and Reinhold Carle - Food Technology Information Service

Betalains e emerging
prospects for
food scientists
Florian C. Stintzing* and
Reinhold Carle
Institute of Food Technology, Section Plant Foodstuff
Technology, Hohenheim University, August-von-
Hartmann-Straße 3, 70599 Stuttgart, Germany
(Tel.: D49 711 459 22314; fax: D49 711 459 24110;
e-mail: stintzin@uni-hohenheim.de)
Betalains have witnessed swayings of scientific interest in the
past 40 years, but only during the past decade research activities
in many disciplines dealing with breeding, phytochemical,
technological and nutritional aspects have broadened
the hitherto narrow view on betalains. The challenge of bringing
together the knowledge from all these different fields of expertise
is considered to be most fruitful. In the present review,
the focus will be on the technologically related analytical
issues.
Introduction
As opposed to other pigment classes such as the carotenoids,
chlorophylls and anthocyanins, the betalains have
been studied with much less intensity. According to literature,
betalains have experienced peaks of scientific attention
in the 1960s and 1970s through the impressive
phytochemical contributions by Piattelli (1976) in Italy,
Dreiding (1961) and Wyler (1969) in Switzerland, Clement,
Mabry, Wyler, and Dreiding (1994) and Mabry (1966) in
USA, as well as Musso (1979) and Reznik (1975) in
Germany. Technological and also nutritional issues were
considered in pioneering studies by von Elbe and Goldman
(2000) in USA in the 1970s and 1980s, which were the catalyst
for an extensive breeding programme for red beets
conducted by Gabelman and later Goldman (Gaertner &
Goldman, 2005; Goldman & Navazio, 2003). In the
1990s research activities were mainly dedicated to
* Corresponding author.
Review
biosynthetic aspects both at the Leibniz Institute of Plant
Biochemistry in Halle (Saale)/Germany (Strack, Vogt, &
Schliemann, 2003) and at the Laboratory of Cellular Phytogenetics
at Lausanne/Switzerland (Zryd & Christinet,
2004).
With a focus on food, the scarce attention towards betalains
may be due to the fact that red beet has long been considered
the only edible betalainic source. In the past five to
ten years, however, leaf and grain amaranth, cactus fruits,
but also coloured Swiss chard and yellow beet have stimulated
food scientists to study betalains from a technological
and nutritional perspective (Stintzing & Carle, 2004, in
press). The present overview will discuss selected features
of betalain chemistry and their importance to food
scientists.
Betalains e a bunch of colourful structures
To date, the betalains comprise a quite modest number
of about 55 structures including the red-violet betacyanins
and the yellow-orange betaxanthins (Stintzing &
Carle, in press), while up to 550 anthocyanins have
been identified in nature thus far (Andersen & Jordheim,
2006). Although not yet being clarified, the co-occurring
betacyanin C15-stereoisomers are mainly considered isolation
artifacts. In contrast, the analogous C11-isomers for
the betaxanthins have not yet been detected as genuine
compounds. Despite this still small number of structures,
which is expected to grow, betalains are a matter of fascination.
In early days erroneously addressed as flavocyanins
(betaxanthins) and nitrogenous anthocyanins
(betacyanins), it was Mabry and Dreiding (1968) who
coined the term ‘‘betalain’’ for both pigment types.
Only slightly earlier, betanin from red beet was the first
betacyanin (Wyler, Mabry, & Dreiding, 1963) and indicaxanthin
from cactus pear the first yellow-orange betaxanthin
structurally characterised (Piattelli, Minale, &
Prota, 1964; Fig. 1). Although the substitution pattern
of betacyanins with respect to sugars and additional acylation
resembles part of the structural design of anthocyanins,
distinct differences exist. The uniqueness of
betalains is their N-heterocyclic nature with betalamic
acid being their common biosynthetic precursor. Aldimine
formation with cyclo-Dopa yields the betanidin
aglycone which is usually conjugated with glucose and
sometimes additionally with glucuronic acid, and may
also be further modified through aliphatic and aromatic

vulgaxanthin I
[glutamine-betaxanthin]
HO
H
HOOC
H
H
HOOC
OH
11
indicaxanthin
[proline-betaxanthin]
H 2 N
H
H
HOOC
+
N
+ N
11
+
N
N
H
11
O
N H
N
H
H
H
COO -
COOH
COO -
COOH
COOH
miraxanthin V
[dopamine-betaxanthin]
HOOC
HO
HO
acid esterifications. In comparison with the anthocyanins
(Andersen & Francis, 2004), a much smaller number of
substituents have been reported for the betalains: glucose,
glucuronic acid and apiose are the typical sugar monomers,
while malonic and 3-hydroxy-3-methyl-glutaric
acids as well as caffeic-, p-coumaric, and ferulic acids
represent typical acid substituents (Strack et al., 2003).
HO
O
C
H
OH
O
O
C
H
C H
O
O
HO
HO
C
CH3 O
3"
CH2 C CH2 C
OH
H
H
O
HO
HO
O
HO
O
OH
HOOC
O
HO
H
6'
C H
O
OH
H
+ COO
N
-
15
O
2' 1' 5
HO
N COOH
H
betanin, isobetanin
HOOC
H
+ COO
N
-
15
N
H
HOOC
H
+ COO
N
-
Noteworthy, sinapic acid has been rarely reported for
the betalains (Kugler, Stintzing, & Carle, 2007; Wybraniec,
Nowak-Wydra, Mitka, Kowalski, & Mizrahi,
2007), while inversely 3-hydroxy-3-methyl-glutaric acid
has never been found as a structural feature in anthocyanins.
