Betalains – emerging prospects for food scientists

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

Review

Betalains e emerging prospects for food scientists Florian C. Stintzing* and Reinhold Carle Institute of Food Technology, Section Plant Foodstuff Technology, Hohenheim University, August-vonHartmann-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 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. 0924-2244/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2007.04.012

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

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

515

H H

O

H

C

HO + N

H

HO

H

O

O

H

OH HO

COO-

11

15

N H

HOOC

COOH

HO

H2N

O

O

C

C

H O

H

C

HO

H

H

O O

HO

COO-

15

N H

COOH

phyllocactin, isophyllocactin [malonyl-(iso)-betanin]

O

CH3 3"

HOOC

HO

N H

HOOC

COOH

OH

CH2

C OH

CH2

C

O

HO HO

HOOC

COO-

+ N

HO

vulgaxanthin I [glutamine-betaxanthin]

H

H

OH

11

HOOC

H

COOH

betanin, isobetanin

O

+N

N H

HOOC

indicaxanthin [proline-betaxanthin]

H

COO-

+ N

+ N

6'

C

H H

O

2'

1'

O

5

OH

H + N

HO

COO-

15

11

N H

COOH

miraxanthin V [dopamine-betaxanthin]

HOOC

N H

COOH

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.

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).

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

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516

(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, Jı´menez, & Paredes-Lo´pez, 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 Miraxanthin V

Betanin Isobetanin

Cactaceae Cactus pear Indicaxanthin

Betanin Isobetanin

Purple Pitayaa,b Betanin Isobetanin Phyllocactin Isophyllocactin Hylocerenin Isohylocerenin a b

Not genuinely present. Presence restricted to certain genotypes.

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

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

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).

517

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,

518

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

1992). To remove this odorant best, a membrane process (Behr, Go¨bel, & 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

HO

C

H

O

2'

HO

1'

O

3 5

OH

+ N

HO

O

H C

HO

2

19

H

O

C O-

14 15

N H

O

17

C

20

OH

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

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

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 cycloDopa 5-O-glucoside (Schwartz & von Elbe, 1983) with concomitant fading, decarboxylation and then combined decarboxylation/dehydrogenation reactions were the predominant degradation paths for hylocerenin (60 -O-[300 -hydroxy-300 -methyl-glutaryl]-betanin) yielding a red and a yellow-orange compound of superior stability. Phyllocactin (60 -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.

519

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,

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

Colour change

Spectrophotometric assessment

HPLC assessment

Applicable to

Total betalain content Betaxanthin/betacyanin ratio (colour shade) Betanin/isobetanin ratio

 , hypsochromic or bathochromic shift 

þ þ

þ þ

All betalainic samples All betalainic samples



þ

Betanin/vulgaxanthin I ratio

, hypsochromic or bathochromic shift

þ

þ

Betanin/indicaxanthin ratio

þ

þ

Betanin/phyllocactin ratio 14,15-Dehydrogenated betacyanin

, hypsochromic or bathochromic shift  þ, hypsochromic shift

All betacyanin containing samples Red beet samples, Swiss chard samples Cactus pear samples

þ þ

2,3-Dehydrogenated betacyanin



 þ, pretends betaxanthin presence 

þ

2-Decarboxybetacyanin





þ

15-Decarboxybetacyanin





þ

17-Decarboxybetacyanin

, hypsochromic shift



þ

Deglycosylation

, bathochromic shift

þ

þ

Indicaxanthin

þ, hypsochromic shift

þ

Isoindicaxanthin Indicaxanthin/isoindicaxanthin-ratio

 

þ, if original sample does not contain this compound  

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

þ þ

Purple pitaya samples All betacyanin containing samples All betacyanin containing samples All betacyanin containing samples All betacyanin containing samples All betacyanin containing samples All betacyanin containing samples Cactus pear samples, purple pitaya samples Cactus pear samples Cactus pear samples

520

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

but 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., Havlı´kova´, ´ Mıkova´, & 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

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betaxanthins) 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

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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 (C3 plant) were acquired (Herbach, Stintzing, Elss, et al., 2006). Because of different CO2 fixation mechanisms with C3 plants being more depleted in the heavy 13C isotope (Winkler & Schmidt, 1980), differentiation was possible yielding d13Cv-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, d2Hv-SMOW were not meaningful by themselves but supported the d13C-data when plotted in a correlation chart (Herbach, Stintzing, Elss, et al., 2006). Since d13C-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

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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).

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.

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.

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 (Fu¨gel, 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

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

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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. References Andersen, O. M., & Francis, G. W. (2004). Techniques of pigment identification. In K. Davies (Ed.), Plant pigments and their manipulation. Annual Plant Reviews, 14 (pp. 293e341). Oxford/UK-Victoria/Australia: CRC Press/Blackwell Publishing. Andersen, O. M., & Jordheim, M. (2006). The anthocyanins. In O. M. Andersen, & K. R. Markham (Eds.), Flavonoids: Chemistry, biochemistry and application (pp. 471e489). Boca Raton/London/ New York: Taylor & Francis Group. Behr, N., Go¨bel, G., & Pfeiffer, H. (1984). Patentanmeldung der Fa. Henkel KGaA, Du¨sseldorf. Herstellung eines Rote-BeeteSaftkonzentrats mit besserer Geschmacksneutralita¨t und Haltbarkeit. Industrielle Obst- und Gemu¨severwertung, 59, 238e239. Bentley, R., & Meganathan, R. (1981). Geosmin and methylisoborneol biosynthesis in Streptomycetes. FEBS Letters, 125, 220e222. Cai, Y.-Z., & Corke, H. (1999). Amaranthus betacyanin pigments applied in model food systems. Journal of Food Science, 64, 869e873. Cai, Y.-Z., Sun, M., & Corke, H. (2005). Characterization and application of betalain pigments from plants of the Amaranthaceae. Trends in Food Science & Technology, 16, 370e376. Chessa, I., & Nieddu, G. (2002). Investigations on variability in the genus Opuntia as fruit crop for genetic improvement. Acta Horticulturae, 575, 345e353. Clement, J. S., Mabry, T. J., Wyler, H., & Dreiding, A. S. (1994). Chemical review and evolutionary significance of the betalains. In H.-D. Behnke, & T. S. Mabry (Eds.), Caryophyllales: Evolution and systematics, Vol. XIV (pp. 247e261). Berlin/Heidelberg: Springer (Chapter 10). Czapski, J. (1985). The effect of heating conditions on losses and regeneration of betacyanins. Zeitschrift fu¨r Lebensmittel Untersuchung und -Forschung, 180, 21e25. Czapski, J., Maksymiuk, M., & Grajek, W. (1998). Analysis of biodenitrification conditions of red beet juice using the response surface method. Journal of Agricultural and Food Chemistry, 46, 4702e4705. _ Czyzowska, A., Klewicka, E., & Libudzisz, Z. (2006). The influence of lactic acid fermentation process of red beet juice on the stability of biologically active colorants. European Food Research and Technology, 223, 110e116. Delgado-Vargas, F., Jime´nez, A. R., & Paredes-Lo´pez, O. (2000). Natural pigments: carotenoids, anthocyanins and betalains. Characteristics, biosynthesis, processing, and stability. Critical Reviews in Food Science and Nutrition, 40, 173e289.

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