin generally comes along with a hypsochromic
shift of about 6 nm, while a second sugar moiety
attached to the first one apparently did not greatly affect
the colour (Cai et al., 1998; Stintzing & Carle, 2004).
Acylation with hydroxycinnamic acids results in a third
maximum (300–330 nm), whereas aliphatic acyl moieties
do not alter the spectrum (Stintzing & Carle, 2004).
Betaxanthins, on the other hand, contain different
amino acid or amine side chains (Strack et al., 2003).
Structural modifications in betaxanthins produce hypso-
or bathochromic shifts. Amine conjugates display a
lower absorption maximum than their respective amino
acid counterparts (Stintzi ng et al., 2002b).
Betalains have several applications in foods, such as
desserts, confectione ries, dry mixes, dairy and meat
products. The concentration of pure pigment required to
obtain the desired hue is relatively small, rarely exceed-
ing 50 mg
)1
kg, calculated as betanin (Delgado-Vargas
et al., 2000). According to the Codex Alimentarius
Commission (2004), betalain use is limited only by Good
Manufacturing Practice. The food colourant known as
‘beetroot red’, extracted from beetroots, is commercia-
lised in European Union and USA as food colourant
(Castellar et al., 2006). Commercial beet colourants are
available as either juice concentrates (produced by
vacuum-concentration of beet juice to 60–65% total
solids) or powders (produced by freeze- or spray-
drying), containing from 0.3% to 1% of pigment
(Cerezal et al., 1994; Cerezal & Nu´ n
˜
ez, 1996).
Cai & Corke (1999) compared Amaranthus betacya-
nins and commercial colorants in terms of their colour
characteristics and stability at different temperatures in
model food systems. Betacyanins exhibited brighter red
colour than the red radish anthocyanin. Both pigments
showed similar colour stability at 14 Cand25C, but
betacyanin colour was less stable than red radish
anthocyanin at 37 C. A synthetic colourant was also
tested, and was more stable than betacyanins under
most storage conditions.
Plant sources of betalains
Anthocyanins and betalains have never been reported in
the same plant, seeming to be mutually exclusive in the
plant kingdom (Stafford, 1994). Betalains can be found
in roots, fruits and flowers (Strack et al., 2003). They
absorb visible radiation over the range of 476–600 nm
with a maximum at 537 nm at pH 5.0. The few edible
known sources of betalains are red and yellow beetroot
(Beta vulgaris L. ssp. vulgaris), coloured Swiss chard
(Beta vulgaris L. ssp. cicla), grain or leafy amaranth
(Amaranthus sp.) and cactus fruits, such as those of
Opuntia and Hylocereus genera (Cai et al., 1998; Stint-
zing et al., 2002b; Kugler et al., 2004; Vaillant et al.,
2005).
The major commercially exploited betalain crop is red
beetroot (Beta vulgaris), which contains two major
soluble pigments, betanin (red) and vulgaxanthine I
(yellow). According to Nilsson (1970) , the betacyanin
and betaxanthin contents of red beetroots vary within
the ranges 0.04–0.21% and 0.02–0.14%, respectively,
depending on the cultivar (Von Elbe, 1975), although
some new varieties produce higher betalain contents
(Pszczola, 1998; Gaertner & Goldman, 2005).
Gasztonyi et al. (2001) analysed five red beet varieties
(‘Bonel’, ‘Nero’, ‘Favo rit’, ‘Rubin’ and ‘Detroit’) in
terms of their pigment composition. In all cases, the
major red-violet pigments were betanin, isobetanin,
betanidin and isobetanidin, and the major yellow
components were vulgaxanthin I and vulgaxanthin II.
Figure 1 General structures of betalamic acid
(a), betacyanins (b) and betaxanthins (c).
Betanin: R
1
=R
2
=H.R
3
= amine or
amino acid group (Strack et al., 2003).
Betalains – a review H. M. C. Azeredo2366
International Journal of Food Science and Technology 2009, 44, 2365–2376 2008 Institute of Food Science and Technology
‘Bonel’, ‘Favorit’ and ‘Rubin’ exhibited the highest
betacyanin contents (around 0.08%), while the ‘Nero’
variety showe d the poorer betacyanin content (near
0.06%). The variety ‘Rubin’ showed the highest beta-
cyanin ⁄ betaxanthin ratio (2.08), so was considered as
the most suitable for food colourant production.