The yellowish counterpart to the acyanic flavonoids,
the so-called anthoxanthins, are the betaxanthins
15
COOH
phyllocactin, isophyllocactin
[malonyl-(iso)-betanin]
N
H
hylocerenin, isohylocerenin
[3-hydroxy-3-methylglutaryl-(iso)-betanin]
Fig. 1. Predominant betaxanthins (left) and betacyanins (right) in fruits and vegetables from the Chenopodiaceae and Cactaceae.
COOH

(Kremer, 2002), the conjugates of betalamic acid with
amino acids or amines (Strack et al., 2003; Fig. 1).
Betalains in food
Besides these biochemically related distinctions, there
are also chemical divergences essential to the food chemist
and technologist. In the first place, the betalains are
more water-soluble than the anthocyanins (Stintzing,
Trichterborn, & Carle, 2006) and exhibit a tinctorial
strength up to three times higher than the anthocyanins
(Stintzing & Carle, in press). The most interesting applicative
feature, however, is the pH stability in the range
from 3 to 7 which makes betalains particularly suitable
for their application in a broad palette of low-acid and
neutral foods. Thus, betalains may be considered substitutes
for the less hydrophilic anthocyanins which under
the same conditions lose their performance through fading
and changing their tint (Stintzing & Carle, 2004).
The characteristic pigments in members from the Chenopodiaceae
and Cactaceae governing the respective
nuances are compiled in Table 1. A particular ratio of
the yellow-orange betaxanthins and the red-violet betacyanins
will determine the colour shade of the particular
plant (Delgado-Vargas, Jímenez, & Paredes-López, 2000;
Stintzing & Carle, 2004; Stintzing, Herbach, Moßhammer,
Kugler, & Carle, in press), so the broader range
of tints may be achieved by the sole presence of betalains,
irrespective of the particular pH value. While this
Table 1. Main betaxanthins (left) and betacyanins (right) in edible
fruit and vegetables from the Chenopodiaceae and Cactaceae
Chenopodiaceae
Red beet
Vulgaxanthin I Betanin
Isobetanin
Yellow beet
Vulgaxanthin I a
Swiss Chard
Vulgaxanthin I Betanin
Miraxanthin V Isobetanin
Cactaceae
Cactus pear
Indicaxanthin Betanin
Isobetanin
Purple Pitaya a,b
a Not genuinely present.
b Presence restricted to certain genotypes.
Betanin
Isobetanin
Phyllocactin
Isophyllocactin
Hylocerenin
Isohylocerenin
paintbox principle only recently demonstrated for coloured
Swiss chard petioles (Kugler, Stintzing, & Carle,
2004), cactus pears (Stintzing et al., 2005), and inflorescences
from Gomphrena globosa and Bougainvillea sp.
(Kugler et al., 2007) is comprehensible, its transfer to
food application has not been pursued with much vigour
(Stintzing & Carle, in press). Even more important, the
multiple reactions occurring during processing of betalainic
food have been scarcely understood until lately.
This may be due to the comparatively restricted number
of edible betalainic food crops known and the little related
efforts.
Analyses of betalains in food
The most straightforward approach to quantify betalains
is spectrophotometry. Nilsson (1970) established a method
for fresh red beets while their application to heat-treated
products was early questioned by Schwartz and von Elbe
(1980) who proposed a time-consuming isolation of crystalline
reference substances for quantification purposes.
Moreover, it was demonstrated that the spectrophotometric
approach would overestimate colour contents and that
HPLC would be indispensable for heat-treated samples
(Schwartz, Hildenbrand, & von Elbe, 1981). Disregarding
these findings, future studies on betalains chiefly relied
on Nilsson’s method, even when studying thermal degradation
kinetics (Herbach, Stintzing, & Carle, 2006). For
Amaranthaceae plants, Cai and Corke (1999) proposed
methods of betalain quantification, however, not considering
co-absorbing substances. Later, it was pointed out
that betalain quantification as proposed by Nilsson would
not be adoptable to cactus fruit betalains either and consequently
a new approach combining spectrophotometric and
HPLC data was suggested (Stintzing, Schieber, & Carle,
2003) which was also successfully applied to Swiss chard
petioles, red and yellow beets (Kugler, Graneis, Stintzing,
& Carle, in press).
