betalains – emerging prospects for food scientists

Trends in Food Science & Technology 18 (2007) 514e525

Betalains e emerging

prospects for

food scientists

Florian C. Stintzing* andReinhold 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: [email protected])

Betalains have witnessed swayings of scientific interest in the

past 40 years, but only during the past decade research activ-

ities in many disciplines dealing with breeding, phytochemi-

cal, technological and nutritional aspects have broadened

the hitherto narrow view on betalains. The challenge of bring-

ing together the knowledge from all these different fields of ex-

pertise is considered to be most fruitful. In the present review,

the focus will be on the technologically related analytical

issues.

IntroductionAs opposed to other pigment classes such as the caroten-

oids, chlorophylls and anthocyanins, the betalains havebeen studied with much less intensity. According to litera-ture, betalains have experienced peaks of scientific atten-tion in the 1960s and 1970s through the impressivephytochemical contributions by Piattelli (1976) in Italy,Dreiding (1961) and Wyler (1969) in Switzerland, Clement,Mabry, Wyler, and Dreiding (1994) and Mabry (1966) inUSA, as well as Musso (1979) and Reznik (1975) inGermany. Technological and also nutritional issues wereconsidered in pioneering studies by von Elbe and Goldman(2000) in USA in the 1970s and 1980s, which were the cat-alyst for an extensive breeding programme for red beetsconducted by Gabelman and later Goldman (Gaertner &Goldman, 2005; Goldman & Navazio, 2003). In the1990s research activities were mainly dedicated to

* Corresponding author.

0924-2244/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2007.04.012

Review

biosynthetic aspects both at the Leibniz Institute of PlantBiochemistry in Halle (Saale)/Germany (Strack, Vogt, &Schliemann, 2003) and at the Laboratory of Cellular Phyto-genetics at Lausanne/Switzerland (Zryd & Christinet,2004).

With a focus on food, the scarce attention towards beta-lains may be due to the fact that red beet has long been con-sidered the only edible betalainic source. In the past five toten years, however, leaf and grain amaranth, cactus fruits,but also coloured Swiss chard and yellow beet have stimu-lated food scientists to study betalains from a technologicaland nutritional perspective (Stintzing & Carle, 2004, inpress). The present overview will discuss selected featuresof betalain chemistry and their importance to foodscientists.

Betalains e a bunch of colourful structuresTo date, the betalains comprise a quite modest number

of about 55 structures including the red-violet betacya-nins and the yellow-orange betaxanthins (Stintzing &Carle, in press), while up to 550 anthocyanins havebeen identified in nature thus far (Andersen & Jordheim,2006). Although not yet being clarified, the co-occurringbetacyanin C15-stereoisomers are mainly considered isola-tion artifacts. In contrast, the analogous C11-isomers forthe betaxanthins have not yet been detected as genuinecompounds. Despite this still small number of structures,which is expected to grow, betalains are a matter of fas-cination. In early days erroneously addressed as flavo-cyanins (betaxanthins) and nitrogenous anthocyanins(betacyanins), it was Mabry and Dreiding (1968) whocoined the term ‘‘betalain’’ for both pigment types.Only slightly earlier, betanin from red beet was the firstbetacyanin (Wyler, Mabry, & Dreiding, 1963) and indi-caxanthin from cactus pear the first yellow-orange betax-anthin structurally characterised (Piattelli, Minale, &Prota, 1964; Fig. 1). Although the substitution patternof betacyanins with respect to sugars and additional acyl-ation resembles part of the structural design of anthocy-anins, distinct differences exist. The uniqueness ofbetalains is their N-heterocyclic nature with betalamicacid being their common biosynthetic precursor. Aldi-mine formation with cyclo-Dopa yields the betanidinaglycone which is usually conjugated with glucose andsometimes additionally with glucuronic acid, and mayalso be further modified through aliphatic and aromatic

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515F.C. Stintzing, R. Carle / Trends in Food Science & Technology 18 (2007) 514e525

N

H

HOOC

+ COO-

HO

O

15

OHO

HO

C

H

HO

OH

N COOHH

H

H COOHHOOC

N

H+ COO-

HO

O

15

OHO

HO

C

H

HO

OH

N

CH3 O

OH

CH2HOOC3"

6'

2' 1' 5

C CCH2

betanin, isobetanin

HO

O

C

O

C

N

H

HOOC COOH

+ COO-

HO

O

15

H

OHO

HO

C

H

HO

OH

N

phyllocactin, isophyllocactin

[malonyl-(iso)-betanin]

hylocerenin, isohylocerenin

[3-hydroxy-3-methyl-

glutaryl-(iso)-betanin]

vulgaxanthin I

[glutamine-betaxanthin]

miraxanthin V

[dopamine-betaxanthin]

HO

OH

H +N

COOH11

H

H

HOOC N

11

indicaxanthin

[proline-betaxanthin]

NH

COOHHOOC

H

N+

COO-

H

H2N

O

+

H

COO-H

N

NH COOHHOOC

H11

Fig. 1. Predominant betaxanthins (left) and betacyanins (right) in fruits and vegetables from the Chenopodiaceae and Cactaceae.

acid esterifications. In comparison with the anthocyanins(Andersen & Francis, 2004), a much smaller number ofsubstituents have been reported for the betalains: glucose,glucuronic acid and apiose are the typical sugar mono-mers, while malonic and 3-hydroxy-3-methyl-glutaricacids as well as caffeic-, p-coumaric, and ferulic acidsrepresent typical acid substituents (Strack et al., 2003).