Since the betalain spectrum of red beets is restricted
mainly to betanin, colour variability is poor. Moreover,
an adverse earthy-like flavour because of geosmin and
some pyrazines is undesirable when applying beet
extracts to, for instance, dairy products (Lu et al.,
2003; Stintzing & Carle, 2004). Therefore, attempts
have been made to explore alternative sources of
betalains. The most promising family among betalain-
bearing plants is the Cactaceae. Among those, cactus
pears (genus Opuntia) and pitayas (genera Cereus,
Hylocereus and Selenicereus) are most commonly
cultivated as fruit crops and best suited to be studied
as betalain sources for colouring food (Mizrahi et al.,
1997; Stintzing et al., 2003; Stintzing & Carle, 2006).
Cactus fruits, in contrast to red beetroot, may be used
in food without negative flavour impacts as those
derived from beetroot extracts. On the contrary, the
faint flavour tends to impair the market potential for
plain cactus fruits, making their utilisation for colour-
ing applications more promising. The betalains in
cactus fruits also cover a broader colour spectrum
from yellow–orange (Opuntia sp.) to red–violet (Hylo-
cereus sp.) compared to red beet and thus may open
new windows of colour diversification. The yellow-
orange cactus fruits are of particular interest, because
of the scarceness of yellow water-soluble pigments
(Mobhammer et al., 2005). Moreover, the low levels of
colourless phenolic compounds in cactus fruits make
them very promising, since potential interactions of
betalains with these phenolics are avoided (Stintzing
et al., 2001). An additional advantage of cactus fruits
are their minimal soil and water requirements, being
regarded as alternative cultures for the agricultural
economy of arid and semi-arid regions (Castellar et al.,
2006). Fruit pulps of Hylocereus cacti contain high
concentrations of betacyanins (0.23–0.39%), both non-
acylated and acylated, and (in contrast with beetroots)
contain no detectable betaxanthins (Wybraniec et al.,
2001; Stintzing et al., 2002a; Vaillant et al., 2005). On
the other hand, Opuntia fruits co ver a broad colouring
range, from bright yellow to red-violet, depending both
on the betacyanin ⁄ betaxathin ratio and their absolute
concentrations (Mobhammer et al., 2005; Stintzing
et al., 2005). Stintzing et al. (2005) reported broad
ranges of betacyanin (0.001–0.059%) and betaxanth in
(0.003–0.055%) contents in different Opuntia clones.
The betaxanthin ⁄ betacyanin ratios vary widely in
cactus pears (Butera et al., 2002; Stintzing et al.,
2003), but fruits containing exclusively betaxanthins
are not known (Stintzing & Carle, 2006).
Castellar et al. (2003) analysed the betalains of three
Opuntia species. Both betacyanins and betaxanthins
were identified in Opuntia undulata and O. ficus-indica,
while in O. stricta only betacyanins were detected.
Among the three species, O. stricta seemed to be the
most promising one, with the highest betacyanin content
(0.08%). Moreover, O. stricta has thinner peel and less
seeds than the other species, making pigment extraction
easier.
Biotechnological production of betalains
Some authors have investigated beet cell cultures for
producing betalains (Leathers et al., 1992; Akita et al.,
2000). With this technology, it would be easier to
control quality and availability of pigments indepen-
dently of environmental changes (Do
¨
rnenburg & Knorr,
1997). However, these cultures are unable to compete
with the beetroot, which is an abundant and inexpensive
crop which may prod uce up to 0.5 g of betanin per kg of
roots (Gasztonyi et al., 2001). The low productivity of
the current bioreactor systems available and the high
cost of the process impair its economic feasibility. Then,
the selection of a bioreactor and cultivation techniques
for optimal culture growth and betalain production is
one of the most important issues to be solved (Jimenez-
Aparicio and Gutierrez-Lopez, 1999).