The betalains have been reviewed earlier (Steglich &
Strack, 1990; Strack et al., 2003). Since then, the following
genuine betaxanthins (bx) have been assigned by coinjection
experiments with semi-synthesised reference
compounds and mass spectrometric support: alanine-bx
and histamine-bx, ethanolamine-bx and threonine-bx in
Swiss chard petioles (Kugler et al., in press;
Kugler et al., 2004), the methionine-bx in cactus pear
(Stintzing et al., 2005), and the arginine-, lysine- and putrescine-conjugates
in Bougainvillea sp. and G. globosa
inflorescences, respectively (Kugler et al., 2007) and
ethanolamine-bx and threonine-bx in red and yellow beets
(Kugler et al., in press). Structure elucidation of betacyanins
is more complicated than that of betaxanthins since
partial synthesis as in the case of betaxanthins is not possible
and thus co-injection experiments cannot be as easily
carried out. However, the pseudomolecular ions and particular
fragmentation patterns during mass spectrometric

analyses together with UVevis data are instructive for assignments
as demonstrated for a multitude of structures
generated upon thermal exposure of betalainic samples
(Herbach, Stintzing, et al., 2006 and refs cited therein)
or 17-decarboxy-amaranthin and various sinapoyl-adducts
in G. globosa inflorescences (Kugler et al., 2007). Inversely,
compounds with identical masses are difficult to
differentiate, because different decarboxylation sites are
conceivable (Herbach, Stintzing, et al., 2006), but also positional
and cis/trans-isomers of acylated betacyanins may
occur (Heuer et al., 1994; Kugler et al., 2007). In food
crops hitherto investigated, the situation was easier because
the structures detected turned out to be less complex
and especially aromatic acylation appeared to be a rare
event (Stintzing & Carle, 2004, in press; Strack et al.,
2003). Still, unambiguous structural evidence can only
be supplied by nuclear magnetic resonance (NMR) measurements
(Strack, Steglich, & Wray, 1993; Strack &
Wray, 1994), requiring tedious isolation and a solid experimental
set-up. Allowing for full structural assignments,
13 C NMR data are needed. Corresponding investigations
exemplified with known betacyanins from red beet and
purple pitaya as well as betaxanthins from cactus pear
and yellow Swiss chard have therefore been established
only recently (Stintzing, Conrad, Klaiber, Beifuss, &
Carle, 2004; Stintzing, Kugler, Carle, & Conrad, 2006)
and successfully applied to partially degraded betacyanins
(Wybraniec, Nowak-Wydra, & Mizrahi, 2006) thus presenting
a dependable tool for future investigations.
Colour stability
For the food technologist, maximising pigment yield
during extraction and processing is a prerequisite. Hence,
starting with a highly pigmented crop is fundamental.
Therefore, careful selection of appropriate plants and sound
technological concepts is crucial and will decide about the
success of the respective commercial commodity.
The most comprehensive data are available for red
beet. Crop colour quality was found to be affected by
the edaphic factors at the cultivation site, the date of
planting and harvest time, but was also dependent on
the respective cultivar (Stintzing, Herbach, et al., in press).
During processing, the betalains will be released from
their protective compartment and affected by multiple factors
such as the particular pH, water activity, exposure to
light, oxygen, metal ions, temperature and enzymatic activities
(Delgado-Vargas et al., 2000; Herbach, Stintzing,
et al., 2006; Stintzing & Carle, 2004). However, within
the optimal area of pH stability, temperature will be the
most decisive factor for betalain decomposition. In general,
degradation is associated with colour fading or
browning due to subsequent polymerisation, but many
more reaction pathways require consideration, some of
which have only recently been scrutinised (Herbach,
Stintzing, et al., 2006; Stintzing & Carle, in press).
Adaption of pH to about 4 has turned out to be recommendable
during red beet processing, for protein precipitation
of colloidal substances but also allowing
pasteurisation instead of sterilisation treatment with temperatures
below 100 C (Stintzing & Carle, in press).
Most important, a time of cool storage as recommended
by von Elbe and co-workers to allow regeneration of
betacyanin colour following thermal exposure has been
recognised as a prerequisite when processing beets (von
Elbe & Goldman, 2000; von Elbe, Schwartz, & Hildenbrand,
1981). While the knowledge from investigations
on beets is highly relevant to other betalainic foods, there
are also distinct differences that need to be considered and
optimised for each colour crop. The most straightforward
way is to conduct experiments with whole food matrices
because the results obtained can be readily transferred to
real-term manufacture. To understand specific degradation
mechanisms, model experiments with purified pigments
may be scrutinised afterwards.
Processing technologies for betalainic crops
Red beet
In the first place, betalains are associated with red
beet because it is not only rich in betacyanins but also
the exclusive commercially exploited betalain crop. It
was Pasch and von Elbe (1977) who proposed to substitute
synthetic colours banned by the FDA pointing out
that FD&C Red No. 2 and No. 40 exhibited half of
the tinctorial strength provided by red beet at the same
concentration level. At that time, red beet was proposed
to be included in low-acid food items such as meat and
dairy products (von Elbe, Klement, Amundson, Cassens,
& Lindsay, 1974; Pasch, von Elbe, & Sell, 1975) and
therefore techniques to process beets into juice were
also developed (Wiley & Lee, 1978; Wiley, Lee, Saladini,
Wyss, & Topalian, 1979). The main topics that
needed to be addressed were the fast browning through
polyphenoloxidase activities and the reduction of the naturally
high nitrate content. While the first was controlled
by heat inactivation and oxygen removal, the latter were
reduced by fermentation strategies (Czapski, Maksymiuk,
& Grajek, 1998; Grajek & Walkowiak-Tomczak, 1997;
Wiley & Lee, 1978). The selection of appropriate crops
with a high colour content rather than high weight was
concomitantly addressed (Ng & Lee, 1978; Nilsson,
1973; Wolyn & Gabelman, 1990). Another topic was
the unpleasant flavour of beet due to geosmin and pyrazine
derivatives that needed to be considered for commercial
red beet application, especially to sensorially
delicate foods (Murray & Bannister, 1975; Pasch &
von Elbe, 1978). Until lately, when the endogenous biosynthesis
of red beet to produce geosmin was unambiguously
proven (Lu, Edwards, Fellman, Mattinson, &
Navazio, 2003a, 2003b), it was believed that geosmin
was due to earth-bound Streptomycetes (Bentley & Meganathan,
1981; Dionigi, Millie, Spanier, & Johnson,

1992). To remove this odorant best, a membrane process
(Behr, Göbel, & Pfeiffer, 1984) or a simple destillative
removal during juice concentration is applied. Since beets
grow underground, carry-over of earth-bound germs presents
a safety issue (Stintzing & Carle, in press). Finally,
red beets are afflicted with a narrow colour spectrum
(Stintzing, Herbach, et al., in press). Thus, alternative
pigment sources have been searched for a long time.