Noteworthy, sinapic acid has been rarely reported forthe betalains (Kugler, Stintzing, & Carle, 2007; Wybra-niec, Nowak-Wydra, Mitka, Kowalski, & Mizrahi,2007), while inversely 3-hydroxy-3-methyl-glutaric acidhas never been found as a structural feature in anthocy-anins. The yellowish counterpart to the acyanic flavo-noids, the so-called anthoxanthins, are the betaxanthins

516 F.C. Stintzing, R. Carle / Trends in Food Science & Technology 18 (2007) 514e525

(Kremer, 2002), the conjugates of betalamic acid withamino acids or amines (Strack et al., 2003; Fig. 1).

Betalains in foodBesides these biochemically related distinctions, there

are also chemical divergences essential to the food chem-ist and technologist. In the first place, the betalains aremore water-soluble than the anthocyanins (Stintzing,Trichterborn, & Carle, 2006) and exhibit a tinctorialstrength up to three times higher than the anthocyanins(Stintzing & Carle, in press). The most interesting appli-cative feature, however, is the pH stability in the rangefrom 3 to 7 which makes betalains particularly suitablefor their application in a broad palette of low-acid andneutral foods. Thus, betalains may be considered substi-tutes for the less hydrophilic anthocyanins which underthe same conditions lose their performance through fad-ing and changing their tint (Stintzing & Carle, 2004).The characteristic pigments in members from the Cheno-podiaceae and Cactaceae governing the respectivenuances are compiled in Table 1. A particular ratio ofthe yellow-orange betaxanthins and the red-violet beta-cyanins will determine the colour shade of the particularplant (Delgado-Vargas, Jımenez, & Paredes-Lopez, 2000;Stintzing & Carle, 2004; Stintzing, Herbach, Moßham-mer, Kugler, & Carle, in press), so the broader rangeof tints may be achieved by the sole presence of beta-lains, irrespective of the particular pH value. While this

Table 1. Main betaxanthins (left) and betacyanins (right) in ediblefruit and vegetables from the Chenopodiaceae and Cactaceae

ChenopodiaceaeRed beet

Vulgaxanthin I BetaninIsobetanin

Yellow beetVulgaxanthin I a

Swiss ChardVulgaxanthin I BetaninMiraxanthin V Isobetanin

CactaceaeCactus pear

Indicaxanthin BetaninIsobetanin

Purple Pitayaa,b

BetaninIsobetaninPhyllocactinIsophyllocactinHylocereninIsohylocerenin

a Not genuinely present.b Presence restricted to certain genotypes.

paintbox principle only recently demonstrated for col-oured Swiss chard petioles (Kugler, Stintzing, & Carle,2004), cactus pears (Stintzing et al., 2005), and inflores-cences from Gomphrena globosa and Bougainvillea sp.(Kugler et al., 2007) is comprehensible, its transfer tofood application has not been pursued with much vigour(Stintzing & Carle, in press). Even more important, themultiple reactions occurring during processing of beta-lainic food have been scarcely understood until lately.This may be due to the comparatively restricted numberof edible betalainic food crops known and the little re-lated efforts.

Analyses of betalains in foodThe most straightforward approach to quantify betalains

is spectrophotometry. Nilsson (1970) established a methodfor fresh red beets while their application to heat-treatedproducts was early questioned by Schwartz and von Elbe(1980) who proposed a time-consuming isolation of crys-talline reference substances for quantification purposes.Moreover, it was demonstrated that the spectrophotometricapproach would overestimate colour contents and thatHPLC would be indispensable for heat-treated samples(Schwartz, Hildenbrand, & von Elbe, 1981). Disregardingthese findings, future studies on betalains chiefly reliedon Nilsson’s method, even when studying thermal degrada-tion kinetics (Herbach, Stintzing, & Carle, 2006). ForAmaranthaceae plants, Cai and Corke (1999) proposedmethods of betalain quantification, however, not consider-ing co-absorbing substances. Later, it was pointed outthat betalain quantification as proposed by Nilsson wouldnot be adoptable to cactus fruit betalains either and conse-quently a new approach combining spectrophotometric andHPLC data was suggested (Stintzing, Schieber, & Carle,2003) which was also successfully applied to Swiss chardpetioles, 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 follow-ing genuine betaxanthins (bx) have been assigned by co-injection experiments with semi-synthesised referencecompounds and mass spectrometric support: alanine-bxand histamine-bx, ethanolamine-bx and threonine-bx inSwiss 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 pu-trescine-conjugates in Bougainvillea sp. and G. globosainflorescences, respectively (Kugler et al., 2007) andethanolamine-bx and threonine-bx in red and yellow beets(Kugler et al., in press). Structure elucidation of betacya-nins is more complicated than that of betaxanthins sincepartial synthesis as in the case of betaxanthins is not pos-sible and thus co-injection experiments cannot be as easilycarried out. However, the pseudomolecular ions and par-ticular fragmentation patterns during mass spectrometric


