Eur Food Res Technol (2007) 224: 649658 DOI 10.

1007/s00217-006-0354-5

ORIGINAL PAPER

Kirsten M. Herbach Christine Maier Florian C. Stintzing Reinhold Carle

Effects of processing and storage on juice colour and betacyanin stability of purple pitaya (Hylocereus polyrhizus) juice

Received: 12 December 2005 / Accepted: 7 April 2006 / Published online: 4 May 2006 C Springer-Verlag 2006

Abstract Purple pitaya (Hylocereus polyrhizus) fruits have been recently proposed as a promising source of betalains. In the present study, purple pitaya juice processing at pilot-plant scale is reported for the rst time. Each processing step was examined in terms of juice colour and betacyanin retention. Lightness (L ) and hue angle (h ) values were strongly inuenced by juice ltration, while chroma (C ) and betacyanin retention were most strongly altered by pasteurisation. Three systems of juice pasteurisation were evaluated. Betacyanin loss and colour alteration were minimal upon pasteurisation in an HTST system and a standard tubular heat exchanger, respectively. Additionally, storage experiments using mucilage-free purple pitaya juice were conducted. Whereas light exposure of unsupplemented samples resulted in signicant pigment degradation, detrimental effects of light exposition were completely prevented by the addition of 1% ascorbic acid prior to storage. After 6 months, about 70% of the initial betacyanin content was retained in the presence of ascorbic acid, irrespective of illumination. Keywords Purple pitaya . Hylocereus polyrhizus . Betacyanins . Stability . Juice processing . Storage . Colouring foodstuff Introduction Due to strong consumer demand for more natural products, new plant-derived colourants for the food industry are being developed [1]. Therefore, natural food colouring by application of intensively pigmented plant products is increasingly superseding articial dyes. Moreover, colour of fruit and vegetable juices is not just natural, but can add
K. M. Herbach C. Maier F. C. Stintzing ( ) R. Carle Section Plant Foodstuff Technology, Institute of Food Technology, Hohenheim University, August-von-Hartmann-Str. 3, Stuttgart 70599, Germany e-mail: stintzin@uni-hohenheim.de Tel.: +49-711-459-2318 Fax: +49-711-459-4110

some health benets and may be labelled, e.g. fruit juice for colour, thus meeting both requirements of children soliciting coloured food and confectionary as well as of health-conscious parents [2]. Betalainic foodstuff like red beet (Beta vulgaris L. ssp. vulgaris) is particularly suitable for colouring low acid food, as anthocyanins visibly degrade at pH values above 3 [3]. Nevertheless, application of red beet is restricted due to considerable nitrate levels [4] and its earthy smell caused by geosmin and pyrazine derivatives [3, 5, 6]. The search for new betalain-containing plants triggered interest in cactus fruits which are devoid of the above-mentioned disadvantages and additionally offer a broad range of different hues [3]. Several studies described the processing of cactus pears (Opuntia sp.) into fruit juices and related products [79], while the production of purple pitaya (Hylocereus polyrhizus [Weber] Britton & Rose) juice at semiindustrial scale has not been described, so far. Compared to most cactus pears, pitayas are devoid of glochids, exhibit an exceptional red-purple hue and feature signicantly higher betalain contents [10]. Thus, the colouring foodstuff portion required to achieve the desired tinctorial strength can be considerably reduced. Additionally, by blending purple pitaya and yellow-orange cactus pear juice, a broad colour range can be obtained [11]. Therefore, in this study, a process previously established for cactus pear juice [7] was adapted to purple pitayas, focusing on betalain retention and colour development during juice production. In order to monitor structural changes, HPLC-DAD analyses were performed, comparing peak areas of the characteristic betalains throughout the process. A second objective of this study was the evaluation of the storage stability of betalains in purple pitaya juice, which is a crucial factor for the application as colouring foodstuff. Since light was reported previously to accelerate betalain degradation [1214], chromatic and structural stability of betalains in purple pitaya juice was investigated under dark and light storage. Supplementation with 1% ascorbic acid has been recently reported to delay betacyanin degradation during thermal treatment of purple pitaya juice [15]. Consequently, the effect of ascorbic acid

650

addition on storage stability of pitaya betacyanins was also examined. Materials and methods Plant material For pitaya juice production at pilot-plant scale, fresh purple pitaya (Hylocereus polyrhizus [Weber] Britton & Rose) fruits were purchased from Israel in 2004. The fruits were cut in halves and peeled manually. For removal of seeds and adherent mucilaginous material, the fruit pulp was strained through a 0.4 mm sieve of a nisher (Bertuzzi Food Technology, Brugherio, Italy) at a rotational speed of 400 rpm. The strained pur e was ushed with nitrogen and frozen at e 26 C until further processing. For storage experiments, fresh purple pitaya fruits originating from Israel were purchased in 2003. Purple pitaya pulp was obtained as described above. After nishing, the fruit pulp was centrifuged at 9750 rpm for 20 min to completely remove pectin-like material. The supernatant juice was ushed with nitrogen and stored at 26 C until use. Solvents and reagents All reagents and solvents were purchased from VWR (Darmstadt, Germany) and were of analytical or HPLC grade. Deionised water was used throughout. Enzyme screening In preliminary tests, various enzyme preparations at different dosages were studied for their applicability to degrade the mucilaginous material of purple pitaya juice
Table 1 Enzyme preparations tested for purple pitaya juice processing Enzyme Dextrozym DX Dextrozym Plus L Fructozym BE Fructozym Color Fructozym Flux Fructozym MA-X-Press Fructozym TF Clear Fructozym UF Fungamyl Gammapect CPL Glucanase 5 XL-G015L Pectinase 444L-P444L Pectinex 5 XL Pectinex Smash XXL Pectinex Ultra SP-L Protease NS 33964 Rapidase Tropical Cloud Rohapect B1L