Amaranth, Swiss chard and yellow beet
A thorough line of investigations was conducted by Cai
and co-workers in a selection programme on Amaranthaceae
plants. Besides pigment pattern characterisation and applicational
issues, the broad genetic variability of grain and
leaf amaranth was addressed (Cai, Sun, & Corke, 2005).
However, the limited colour range known from red beet
could not be extended. Hence, further edible plant sources
were addressed among which coloured Swiss chard (Kugler
et al., 2004) and also yellow beet (Kugler et al., in press;
Stintzing, Bretag, Moßhammer, & Carle, 2006; Stintzing,
Schieber, & Carle, 2002) were investigated. While pigment
yields of Swiss chard amounting to 4e8 mg/100 g stem
fresh weight stayed far behind those of common red beet
cultivars with 40e160 mg/100 g fresh weight, another forgotten
crop, i.e. the yellow beet appeared to be a promising
source to be studied further. Therefore, a process to obtain
a highly brilliant juice was developed and application to
dairy samples proved to be successful. Antioxidant addition
as well as acidification was crucial to counteract dopamine
oxidation during processing of yellow beet. Notably, blending
red and yellow beet juices offered a feasible way to
broaden the narrow colour range of red beet preparations
(Stintzing, Bretag, et al., 2006; Stintzing, Herbach, et al.,
in press).
Cactus fruits
Both being devoid of unpleasant ingredients and at the
same time offering a broad range of colour nuances, cactus
fruits appear to be the most seminal betalainic colour
crops (Stintzing & Carle, 2006) and thus have been investigated
in detail. For the first time, a thorough study to
produce highly brilliant juices from cactus pears (Opuntia
ficus-indica [L.] Mill.) was pursued. Moreover, the manufacture
of concentrates and spray-dried products was also
successful (Moßhammer, Stintzing, & Carle, 2006 and
refs cited therein). This was of notable importance because
it was earlier suspected that the simultaneous presence
of reducing sugars and free amino acids would
trigger detrimental Maillard browning during processing.
Against all odds, colour remained stable and betaxanthin
retention was admissible. Based on these promising results,
a process for red-purple pitaya (Hylocereus polyrhizus
[Weber] Britton & Rose] fruits was established with
reasonable success (Herbach, Maier, Stintzing, & Carle,
2007) presenting a solid basis for future technological
optimisation.
Structural alterations and colour changes during
processing and storage
Betacyanins
Early studies on red beets demonstrated that betanin
may degrade by hydrolytic cleavage to yield the biogenetic
precursors betalamic acid and cyclo-Dopa 5-O-glucoside
from betanin while deglucosylation yielded the
respective aglycone accompanied by a bathochromic shift
(Schwartz et al., 1981). Furthermore, betanin was found to
regenerate to a certain extent by recondensation of the hydrolysis
products associated with a colour regain after
cold storage of the heated extracts. Upon thermal exposure,
also isomerisation and decarboxylation of betanin
to yield its C15-stereoisomer isobetanin and 15-descarboxybetanin,
respectively, were observed without affecting
overall appearance (Schwartz & von Elbe, 1983; von
Elbe et al., 1981). Therefore, monitoring total betalain
contents has long been considered adequate to track pigment
loss. According to a series of most recent investigations,
this concept requires revision, because a complex
spectrum of hitherto unknown degradation products was
found (Fig. 2). These compounds were characterised by
one- or more-fold decarboxylation and/or dehydrogenation
of the genuine pigments. Dehydrogenation of betacyanins
at C-14/C-15 to yield the corresponding neo-compounds
entailed by a yellowish colour shift was unambiguously
demonstrated for betacyanins from red beet and also purple
pitaya. Even more important, decarboxylation at C-17
and/or C-2 and dehydrogenation at C-14/C-15 were found
to modify appearance and stability of the genuine pigments
(Herbach, Stintzing, et al., 2006 and refs cited
therein).
Thus it was concluded that both quantification and
colour measurements should be carried out to adequately
monitor pigment alterations caused during processing.
H
R O
C H
O
HO
2'
1'
HO
OH
O
HO
5
O
HO
H
C
19
3
2
+
N
14
15
N
H
H
C
O
O -
17
C
20
Fig. 2. Possible sites of decarboxylation (oval, dotted line), dehydrogenation
(square, solid line) and deglycosylation (circle; dotted-dashed
line) in betacyanins.