analyses together with UVevis data are instructive for as-signments as demonstrated for a multitude of structuresgenerated upon thermal exposure of betalainic samples(Herbach, Stintzing, et al., 2006 and refs cited therein)or 17-decarboxy-amaranthin and various sinapoyl-adductsin G. globosa inflorescences (Kugler et al., 2007). In-versely, compounds with identical masses are difficult todifferentiate, because different decarboxylation sites areconceivable (Herbach, Stintzing, et al., 2006), but also po-sitional and cis/trans-isomers of acylated betacyanins mayoccur (Heuer et al., 1994; Kugler et al., 2007). In foodcrops hitherto investigated, the situation was easier be-cause the structures detected turned out to be less complexand especially aromatic acylation appeared to be a rareevent (Stintzing & Carle, 2004, in press; Strack et al.,2003). Still, unambiguous structural evidence can onlybe supplied by nuclear magnetic resonance (NMR) mea-surements (Strack, Steglich, & Wray, 1993; Strack &Wray, 1994), requiring tedious isolation and a solid exper-imental set-up. Allowing for full structural assignments,13C NMR data are needed. Corresponding investigationsexemplified with known betacyanins from red beet andpurple pitaya as well as betaxanthins from cactus pearand yellow Swiss chard have therefore been establishedonly 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 pre-senting a dependable tool for future investigations.

Colour stabilityFor 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 soundtechnological concepts is crucial and will decide about thesuccess of the respective commercial commodity.

The most comprehensive data are available for redbeet. Crop colour quality was found to be affected bythe edaphic factors at the cultivation site, the date ofplanting and harvest time, but was also dependent onthe respective cultivar (Stintzing, Herbach, et al., in press).During processing, the betalains will be released fromtheir protective compartment and affected by multiple fac-tors such as the particular pH, water activity, exposure tolight, oxygen, metal ions, temperature and enzymatic ac-tivities (Delgado-Vargas et al., 2000; Herbach, Stintzing,et al., 2006; Stintzing & Carle, 2004). However, withinthe optimal area of pH stability, temperature will be themost decisive factor for betalain decomposition. In gen-eral, degradation is associated with colour fading orbrowning due to subsequent polymerisation, but manymore reaction pathways require consideration, some ofwhich 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 recom-mendable during red beet processing, for protein precipi-tation of colloidal substances but also allowingpasteurisation instead of sterilisation treatment with tem-peratures below 100 �C (Stintzing & Carle, in press).Most important, a time of cool storage as recommendedby von Elbe and co-workers to allow regeneration ofbetacyanin colour following thermal exposure has beenrecognised as a prerequisite when processing beets (vonElbe & Goldman, 2000; von Elbe, Schwartz, & Hilden-brand, 1981). While the knowledge from investigationson beets is highly relevant to other betalainic foods, thereare also distinct differences that need to be considered andoptimised for each colour crop. The most straightforwardway is to conduct experiments with whole food matricesbecause the results obtained can be readily transferred toreal-term manufacture. To understand specific degradationmechanisms, model experiments with purified pigmentsmay be scrutinised afterwards.

Processing technologies for betalainic cropsRed beet

In the first place, betalains are associated with redbeet because it is not only rich in betacyanins but alsothe exclusive commercially exploited betalain crop. Itwas Pasch and von Elbe (1977) who proposed to substi-tute synthetic colours banned by the FDA pointing outthat FD&C Red No. 2 and No. 40 exhibited half ofthe tinctorial strength provided by red beet at the sameconcentration level. At that time, red beet was proposedto be included in low-acid food items such as meat anddairy products (von Elbe, Klement, Amundson, Cassens,& Lindsay, 1974; Pasch, von Elbe, & Sell, 1975) andtherefore techniques to process beets into juice werealso developed (Wiley & Lee, 1978; Wiley, Lee, Salad-ini, Wyss, & Topalian, 1979). The main topics thatneeded to be addressed were the fast browning throughpolyphenoloxidase activities and the reduction of the nat-urally high nitrate content. While the first was controlledby heat inactivation and oxygen removal, the latter werereduced by fermentation strategies (Czapski, Maksymiuk,& Grajek, 1998; Grajek & Walkowiak-Tomczak, 1997;Wiley & Lee, 1978). The selection of appropriate cropswith a high colour content rather than high weight wasconcomitantly addressed (Ng & Lee, 1978; Nilsson,1973; Wolyn & Gabelman, 1990). Another topic wasthe unpleasant flavour of beet due to geosmin and pyra-zine derivatives that needed to be considered for com-mercial red beet application, especially to sensoriallydelicate foods (Murray & Bannister, 1975; Pasch &von Elbe, 1978). Until lately, when the endogenous bio-synthesis of red beet to produce geosmin was unambigu-ously proven (Lu, Edwards, Fellman, Mattinson, &Navazio, 2003a, 2003b), it was believed that geosminwas due to earth-bound Streptomycetes (Bentley & Meg-anathan, 1981; Dionigi, Millie, Spanier, & Johnson,


1992). To remove this odorant best, a membrane process(Behr, Gobel, & Pfeiffer, 1984) or a simple destillativeremoval during juice concentration is applied. Since beetsgrow underground, carry-over of earth-bound germs pres-ents a safety issue (Stintzing & Carle, in press). Finally,red beets are afflicted with a narrow colour spectrum(Stintzing, Herbach, et al., in press). Thus, alternativepigment sources have been searched for a long time.