Betalain extraction
Betalain-containing materials are generally macerated
or ground. Pigments can be water-extracted, although,
in most cases, the use of methanol or ethanol solutions
(20–50%) is required to complete extraction (Delgado-
Vargas et al., 2000). Nevertheless, Castellar et al. (2006)
reported that water extracted higher levels of pigments
from Opuntia fruits than ethanol:water. Garcı
´
a Barrera
et al. (1998) reported higher betalain extraction with
ethanol–HCl (v ⁄ v ratio, 99:1) than that with water.
However, the aqueous extraction promoted better sta-
bility of the pigments. Slight acidification of the extrac-
tion medium enhances betacyanin stability and avoids
oxidation by polypheno loxidases (Schliemann et al.,
1999; Strack et al., 2003).
A previous enzyme inactivation by a short heat
treatment of the extract is desirable, to avoid betalain
enzymatic degradation (Delgado-V argas et al., 2000).
Enzymatic treatments for degradation of hydrocolloids
may also favour the pigment extraction (Mobhammer
et al., 2005). Fermentation of extracts may reduce free
sugars, increasing the betacya nin content (Pourrat et al.,
1988).
The degree of cell membrane permeabilisation is a
major factor to determine the extraction efficiency.
Pulsed electric field treatments increase cell permeability
(Rastogi et al., 1999; Ade-Omowaye et al., 2001),
Betalains – a review H. M. C. Azeredo 2367
2008 Institute of Food Science and Technology International Journal of Food Science and Technology 2009, 44, 2365–2376
enhancing betalain extraction efficiency (Chalermchat
et al., 2004; Fincan et al., 2004), with relatively low
levels of tissue damage and low energy consumption
(Fincan et al., 2004). Nayak et al. (2006), using gamma-
irradiation as a pre-treatment to a solid–liquid extrac-
tion of betanin from red beets, observed that the
extraction efficiency increased with the irradiation doses
(0–10 kGy); this effect was attributed to the cell perme-
abilisation. On the other hand, irradiation also increased
betanin degradation rates .
On a laboratory scale, betalains may be extracted by
various methods, such as diffusion-extraction (Wiley &
Lee, 1978), solid–liquid extraction (Lee & Wiley, 1981),
reverse osmosis (Lee et al., 1982) and ultrafiltration
(Bayindirli et al., 1988; Real & Cerezal, 1995). These
processes are more efficient on recovering betalains from
beet tissue when compared to conventional hydraulic
techniques (Real & Cerezal, 1995). Since approximately
80% of beet juice solids consist of fermentable
carbohydrates and nitrogenous compounds, fermenta-
tion process es have been applied to remove these
materials (Drda
´
k et al., 1992), thus increasing betalain
concentrations.
Betalain analysis
Betalain analysis has been carried out basically on UV-
visible spectroscopy. Betacyanins absorb around
k
ma
´
x
= 540 nm, and betaxanthins, at k
ma
´
x
= 480 nm.
The first studies of betalain identification were based on
this methodology. Structural modifications of betalains
have also been followed by UV-visible spectroscopy
(Piattelli, 1981). Nowadays, chemical characterisation
must be carried out considering at least HPLC separa-
tion and UV-visible, mass spectrometry, and NMR
spectroscopy, for identification of individual com-
pounds (Strack et al., 1993; Stintzing et al., 2004).
Stintzing et al. (2004) developed a solvent system
which improved data acquisition at almost neutral pH.
The authors were successful in submitting four
non-carboxylated betacyanins (betanin, isobetanin,
phyllocactin and hylocerenin) to
1
H and
13
CNMR
characterisation. Later, Wybraniec et al. (2006) eluci-
dated structures of mono- and di-decarboxylated beta-
cyanins by using both techniques. Until then, a highly
acidic media was considered as necessary for an adequate
data acquisition during NMR measuring. Since betalains
are unstable under such conditions, betalain structure
elucidation by NMR spectroscopy was scarce and
limited to
1
H NMR (Wybraniec et al., 2001; Stintzing
et al., 2002a). The only
13
C NMR betacyanin spectrum
then available (Alard et al., 1985) was that of neobetanin
(14,15-dehydrobetanin), thanks to its higher stability to
acidic conditions when compared to other betacyanins.