O
OH

Not being noticed by simple spectrophotometric readings,
structural pigment alterations may be accurately monitored
by high-performance liquid chromatography with diode-array
detection and mass spectrometric investigations
(Table 2): While betanin was mainly hydrolysed into
its biosynthetic precursors betalamic acid and cyclo-
Dopa 5-O-glucoside (Schwartz & von Elbe, 1983) with
concomitant fading, decarboxylation and then combined
decarboxylation/dehydrogenation reactions were the predominant
degradation paths for hylocerenin (6 0 -O-[3 00 -hydroxy-3
00 -methyl-glutaryl]-betanin) yielding a red and
a yellow-orange compound of superior stability. Phyllocactin
(6 0 -O-malonyl-betanin) afforded betanin and various
yellow and red dehydrogenated and decarboxylated
derivatives. Both for phyllocactin and especially for hylocerenin,
hydrolytic cleavage was a minor event. Thus, the
improved stability of pitaya as compared to red beet
juice upon heating found earlier was not due to the genuine
acylated betacyanins in pitaya, but rather due to the
higher stability of the heat-induced artifacts (Herbach,
Stintzing, et al., 2006; Stintzing, Herbach, et al., in
press).
The alleged contribution of the plant matrix to pigment
stability (Singer & von Elbe, 1980; Sapers & Hornstein,
1979) and the effect of pigment isolation in betalainic
preparations has been another interesting research topic.
Table 2. Indicators for assessment of process-induced changes in betalainic samples
On a quantitative basis, the betacyanins from purple pitaya
decomposed faster when isolated from the food
matrix (Herbach, Rohe, Stintzing, & Carle, 2006). The
qualitative approach was even more rewarding: decarboxylation
of betacyanins was found to be more pronounced
in the food matrix (Herbach, Rohe, et al., 2006) than in
a purified solution where hydrolytic cleavage dominated.
In addition, the respective solvent was decisive: ethanolic
solutions promoted decarboxylation at C-17, while a CO2
loss at C-2 was found to be the major event in aqueous
media (Herbach, Rohe, et al., 2006; Moßhammer, Rohe,
Stintzing, & Carle, 2007; Wybraniec, 2005). These findings
demonstrated that not only the extent of pigment
loss, but also the degradation path would be clearly determined
by the presence and nature of the accompanying
matrix. It is suspected that a selective adsorption process
to pectins or proteins of the matrix will alter mobility
of the ingredients and their mutual interactions. However,
the exact mechanism underlying these observations remains
to be clarified.
Betaxanthins
The betaxanthins have received little attention as they
constitute only minor pigments in red beet and have therefore
been addressed much less. Betalainic food crops dominated
by betaxanthins are yellow Swiss chard, yellow beet,
Parameter Colour change Spectrophotometric assessment HPLC assessment Applicable to
Total betalain content þ þ All betalainic samples
Betaxanthin/betacyanin ratio
, hypsochromic þ þ All betalainic samples
(colour shade)
or bathochromic shift
Betanin/isobetanin ratio þ All betacyanin
containing samples
Betanin/vulgaxanthin I ratio , hypsochromic þ þ Red beet
or bathochromic shift
samples, Swiss
chard samples
Betanin/indicaxanthin ratio , hypsochromic
or bathochromic shift
þ þ Cactus pear samples
Betanin/phyllocactin ratio þ Purple pitaya samples
14,15-Dehydrogenated betacyanin þ, hypsochromic shift þ, pretends
þ All betacyanin
betaxanthin presence
containing samples
2,3-Dehydrogenated betacyanin þ All betacyanin
containing samples
2-Decarboxybetacyanin þ All betacyanin
containing samples
15-Decarboxybetacyanin þ All betacyanin
containing samples
17-Decarboxybetacyanin , hypsochromic shift þ All betacyanin
containing samples
Deglycosylation , bathochromic shift þ þ All betacyanin
containing samples
Indicaxanthin þ, hypsochromic shift þ, if original
þ Cactus pear
sample does not contain
samples, purple
this compound
pitaya samples
Isoindicaxanthin þ Cactus pear samples
Indicaxanthin/isoindicaxanthin-ratio þ Cactus pear samples
þ, Possible; , not possible; and , possible, if present in considerable quantities.

ut also yellow-orange cactus fruits (Kugler et al., 2004;
Stintzing & Carle, in press; Stintzing, Herbach, et al., in
press).
The colour variability of genuine betaxanthins is quite
narrow (Stintzing, Herbach, et al., in press) and heatinduced
chemical changes are little understood. Mainly
based on findings from red beet, the yellow betalains
are considered less stable than their red counterparts (Herbach,
Stintzing, et al., 2006). Most recent investigations
on cactus pear juices demonstrated that isomerisation of
the main compound indicaxanthin will be induced by thermal
exposure. Most interestingly, the isomer ratio of the
main cactus pear betalain indicaxanthin turned out to be
a useful parameter to retrospectively calculate the initial
pigment content (Moßhammer, Maier, Stintzing, & Carle,
2006). Moreover, de novo formation of indicaxanthin by
spontaneous condensation of betalamic acid released
upon thermal exposure and the free amino compound proline
from the juice matrix was observed (Herbach, Rohe,
et al., 2006).
Improvement of betalain stability during processing
and storage
Purple pitaya
To prove the suitability of purple pitaya for commercial
exploitation, colour and pigment analyses during processing
and storage were monitored with juices from purple pitaya.