Amaranth, Swiss chard and yellow beetA thorough line of investigations was conducted by Cai

and co-workers in a selection programme on Amaranthaceaeplants. Besides pigment pattern characterisation and appli-cational issues, the broad genetic variability of grain andleaf amaranth was addressed (Cai, Sun, & Corke, 2005).However, the limited colour range known from red beetcould not be extended. Hence, further edible plant sourceswere addressed among which coloured Swiss chard (Kugleret al., 2004) and also yellow beet (Kugler et al., in press;Stintzing, Bretag, Moßhammer, & Carle, 2006; Stintzing,Schieber, & Carle, 2002) were investigated. While pigmentyields of Swiss chard amounting to 4e8 mg/100 g stemfresh weight stayed far behind those of common red beetcultivars with 40e160 mg/100 g fresh weight, another for-gotten crop, i.e. the yellow beet appeared to be a promisingsource to be studied further. Therefore, a process to obtaina highly brilliant juice was developed and application todairy samples proved to be successful. Antioxidant additionas well as acidification was crucial to counteract dopamineoxidation during processing of yellow beet. Notably, blend-ing red and yellow beet juices offered a feasible way tobroaden the narrow colour range of red beet preparations(Stintzing, Bretag, et al., 2006; Stintzing, Herbach, et al.,in press).

Cactus fruitsBoth being devoid of unpleasant ingredients and at the

same time offering a broad range of colour nuances, cac-tus fruits appear to be the most seminal betalainic colourcrops (Stintzing & Carle, 2006) and thus have been inves-tigated in detail. For the first time, a thorough study toproduce highly brilliant juices from cactus pears (Opuntiaficus-indica [L.] Mill.) was pursued. Moreover, the manu-facture of concentrates and spray-dried products was alsosuccessful (Moßhammer, Stintzing, & Carle, 2006 andrefs cited therein). This was of notable importance be-cause it was earlier suspected that the simultaneous pres-ence of reducing sugars and free amino acids wouldtrigger detrimental Maillard browning during processing.Against all odds, colour remained stable and betaxanthinretention was admissible. Based on these promising re-sults, a process for red-purple pitaya (Hylocereus polyrhi-zus [Weber] Britton & Rose] fruits was established withreasonable success (Herbach, Maier, Stintzing, & Carle,2007) presenting a solid basis for future technologicaloptimisation.

Structural alterations and colour changes duringprocessing and storageBetacyanins

Early studies on red beets demonstrated that betaninmay degrade by hydrolytic cleavage to yield the bioge-netic precursors betalamic acid and cyclo-Dopa 5-O-glu-coside from betanin while deglucosylation yielded therespective aglycone accompanied by a bathochromic shift(Schwartz et al., 1981). Furthermore, betanin was found toregenerate to a certain extent by recondensation of the hy-drolysis products associated with a colour regain aftercold storage of the heated extracts. Upon thermal expo-sure, also isomerisation and decarboxylation of betaninto yield its C15-stereoisomer isobetanin and 15-descarbox-ybetanin, respectively, were observed without affectingoverall appearance (Schwartz & von Elbe, 1983; vonElbe et al., 1981). Therefore, monitoring total betalaincontents has long been considered adequate to track pig-ment loss. According to a series of most recent investiga-tions, this concept requires revision, because a complexspectrum of hitherto unknown degradation products wasfound (Fig. 2). These compounds were characterised byone- or more-fold decarboxylation and/or dehydrogenationof the genuine pigments. Dehydrogenation of betacyaninsat C-14/C-15 to yield the corresponding neo-compoundsentailed by a yellowish colour shift was unambiguouslydemonstrated for betacyanins from red beet and also pur-ple pitaya. Even more important, decarboxylation at C-17and/or C-2 and dehydrogenation at C-14/C-15 were foundto modify appearance and stability of the genuine pig-ments (Herbach, Stintzing, et al., 2006 and refs citedtherein).

Thus it was concluded that both quantification andcolour measurements should be carried out to adequatelymonitor pigment alterations caused during processing.

HO

O

C

O-

O

C

15

2

R O H

H

C

1'HO

HOO

H

5

O

HO+

H

H

N

N

OH

20

17

2'

19C

O

OH

3

14

Fig. 2. Possible sites of decarboxylation (oval, dotted line), dehydroge-nation (square, solid line) and deglycosylation (circle; dotted-dashed

line) in betacyanins.