Betaxanthin structure elucidation was also exclusively
based on
1
H NMR data (Piattelli et al., 1964, 1965;
Wyler & Dreiding, 1984; Hilpert et al., 1985; Strack
et al., 1987; Trezzini & Zr, 1991), until Stintzing et al.
(2006), applying only slightly acidic conditions, were
successful in reporting the first
13
C NMR data of two
betaxanthins (indicaxanthin and miraxanthin).
Factors affecting chemical stability of betalains
Betalain degradation may occur by different mecha-
nisms, which were detailed by Herbach et al. (2006b).
Several fact ors, both intrinsic and extrinsic, affect
betalain stability, and need to be considered to ensure
optimum pigment and colour retention in foods con-
taining betalains.
Structure and composition
Concerning structural aspects, betacyanins have been
reported to be more stable than betaxanthins, both at
room temperature (Sapers & Hornstein, 1979) and upon
heating (Singer & von Elbe, 1980; Herbach et al.,
2004a). Comparing stability of different betacyanins,
glycosylated structures are more stable than aglycons,
probably because of the higher oxidation–reduction
potentials of the former (von Elbe & Attoe, 1985).
However, stabi lity does not seem to be enhanced by
further glycosylation (Huang & von Elbe, 1986). Some
studies have indicated increasing betacyanin stability
resulting from ester ification with aliphatic acids (Reyn-
oso et al., 1997; Garcı
´
a Barrera et al., 1998), as well with
aromatic acids, especially at the 6-O position (Heuer
et al., 1994; Schliemann & Strack, 1998). However,
Herbach et al. (2006c) found interesting results when
monitoring thermal degradation of betanin and acylated
betacyanins (phyllocactin and hylocerenin) by spectro-
photometric and high-performance liquid chromato-
graphy-diode array detection (HPLC-DAD) analyses.
They observed that betanin were more stable than the
acylated structures, but the tinctorial stability of phyl-
locactin and especially hylocerenin solutions was fa-
voured by the formation of red degradation products
with high colour retention. Hence, spectrophotometric
analyses were reported to be insufficient to assess
structure-related stability characteristics of betacyanins.
Red beets have several endogenous enzymes such as
b-glucosidases, polyphenoloxidases and peroxidases,
which if not properly inactivated by blanching may
account for betalain degradat ion and colour losses
(Lee & Smith, 1979; Martı
´
nez-Parra & Mun
˜
oz, 2001;
Escribano et al., 2002). The optimum pH for enzymatic
degradation of both betacyanins and betaxanthins was
reported to be around 3.4 (Shih & Wiley, 1981). The
degradation products are similar to those of thermal,
acid or alkaline degradation (Martı
´
nez-Parra & Mun
˜
oz,
2001; Escribano et al., 2002; Stintzing & Carle, 2004).
Betacyanins are more susceptible than betaxanthins to
Betalains – a review H. M. C. Azeredo2368
International Journal of Food Science and Technology 2009, 44, 2365–2376 2008 Institute of Food Science and Technology
degradation by pe roxidases, while the latter are more
oxidised by hydrogen peroxide, since the presence of
catalase almost thoroughly suppressed betaxanthin oxi-
dation (Wasserman et al., 1984). Some attempts have
been made to take advantage of endogenous b-glucosi-
dase activity to extend available shades offered by red
beets. The transformation of betanin glycos ides into
their respective aglycones produces a bathochromic shift
of 4–6 nm. However, these aglycones are more labile
and prone to further oxidation which results in red
colour losses and subsequent browning (Stintzing &
Carle, 2004).
pH
Although altering their charge upon pH changes,
betalains are not as susceptible to hydrolytic cleavage
as the anthocyanins. Betalains is relatively stable over
the broad pH range from 3 to 7 (Jackman & Smith,
1996), which allows their application to low acidity
foods. Below pH 3.5, the absorption maximum shifts
toward lower wavelengths, and above pH 7 the change is
toward upper ones; out of the pH range 3.5–7.0 the
intensity of the visible spectra decreases. Optimal pH
range for maximum betanin stability is 5–6 (Huang &
von Elbe, 1985, 1987; Castellar et al., 2003; Vaillant
et al., 2005). Alkaline conditions cause aldimine bond
hydrolysis, while acidification induces recondensation of
betalamic acid with the amine group of the addition
residue (Schwartz & von Elbe, 1983). At low pH values,
C
15
isomerisation (Wyler & Dreiding, 1984) and dehy-
drogenation (Mabry et al., 1967) were observed. Von
Elbe et al. (1974) observed that, under fluorescent light,
betanin degradation rate was three-fold higher at pH 3
than at pH 5. However, according to Herbach et al.