Since early studies on red beet had shown that betalain
regeneration after processing increased overall colour retention
(Czapski, 1985; von Elbe et al., 1981), the betalain
content development of the obtained juices was registered
over 72 h at 4 C. Completion of betacyanin regeneration
was found after 24 h and was considered crucial to maximise
pigment yield. While a gain of 3% was found for
unheated juice, up to 10% colour was regenerated in
heat-treated samples (Herbach, Stintzing, et al., 2006 and
refs cited therein). As earlier reports concerning the stabilising
effects of common food additives were found to be
contradictory, organic acids such as citric, ascorbic and isoascorbic
acids were added to juices and pigment preparations
from 0.1 to 1% prior to heating (Herbach, Rohe,
et al., 2006). Although pigment regeneration and stabilisation
differed between pH 4 and pH 6, the study focused on
pH 4 being relevant for industrial processes. Noteworthy,
purified pigment samples devoid of matrix were less effectively
stabilised than unpurified juice samples and a dosage
of 1% ascorbic acid was found to significantly reduce betacyanin
degradation (Herbach, Rohe, et al., 2006; Herbach,
Stintzing, et al., 2006). After high temperature-short time
(HTST) treatment at semi-industrial scale, up to two-thirds
of the initial betacyanin content were retained. Hence, pitaya
juice processing was considered feasible if adequate
stabilisation measures were applied to strongly enhance
overall pigment yield.
After 6 months of storage under light or in the dark,
70% of the initial betacyanins remained intact when
ascorbic acid was applied. In contrast, pigment losses
amounting to 60 and 90% upon dark and light storage
at 20 C, respectively, were registered without ascorbic
acid addition (Herbach et al., 2007). Besides quantitative
data, qualitative colour alterations could be readily
monitored by the DE*-value comprising all chromatic
parameters, i.e. lightness L*, green-redness a*, and blueyellowness
b*.
Yellow-orange cactus pear
In agreement with previous findings (e.g., Havlíková,
Míková, & Kyzlink, 1983; Singer & von Elbe, 1980;
von Elbe, Maing, & Amundson, 1974), organic acids
slowed down betalain degradation upon thermal exposure.
Betaxanthins were considerably more stable at pH 6 as
opposed to pH 4, whereas the pH stability of betacyanins
depended on the respective acid applied. The most promising
results were obtained with 0.1% isoascorbic acid at
pH 4 and 0.1% citric acid at pH 6 (Moßhammer et al.,
2007). At pH 4, half-life values for indicaxanthin were increased
by 0.1% isoascorbic acid dosage from 78.8 min to
126.6 min, 31.4 min to 46.5 min, and 13.4 min to 21.7 min
at 75 C, 85 C and 95 C, respectively (Moßhammer,
Stintzing, et al., 2006). Moreover, stabilisation of betalain
preparations devoid of matrix constituents was less effective
compared to juices. Hence, a matrix index was introduced
to express the potential of various organic acids to
improve pigment stability (Moßhammer et al., 2007). Finally,
regeneration of betaxanthins not considered earlier
was found to be an important factor in maximising pigment
yield. Betaxanthin regeneration without additive
was better at pH 6 amounting to 6% compared to only
2.5% at pH 4 without additive, while overall colour retention
was best by addition of 0.1% isoascorbic acid at pH 4
or 0.1% citric acid at pH 6 prior to heating (Moßhammer
et al., 2007).
Upon cactus pear juice processing at pilot-plant scale,
vulgaxanthin I being the predominant compound in red
beet and Swiss chard was found to be less stable than indicaxanthin
(Moßhammer et al., 2007). This lent support to
the fact that cactus pear fruits may be regarded as promising
betalain colour crops.
During a 6-month storage, pigments were again best
protected, if juices were stabilised with 0.1% isoascorbic
acid. The most notable change was registered during the
first month, being less pronounced afterwards. Half-life
values of betaxanthins and betacyanins were around 1
month without additive and could be prolonged to 2.6
and 3.6 months by 0.1% isoascorbic acid addition. The
effect of stabilisation was less pronounced during storage
under light storage when half lives of 0.8 and 1.3 months
were achieved for betaxanthins and betacyanins, respectively
(Moßhammer et al., 2007). These studies on purple
pitaya and orange-yellow cactus pear demonstrated that
the choice of the adequate additive at the proper concentration
will depend both on pigment type (betacyanins and

etaxanthins) and the composition of the respective betalain
source (Herbach, Rohe, et al., 2006; Moßhammer,
Stintzing, et al., 2006). Consequently, the stabilisation strategy
needs to be adjusted for each commodity.
Quality control of betalainic food
Markers for processed betalain samples
Suitable markers to identify particular pigment changes
appear to be valuable for food processors. While the total
betalain colour content and also the particular betaxanthin/
betacyanin ratio, i.e. colour shade have hitherto been
exclusively used for quality assessment, further parameters
may be instrumental, especially to provide evidence of process-induced
alterations. To allow a consistent statement,
knowledge of the genuine pigment pattern of the particular
food source is required first. The parameters as compiled in
Table 2 may be expedient: a higher ratio of isomerisation
in the betalamic acid part is generally associated with extended
heat exposure and storage. Dehydrogenation and decarboxylation
are profound markers for heat exposure, while
deglycosylation presents an indicator for insufficient heat inactivation
of the plant’s b-glucosidase activity and/or fermentation
(Czy_zowska, Klewicka, & Libudzisz, 2006;
Herbach, Rohe, et al., 2006; Herbach, Stintzing, et al.,
2006; Herbach et al., 2007; Moßhammer, Stintzing, et al.,
2006).