Not being noticed by simple spectrophotometric readings,structural pigment alterations may be accurately moni-tored by high-performance liquid chromatography with di-ode-array detection and mass spectrometric investigations(Table 2): While betanin was mainly hydrolysed intoits biosynthetic precursors betalamic acid and cyclo-Dopa 5-O-glucoside (Schwartz & von Elbe, 1983) withconcomitant fading, decarboxylation and then combineddecarboxylation/dehydrogenation reactions were the pre-dominant degradation paths for hylocerenin (60-O-[300-hy-droxy-300-methyl-glutaryl]-betanin) yielding a red anda yellow-orange compound of superior stability. Phyllo-cactin (60-O-malonyl-betanin) afforded betanin and vari-ous yellow and red dehydrogenated and decarboxylatedderivatives. Both for phyllocactin and especially for hylo-cerenin, hydrolytic cleavage was a minor event. Thus, theimproved stability of pitaya as compared to red beetjuice upon heating found earlier was not due to the gen-uine acylated betacyanins in pitaya, but rather due to thehigher stability of the heat-induced artifacts (Herbach,Stintzing, et al., 2006; Stintzing, Herbach, et al., inpress).

The alleged contribution of the plant matrix to pigmentstability (Singer & von Elbe, 1980; Sapers & Hornstein,1979) and the effect of pigment isolation in betalainicpreparations has been another interesting research topic.

On a quantitative basis, the betacyanins from purple pit-aya decomposed faster when isolated from the foodmatrix (Herbach, Rohe, Stintzing, & Carle, 2006). Thequalitative approach was even more rewarding: decarbox-ylation of betacyanins was found to be more pronouncedin the food matrix (Herbach, Rohe, et al., 2006) than ina purified solution where hydrolytic cleavage dominated.In addition, the respective solvent was decisive: ethanolicsolutions promoted decarboxylation at C-17, while a CO2

loss at C-2 was found to be the major event in aqueousmedia (Herbach, Rohe, et al., 2006; Moßhammer, Rohe,Stintzing, & Carle, 2007; Wybraniec, 2005). These find-ings demonstrated that not only the extent of pigmentloss, but also the degradation path would be clearly deter-mined by the presence and nature of the accompanyingmatrix. It is suspected that a selective adsorption processto pectins or proteins of the matrix will alter mobilityof the ingredients and their mutual interactions. However,the exact mechanism underlying these observations re-mains to be clarified.

BetaxanthinsThe betaxanthins have received little attention as they

constitute only minor pigments in red beet and have there-fore been addressed much less. Betalainic food crops dom-inated by betaxanthins are yellow Swiss chard, yellow beet,

Table 2. Indicators for assessment of process-induced changes in betalainic samples

Parameter Colour change Spectrophotometric assessment HPLC assessment Applicable to

Total betalain content � þ þ All betalainic samplesBetaxanthin/betacyanin ratio(colour shade)

�, hypsochromicor bathochromic shift

þ þ All betalainic samples

Betanin/isobetanin ratio � � þ All betacyanincontaining samples

Betanin/vulgaxanthin I ratio �, hypsochromicor bathochromic shift

þ þ Red beetsamples, Swisschard samples

Betanin/indicaxanthin ratio �, hypsochromicor bathochromic shift

þ þ Cactus pear samples

Betanin/phyllocactin ratio � � þ Purple pitaya samples14,15-Dehydrogenated betacyanin þ, hypsochromic shift þ, pretends

betaxanthin presenceþ All betacyanin

containing samples2,3-Dehydrogenated betacyanin � � þ All betacyanin

containing samples2-Decarboxybetacyanin � � þ All betacyanin

containing samples15-Decarboxybetacyanin � � þ All betacyanin

containing samples17-Decarboxybetacyanin �, hypsochromic shift � þ All betacyanin

containing samplesDeglycosylation �, bathochromic shift þ þ All betacyanin

containing samplesIndicaxanthin þ, hypsochromic shift þ, if original

sample does not containthis compound

þ Cactus pearsamples, purplepitaya samples

Isoindicaxanthin � � þ Cactus pear samplesIndicaxanthin/isoindicaxanthin-ratio � � þ Cactus pear samples

þ, Possible; �, not possible; and �, possible, if present in considerable quantities.


but also yellow-orange cactus fruits (Kugler et al., 2004;Stintzing & Carle, in press; Stintzing, Herbach, et al., inpress).

The colour variability of genuine betaxanthins is quitenarrow (Stintzing, Herbach, et al., in press) and heat-induced chemical changes are little understood. Mainlybased on findings from red beet, the yellow betalainsare considered less stable than their red counterparts (Her-bach, Stintzing, et al., 2006). Most recent investigationson cactus pear juices demonstrated that isomerisation ofthe main compound indicaxanthin will be induced by ther-mal exposure. Most interestingly, the isomer ratio of themain cactus pear betalain indicaxanthin turned out to bea useful parameter to retrospectively calculate the initialpigment content (Moßhammer, Maier, Stintzing, & Carle,2006). Moreover, de novo formation of indicaxanthin byspontaneous condensation of betalamic acid releasedupon thermal exposure and the free amino compound pro-line from the juice matrix was observed (Herbach, Rohe,et al., 2006).