(2006b), any of the degradation mechanisms elucidated
so far are able to explain the shift in betanin maximum
absorbance at pH values below 3.0 and the slight
increase in absorbance at 570–640 nm (Jackman &
Smith, 1996).
Although activation energy for betacyanin degrada-
tion decreases with pH, this does not impairs pigment
application to most foods undergoing ordinary thermal
treatments. As an example, betacyanin losses in pitaya
juice acidified to pH 4 is less than 10% during
pasteurisation at 80 C for 5 min (Vaillant et al., 2005).
Some factors affect betalain stability upon pH. Hav-
lı
´
kova
´
et al. (1983) reported that high temperatures
shifted the optimum pH for betacyanin stability toward
6. According to Huang & von Elbe (1987), anaerobic
conditions favour betani n stability at lower pH (4.0–5.0).
Water activity ( a
w
)
Betalain stability is exponentially affected by a
w
, which
is a key factor determining the pigment susceptibility to
aldimine bond cleavage (Saguy et al., 1984; Herbach
et al., 2006b). The a
w
effect on betalain stability may be
attributed to a reduced mobility of reactants or limited
oxygen solubility (Delgado-Vargas et al., 2000).
Kearsley & Katsaboxakis (1980) reported that a
w
reduction improved betanin stability, especially below
0.63. Cohen & Saguy (1983) observed an increase of
about one order of magnitude in betalain degradation
rates when a
w
increased from 0.32 to 0.75. Stability of
betacyanins was reported to increase after being sub-
mitted to methods to reduce water activity, such as
concentration (Castellar et al., 2006) and spray-drying
(Cai & Corke, 2000). In a stability study of encapsulated
beetroot pigments, greatest betanin degradation oc-
curred at a
w
= 0.64 (Serris & Biliaderis, 2001); this
value was attributed by the authors to the decreasing
mobility of reactants at lower and the dilution effects at
higher a
w
values.
Oxygen
Betalains react with molecular oxygen (Attoe & von
Elbe, 1985). The storage of betanin solutions under low
oxygen levels results in decreased pigment degradation
than under air atmosphere, since low oxygen levels
favour the pigment to be partially recovered after
degradation (Von Elbe et al., 1974; Huang & von Elbe,
1987). A deviation from the first- order degradation
kinetics of betanin in absence of oxygen was attributed
to reaction reversibility. Betalain stability has been
reported to be improved by antioxidants (Attoe & von
Elbe, 1985; Altamirano et al., 1992; Han et al., 1998) or
by a nitrogen atmosphere (Attoe & von Elbe, 1982,
1985; Drunkler et al., 2006).
Light
Betalain stability was reported to be impaired by light
exposure (Von Elbe et al., 1974; Attoe & von Elbe, 1981;
Cai et al., 1998). Attoe & von Elbe (1981) showed an
inverse relationship between betalain stability and light
intensity (in the range 2200–4400 lux). UV or visible
light absorption excites p electrons of the pigmen t
chromophore to a more energetic state (p*), increasing
reactivity or lowering activation energy for the molecule
(Jackman & Smith, 1996). Betalain light-induced deg-
radation is oxygen dependent, because the effects of
light exposure are negligible under anaerobic conditions
(Attoe & von Elbe, 1981; Huang & von Elbe, 1986).
Metals
Some metal cations, such as iron, copper, tin and
aluminium were reported to accelerate betanin degra-
dation (Pasch & von Elbe, 1979; Attoe & von Elbe, 1984;
Czapski, 1990; Sobkowska et al., 1991). Metal -pigment
Betalains – a review H. M. C. Azeredo 2369
2008 Institute of Food Science and Technology International Journal of Food Science and Technology 2009, 44, 2365–2376