Differentiation of purple pitaya genotypes
Up to now, pitayas (Hylocereus sp.) were subject to
cultivation and hybridisation experiments to improve fruit
quality (Le Bellec, Vaillant, & Imbert, 2006; Nerd, Gutman,
& Mizrahi, 1999; Raveh, Weiss, Nerd, & Mizrahi,
1993; Tel-Zur, Abbo, Bar-Zvi, & Mizrahi, 2004; Wybraniec
& Mizrahi, 2002). The most interesting purple pitaya
(H. polyrhizus [Weber] Britton & Rose) has been parallelly
investigated for its pigment pattern being composed
of both acylated and nonacylated pigments; and is considered
a viable source for food colouring (Stintzing,
Schieber, et al., 2003; Wybraniec & Mizrahi, 2002).
Since no reliable information on pitaya genotypes of
the Latin American flora where the fruits originally stem
from was available, dependable parameters for their differentiation
were required. Therefore, specific betacyanin
fingerprints of the five Hylocereus genotypes ‘Lisa’, ‘Orejona’,
‘Nacional’, ‘Rosa’, and ‘San Ignacio’ were assessed
(Esquivel, Stintzing, & Carle, in press-a). While individual
ratios of the main pigments were not consistently meaningful,
the ratio of acylated and nonacylated compounds
ranging from 0.9 to 5.6 appeared to be a more worthwhile
parameter (Esquivel et al., in press-a). Considering the
higher stability of heat-induced artefacts from acylated
rather than nonacylated betacyanins (Herbach, Stintzing,
et al., 2006), these data promise a high applicational
value. Noteworthy, the presence of neobetanin, earlier reported
to be not present in pitaya fruits, appeared to be
a valuable tool for genotype differentiation. Another
hitherto unknown betalain in pitaya was indicaxanthin,
that was otherwise found as artifact in heated pitaya juice
samples (Herbach, Stintzing, et al., 2006). Continuing
studies need to prove the abovementioned findings for
their consistency with respect to year of harvest and fruit
maturity.
Detection of red beet admixtures to purple pitaya
Despite their differing pigment pattern, verification of
purple pitaya adulteration with red beet preparations
turned out to be difficult, due to the co-occurrence of
betanin and isobetanin. To afford a reliable distinction
between products based on red beet or cactus fruits,
authenticity control of betalainic preparations with the
aim to identify admixtures of inexpensive red beet to
high-priced pitaya extracts is required. Thus, carbon and
hydrogen isotope ratios of the purified pigments for the
unambiguous discrimination of cactus (CAM plant) and
red beet (C 3 plant) were acquired (Herbach, Stintzing,
Elss, et al., 2006). Because of different CO2 fixation
mechanisms with C3 plants being more depleted in the
heavy 13 C isotope (Winkler & Schmidt, 1980), differentiation
was possible yielding d 13 Cv-PDB-values of 17 to
18 and 27 to 28 for betanin and isobetanin from pitaya
and red beet, respectively. Although CAM plants
should exhibit a greater tendency for deuterium enrichment
if grown under identical conditions (Ting, 1985), hydrogen
isotope equilibria are subject to a range of
metabolic events (Hobbie & Werner, 2004; Schmidt,
2003; Schmidt & Kexel, 1998) and will also depend on
climatic conditions (Martin & Martin, 2003). Hence,
d 2 Hv-SMOW were not meaningful by themselves but supported
the d 13 C-data when plotted in a correlation chart
(Herbach, Stintzing, Elss, et al., 2006). Since d 13 C-values
of betanin and isobetanin were found to be identical, separation
of betanin and isobetanin was not required when
whole samples were addressed. Based on an equivalent total
soluble solid basis, an addition of 6% red beet juice to
purple pitaya could be detected. Future investigations and
extension to a broader set of samples will have to substantiate
these findings.
Admixtures of betalains to anthocyanin preparations
To improve the colouring strength of anthocyanin
preparations at near neutral pH regimes, commixing
with red beet appears to be tempting. Therefore, a method
to discover red beet addition was required to secure authenticity
of the particular anthocyanin preparation. Due
to the similar absorption maxima of anthocyanins and betalains,
mere spectrophotometric readings would not allow
to judge if blends were present. Since betalains
and anthocyanins are mutually exclusive (Stintzing &
Carle, 2004), betalain detection in anthocyanic preparations
is an unambiguous proof of admixtures. While an
earlier attempt was intended to roughly differentiate between
an early betalain and a late anthocyanin eluting

fraction (IFU, 1998), a thorough HPLCeMS method for
simultaneous assessment of betalains and both acylated
and nonacylated anthocyanins proved viable for a number
of commercial extracts and was feasible for routine application
to detect red beet addition to anthocyanin-based
fruit or vegetable preparations (Stintzing, Trichterborn,
et al., 2006).
Future challenges in betalain research
Since markets are increasingly oriented towards natural
colourants, extension of the well-established range of fruit
and vegetable preparations is required. Moreover, the current
colour market demands a high degree of diversification.