Improvement of betalain stability during processingand storagePurple pitaya

To prove the suitability of purple pitaya for commercialexploitation, colour and pigment analyses during process-ing and storage were monitored with juices from purple pit-aya. Since early studies on red beet had shown that betalainregeneration after processing increased overall colour re-tention (Czapski, 1985; von Elbe et al., 1981), the betalaincontent development of the obtained juices was registeredover 72 h at 4 �C. Completion of betacyanin regenerationwas found after 24 h and was considered crucial to maxi-mise pigment yield. While a gain of 3% was found forunheated juice, up to 10% colour was regenerated inheat-treated samples (Herbach, Stintzing, et al., 2006 andrefs cited therein). As earlier reports concerning the stabil-ising effects of common food additives were found to becontradictory, organic acids such as citric, ascorbic and iso-ascorbic acids were added to juices and pigment prepara-tions from 0.1 to 1% prior to heating (Herbach, Rohe,et al., 2006). Although pigment regeneration and stabilisa-tion differed between pH 4 and pH 6, the study focused onpH 4 being relevant for industrial processes. Noteworthy,purified pigment samples devoid of matrix were less effec-tively stabilised than unpurified juice samples and a dosageof 1% ascorbic acid was found to significantly reduce beta-cyanin 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-thirdsof the initial betacyanin content were retained. Hence, pit-aya juice processing was considered feasible if adequatestabilisation measures were applied to strongly enhanceoverall 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 lossesamounting to 60 and 90% upon dark and light storageat 20 �C, respectively, were registered without ascorbicacid addition (Herbach et al., 2007). Besides quantitativedata, qualitative colour alterations could be readilymonitored by the DE*-value comprising all chromaticparameters, i.e. lightness L*, green-redness a*, and blue-yellowness b*.

Yellow-orange cactus pearIn agreement with previous findings (e.g., Havlıkova,

Mıkova, & Kyzlink, 1983; Singer & von Elbe, 1980;von Elbe, Maing, & Amundson, 1974), organic acidsslowed down betalain degradation upon thermal exposure.Betaxanthins were considerably more stable at pH 6 asopposed to pH 4, whereas the pH stability of betacyaninsdepended on the respective acid applied. The most prom-ising results were obtained with 0.1% isoascorbic acid atpH 4 and 0.1% citric acid at pH 6 (Moßhammer et al.,2007). At pH 4, half-life values for indicaxanthin were in-creased by 0.1% isoascorbic acid dosage from 78.8 min to126.6 min, 31.4 min to 46.5 min, and 13.4 min to 21.7 minat 75 �C, 85 �C and 95 �C, respectively (Moßhammer,Stintzing, et al., 2006). Moreover, stabilisation of betalainpreparations devoid of matrix constituents was less effec-tive compared to juices. Hence, a matrix index was intro-duced to express the potential of various organic acids toimprove pigment stability (Moßhammer et al., 2007). Fi-nally, regeneration of betaxanthins not considered earlierwas found to be an important factor in maximising pig-ment yield. Betaxanthin regeneration without additivewas better at pH 6 amounting to 6% compared to only2.5% at pH 4 without additive, while overall colour reten-tion was best by addition of 0.1% isoascorbic acid at pH 4or 0.1% citric acid at pH 6 prior to heating (Moßhammeret al., 2007).

Upon cactus pear juice processing at pilot-plant scale,vulgaxanthin I being the predominant compound in redbeet and Swiss chard was found to be less stable than indi-caxanthin (Moßhammer et al., 2007). This lent support tothe fact that cactus pear fruits may be regarded as promis-ing betalain colour crops.

During a 6-month storage, pigments were again bestprotected, if juices were stabilised with 0.1% isoascorbicacid. The most notable change was registered during thefirst month, being less pronounced afterwards. Half-lifevalues of betaxanthins and betacyanins were around 1month without additive and could be prolonged to 2.6and 3.6 months by 0.1% isoascorbic acid addition. Theeffect of stabilisation was less pronounced during storageunder light storage when half lives of 0.8 and 1.3 monthswere achieved for betaxanthins and betacyanins, respec-tively (Moßhammer et al., 2007). These studies on purplepitaya and orange-yellow cactus pear demonstrated thatthe choice of the adequate additive at the proper concentra-tion will depend both on pigment type (betacyanins and


betaxanthins) and the composition of the respective beta-lain source (Herbach, Rohe, et al., 2006; Moßhammer,Stintzing, et al., 2006). Consequently, the stabilisation strat-egy needs to be adjusted for each commodity.

Quality control of betalainic foodMarkers for processed betalain samples

Suitable markers to identify particular pigment changesappear to be valuable for food processors. While the totalbetalain colour content and also the particular betaxanthin/betacyanin ratio, i.e. colour shade have hitherto beenexclusively used for quality assessment, further parametersmay be instrumental, especially to provide evidence of pro-cess-induced alterations. To allow a consistent statement,knowledge of the genuine pigment pattern of the particularfood source is required first. The parameters as compiled inTable 2 may be expedient: a higher ratio of isomerisationin the betalamic acid part is generally associated with ex-tended heat exposure and storage. Dehydrogenation and de-carboxylation are profound markers for heat exposure, whiledeglycosylation presents an indicator for insufficient heat in-activation of the plant’s b-glucosidase activity and/or fer-mentation (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 genotypesUp to now, pitayas (Hylocereus sp.) were subject to

cultivation and hybridisation experiments to improve fruitquality (Le Bellec, Vaillant, & Imbert, 2006; Nerd, Gut-man, & Mizrahi, 1999; Raveh, Weiss, Nerd, & Mizrahi,1993; Tel-Zur, Abbo, Bar-Zvi, & Mizrahi, 2004; Wybra-niec & Mizrahi, 2002). The most interesting purple pitaya(H. polyrhizus [Weber] Britton & Rose) has been paral-lelly investigated for its pigment pattern being composedof both acylated and nonacylated pigments; and is consid-ered a viable source for food colouring (Stintzing,Schieber, et al., 2003; Wybraniec & Mizrahi, 2002).

Since no reliable information on pitaya genotypes ofthe Latin American flora where the fruits originally stemfrom was available, dependable parameters for their dif-ferentiation were required. Therefore, specific betacyaninfingerprints of the five Hylocereus genotypes ‘Lisa’, ‘Ore-jona’, ‘Nacional’, ‘Rosa’, and ‘San Ignacio’ were assessed(Esquivel, Stintzing, & Carle, in press-a). While individualratios of the main pigments were not consistently mean-ingful, the ratio of acylated and nonacylated compoundsranging from 0.9 to 5.6 appeared to be a more worthwhileparameter (Esquivel et al., in press-a). Considering thehigher stability of heat-induced artefacts from acylatedrather than nonacylated betacyanins (Herbach, Stintzing,et al., 2006), these data promise a high applicationalvalue. Noteworthy, the presence of neobetanin, earlier re-ported to be not present in pitaya fruits, appeared to bea valuable tool for genotype differentiation. Another

hitherto unknown betalain in pitaya was indicaxanthin,that was otherwise found as artifact in heated pitaya juicesamples (Herbach, Stintzing, et al., 2006). Continuingstudies need to prove the abovementioned findings fortheir consistency with respect to year of harvest and fruitmaturity.

Detection of red beet admixtures to purple pitayaDespite their differing pigment pattern, verification of

purple pitaya adulteration with red beet preparationsturned out to be difficult, due to the co-occurrence ofbetanin and isobetanin. To afford a reliable distinctionbetween products based on red beet or cactus fruits,authenticity control of betalainic preparations with theaim to identify admixtures of inexpensive red beet tohigh-priced pitaya extracts is required. Thus, carbon andhydrogen isotope ratios of the purified pigments for theunambiguous discrimination of cactus (CAM plant) andred beet (C3 plant) were acquired (Herbach, Stintzing,Elss, et al., 2006). Because of different CO2 fixationmechanisms with C3 plants being more depleted in theheavy 13C isotope (Winkler & Schmidt, 1980), differenti-ation was possible yielding d13Cv-PDB-values of �17 to�18 and �27 to �28 for betanin and isobetanin from pit-aya and red beet, respectively. Although CAM plantsshould exhibit a greater tendency for deuterium enrich-ment if grown under identical conditions (Ting, 1985), hy-drogen isotope equilibria are subject to a range ofmetabolic events (Hobbie & Werner, 2004; Schmidt,2003; Schmidt & Kexel, 1998) and will also depend onclimatic conditions (Martin & Martin, 2003). Hence,d2Hv-SMOW were not meaningful by themselves but sup-ported the d13C-data when plotted in a correlation chart(Herbach, Stintzing, Elss, et al., 2006). Since d13C-valuesof betanin and isobetanin were found to be identical, sep-aration of betanin and isobetanin was not required whenwhole samples were addressed. Based on an equivalent to-tal soluble solid basis, an addition of 6% red beet juice topurple pitaya could be detected. Future investigations andextension to a broader set of samples will have to substan-tiate these findings.

Admixtures of betalains to anthocyanin preparationsTo improve the colouring strength of anthocyanin

preparations at near neutral pH regimes, commixingwith red beet appears to be tempting. Therefore, a methodto discover red beet addition was required to secure au-thenticity of the particular anthocyanin preparation. Dueto the similar absorption maxima of anthocyanins and be-talains, mere spectrophotometric readings would not al-low to judge if blends were present. Since betalainsand anthocyanins are mutually exclusive (Stintzing &Carle, 2004), betalain detection in anthocyanic prepara-tions is an unambiguous proof of admixtures. While anearlier attempt was intended to roughly differentiate be-tween an early betalain and a late anthocyanin eluting


fraction (IFU, 1998), a thorough HPLCeMS method forsimultaneous assessment of betalains and both acylatedand nonacylated anthocyanins proved viable for a numberof commercial extracts and was feasible for routine ap-plication to detect red beet addition to anthocyanin-basedfruit or vegetable preparations (Stintzing, Trichterborn,et al., 2006).