Besides the chemical stability, a high tinctorial
strength and constancy in appearance within a broad pH
range presents an important criterion. Coloured extracts
are preferred over purified colours because declaration
of the former allows clean labeling. In this respect, the
betalains deserve intense research as they offer hues
and stability characteristics uncommon to anthocyanic
sources.
Horticultural aspects for the improvement of colour
crop quality
From the studies on Swiss chard, it became obvious that
their use on a future colourant market would not be
competitive (Kugler et al., 2004). Knowledge on pigmented
Swiss chard is generally quite fragmentary and thus breeding
and horticultural studies should be enforced to improve
pigment quantity per crop (Stintzing, Herbach, et al., in
press).
Despite their favourite properties, the main drawback to
introduce cactus fruits as common colour crops is their limited
availability and the little efforts hitherto dedicated to
improve specific properties. Because of their high genetic
variability (Chessa & Nieddu, 2002; Felker et al., 2005;
Mizrahi, Nerd, & Nobel, 1997), cactus pears (Opuntia
spp.) appear to be a predestined target though. Preliminary
data from differently coloured cactus pear clones were
promising (Stintzing et al., 2005) and future investigations
will have to address selected cactus fruits with respect to
colour shade, pigment and juice yield both for the fresh
market but also for fruit manufacture.
Technological tasks for maximising yield during cactus
juice production
Despite promising investigations to produce cactus pear
(Moßhammer, Maier, et al., 2006) or pitaya juices (Herbach
et al., 2007), further process optimisation is warranted. The
main obstacle is the pectic substances that need to be degraded
more effectively to facilitate pigment release and allow
improved filtration thus further increasing yield and
reducing processing wastes at the same time. To achieve
this goal, the mucilage composition of both cactus pears
and pitayas needs to be characterised. In addition, the
production design should be extended to the exploitation
of the processing residues to improve the overall economic
balance, such as seed extraction for oil recovery. In this
line, some prospects for a more thorough utilisation together
with current and future uses of cactus pears (Moßhammer,
Maier, et al., 2006) may help scientist to figure
out the most urgent tasks to pursue in their specific field
of interest.
Quality assessment of betalainic preparations
How betalains change their properties when added to
food to improve appearance has not been studied systematically.
In this regard, interactions with the food matrix
need to be addressed because pigments may change and
affect overall appearance. The food matrix may be beneficial
in stabilising pigments, but may also be deleterious
if the expected colour of the food is impaired through enzymatic
and nonenzymatic browning reactions (Moßhammer,
Maier, et al., 2006; Stintzing, Herbach, et al., in
press). Hence, detailed knowledge of the food composition
is required. Moreover, systematic studies on the
pigment composition underlying colour blends from betaxanthins
and betacyanins both in edible and non-edible
plants are needed (Stintzing, Herbach, et al., in press).
Based on these findings the prospective calculation and
exact adjustment of tailor-made hues through blending
of betalainic fruit and vegetable juices as exemplified
for cactus and beet juices are made possible (Moßhammer,
Stintzing, & Carle, 2005; Stintzing, Herbach, et al.,
in press; Stintzing, Kugler, et al., 2006) and simplify adaption
to industrial manufacture.
Robust analytical HPLCeDADeMS techniques allow
to monitor changes induced by processing of betalainic
fruit and vegetables. Selected compounds and pigment
profiles may be valuable markers to assess the heat burden
a particular food has undergone. However, the lack
of commercially available reference substances complicates
analyses, especially with respect to quantitative
determination. Results from thermostability studies on
betacyanins allude to the fact that dehydrogenated and
decarboxylated betacyanins present useful markers to retrospectively
assess intensity and duration of heat processing.
Moreover, specific pigments and pigment ratios may
help to obtain an idea about the possible origin and the
processing technologies applied (Table 2). Continuing
studies will have to substantiate these parameters for
routine application. With an increase of betalaincontaining
preparations on the market, their origin and
authenticity need to be secured. Common techniques
such as UV, near- and mid-infrared, visible and Raman spectroscopies,
electronic nose, polymerase chain reactions,
enzyme-linked immunosorbent assays or thermal analyses,
chromatographic and isotopic analyses are widespread
(Fügel, Carle, & Schieber, 2005; Reid, O’Donnell, &
Downey, 2006). Preliminary steps have been taken by isotope
ratio differentiation based on typical betacyanin pigments
(Herbach, Stintzing, Elss, et al., 2006). These

pioneering studies should be extended to other samples, i.e.
red beet addition to red-purple cactus pear exhibiting the
same pigment pattern thus establishing a comprehensive
data basis of fruits and vegetables from different provenances.
To assure quality and unveal adulteration, further
chemical parameters should be included such as the amino
or phenolic compound spectra (Esquivel, Stintzing, & Carle,
in press-b; Kugler, Graneis, Schreiter, Stintzing, & Carle,
2006).
In summary, there is a bunch of colourful analytical
challenges to be addressed in future betalain research.
The enormous potential of plant breeding to improve
the pigment crop quality and quantity has only been realised
for red beet, but not fully considered for others.
It is up to all disciplines dealing with the food chain
to join their forces to fully exploit the scientific and applicative
potential of betalains, including nutritional
implications.
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