Future challenges in betalain researchSince markets are increasingly oriented towards natural

colourants, extension of the well-established range of fruitand vegetable preparations is required. Moreover, the cur-rent colour market demands a high degree of diversifica-tion. Besides the chemical stability, a high tinctorialstrength and constancy in appearance within a broad pHrange presents an important criterion. Coloured extractsare preferred over purified colours because declarationof the former allows clean labeling. In this respect, thebetalains deserve intense research as they offer huesand stability characteristics uncommon to anthocyanicsources.

Horticultural aspects for the improvement of colourcrop quality

From the studies on Swiss chard, it became obvious thattheir use on a future colourant market would not becompetitive (Kugler et al., 2004). Knowledge on pigmentedSwiss chard is generally quite fragmentary and thus breed-ing and horticultural studies should be enforced to improvepigment quantity per crop (Stintzing, Herbach, et al., inpress).

Despite their favourite properties, the main drawback tointroduce cactus fruits as common colour crops is their lim-ited availability and the little efforts hitherto dedicated toimprove specific properties. Because of their high geneticvariability (Chessa & Nieddu, 2002; Felker et al., 2005;Mizrahi, Nerd, & Nobel, 1997), cactus pears (Opuntiaspp.) appear to be a predestined target though. Preliminarydata from differently coloured cactus pear clones werepromising (Stintzing et al., 2005) and future investigationswill have to address selected cactus fruits with respect tocolour shade, pigment and juice yield both for the freshmarket but also for fruit manufacture.

Technological tasks for maximising yield during cactusjuice production

Despite promising investigations to produce cactus pear(Moßhammer, Maier, et al., 2006) or pitaya juices (Herbachet al., 2007), further process optimisation is warranted. Themain obstacle is the pectic substances that need to be de-graded more effectively to facilitate pigment release and al-low improved filtration thus further increasing yield andreducing processing wastes at the same time. To achievethis goal, the mucilage composition of both cactus pearsand pitayas needs to be characterised. In addition, theproduction design should be extended to the exploitation

of the processing residues to improve the overall economicbalance, such as seed extraction for oil recovery. In thisline, some prospects for a more thorough utilisation to-gether with current and future uses of cactus pears (Moß-hammer, Maier, et al., 2006) may help scientist to figureout the most urgent tasks to pursue in their specific fieldof interest.

Quality assessment of betalainic preparationsHow betalains change their properties when added to

food to improve appearance has not been studied system-atically. In this regard, interactions with the food matrixneed to be addressed because pigments may change andaffect overall appearance. The food matrix may be bene-ficial in stabilising pigments, but may also be deleteriousif the expected colour of the food is impaired through en-zymatic and nonenzymatic browning reactions (Moßham-mer, Maier, et al., 2006; Stintzing, Herbach, et al., inpress). Hence, detailed knowledge of the food composi-tion is required. Moreover, systematic studies on thepigment composition underlying colour blends from be-taxanthins and betacyanins both in edible and non-edibleplants are needed (Stintzing, Herbach, et al., in press).Based on these findings the prospective calculation andexact adjustment of tailor-made hues through blendingof betalainic fruit and vegetable juices as exemplifiedfor cactus and beet juices are made possible (Moßham-mer, Stintzing, & Carle, 2005; Stintzing, Herbach, et al.,in press; Stintzing, Kugler, et al., 2006) and simplify adap-tion to industrial manufacture.

Robust analytical HPLCeDADeMS techniques allowto monitor changes induced by processing of betalainicfruit and vegetables. Selected compounds and pigmentprofiles may be valuable markers to assess the heat bur-den a particular food has undergone. However, the lackof commercially available reference substances compli-cates analyses, especially with respect to quantitativedetermination. Results from thermostability studies onbetacyanins allude to the fact that dehydrogenated anddecarboxylated betacyanins present useful markers to ret-rospectively assess intensity and duration of heat process-ing. Moreover, specific pigments and pigment ratios mayhelp to obtain an idea about the possible origin and theprocessing technologies applied (Table 2). Continuingstudies will have to substantiate these parameters forroutine application. With an increase of betalain-containing preparations on the market, their origin andauthenticity need to be secured. Common techniquessuch as UV, near- and mid-infrared, visible and Raman spec-troscopies, electronic nose, polymerase chain reactions,enzyme-linked immunosorbent assays or thermal analyses,chromatographic and isotopic analyses are widespread(Fugel, Carle, & Schieber, 2005; Reid, O’Donnell, &Downey, 2006). Preliminary steps have been taken by iso-tope ratio differentiation based on typical betacyanin pig-ments (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 thesame pigment pattern thus establishing a comprehensivedata basis of fruits and vegetables from different prove-nances. To assure quality and unveal adulteration, furtherchemical parameters should be included such as the aminoor phenolic compound spectra (Esquivel, Stintzing, & Carle,in press-b; Kugler, Graneis, Schreiter, Stintzing, & Carle,2006).

In summary, there is a bunch of colourful analyticalchallenges to be addressed in future betalain research.The enormous potential of plant breeding to improvethe pigment crop quality and quantity has only been re-alised for red beet, but not fully considered for others.It is up to all disciplines dealing with the food chainto join their forces to fully exploit the scientific and ap-plicative potential of betalains, including nutritionalimplications.

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betalains – emerging prospects for food scientists

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