(Table 1). After enzymatic treatment at 40 C for 2 h and enzyme inactivation by heating to 90 C, juice yield after ltration at laboratory scale proved to be a viable parameter for degradation of the pectin-like material. The combined application of 1000 ppm Rohapect B1L (AB Enzymes, Darmstadt, Germany) and 1000 ppm Fructozym BE (Erbsl h Geisenheim Getr nketechnologie, o a Geisenheim, Germany) was found to be most suitable. Juice production at pilot-plant scale Production of claried purple pitaya juice was accomplished by adapting a process previously established for cactus pear juice (Fig. 1) [7]. After thawing purple pitaya pur e (see above), a part of the mucilaginous material was e removed with a decanter centrifuge (Type CA 150-01-33; rotational speed of the core drum: 6700 rpm, rotational speed difference: 6 rpm; Westfalia Separator, Oelde, Germany). Subsequently, enzymatic treatment using 1000 ppm Rohapect B1L (AB Enzymes, Darmstadt, Germany) and 1000 ppm Fructozym BE (Erbsl h Geisenheim o Getr nketechnologie, Geisenheim, Germany) was carried a out for 2 h at 40 C in a 48 l processing vessel (Type LRS III; Wolf, Lage, Germany). For enzyme inactivation, the macerated pulp was subsequently heated to 92 C and cooled to room temperature in a tubular heat exchanger (Ruland Engineering and Consulting, Neustadt, Germany). Juice ltration was carried out in a sheet lter system (Pall Seitz Schenk, Waldstetten, Germany) equipped with T2600 and K300 lter sheets (Pall Seitz Schenk Filtersystems, Bad Kreuznach, Germany). To determine the inuence of different heat treatments on purple pitaya betacyanins, the ltered juice was splitted and pasteurisation was carried out in an HTST system (Sample 6A; T = 92 C, ow rate: 100 l/h, preheating: 7 s, holding time: 26 s; Ruland Engineering and Consulting, Neustadt, Germany), in a
Tested enzyme concentrations (ppm) 1000 1000 750; 1000 100; 500 1000 100; 500 1000 100; 500 1000 1000 1000 1000 1000 500 100; 500; 1000 500; 1000 100; 500 100; 500; 750; 1000; 5000; 10000

Producer Novozymes, Dittingen, Switzerland Novozymes, Dittingen, Switzerland Erbsl h Geisenheim, Germany o Erbsl h Geisenheim, Germany o Erbsl h Geisenheim, Germany o Erbsl h Geisenheim, Germany o Erbsl h Geisenheim, Germany o Erbsl h Geisenheim, Germany o Novozymes, Dittingen, Switzerland AB Enzymes, Darmstadt, Germany Biocatalysts, Cardiff, UK Biocatalysts, Cardiff, UK Novozymes, Dittingen, Switzerland Novozymes, Dittingen, Switzerland Novozymes, Dittingen, Switzerland Novozymes, Dittingen, Switzerland DSM, Seclin, France AB Enzymes, Darmstadt, Germany

651 Fig. 1 Production scheme of purple pitaya juice at pilot-plant scale (adapted from [7])
Frozen pitaya pure

Thawing

Sample 1

Mucilaginous material

Separation

Sample 2

Enzymation

Sample 3

Enzyme inactivation

Sample 4

Filtration

Sample 5

Pasteurisation

HTST system

Tubular heat exchanger

Processing vessel

Hot filling and cooling

Hot filling and cooling

Hot filling and cooling

Pasteurised juice A

Pasteurised juice B

Pasteurised juice C

Sample 6 A

Sample 6 B

Sample 6 C

tubular heat exchanger system (Sample 6B; T = 92 C, ow rate: 120 l/h, preheating: 53 s, holding time: 26 s; Ruland Engineering and Consulting, Neustadt, Germany), and in a 10 l vapour heated double jacketed processing vessel (Sample 6C; T = 92 C, preheating: 85 min; no holding time; ESCO-Labor, Riehen, Switzerland), respectively (Fig. 1). The pasteurised juices (Samples 6AC) were bottled in 0.5 l wide-necked bottles under steam injection and cooled to room temperature. To monitor the effect of the individual processing steps on betacyanin retention and colour of the purple pitaya juice, samples were taken after each step (Fig. 1) and stored at 26 C until analysis. Juice storage For storage experiments, samples with and without the addition of 1% ascorbic acid were stored in light and darkness, respectively. Purple pitaya juice obtained by centrifugation of strained fruit pulp (see above) was used. After thawing, all juice samples were adjusted to pH 4.0 with HCl (0.1 mol/l) and NaOH (0.2 mol/l), irrespective of prior supplementation with 1% ascorbic acid. Aliquots of 8 ml were lled in test tubes, sealed with screw caps and pasteurised in a water bath at 95 C for 5 min before cooling in an ice bath for 90 s. For dark storage, samples were wrapped in aluminium foil and placed in a dark room, while illuminated test tubes were exposed to uorescent light at 4500 lx (Flu-

orescent tube L36W/21-840; Osram, Munich, Germany). For all samples, temperature was kept at 20 2 C. After 1 day and 16 months, respectively, two test tubes of each variant were sampled. An aliquot of each test tube was used for spectrophotometric analyses, while the remainder was ltered (0.45 m) and stored in a vial at 20 C until HPLC analyses. Spectrophotometric analyses All spectrophotometric analyses were performed using a UV-Vis photometer (Perkin-Elmer, Uberlingen, Germany) equipped with a UV-Vis (UVWinLab V 2.85.04) and a colour (Wincol V 2.05) software (Perkin-Elmer Instruments, Norwalk, CT, USA). Colour analyses For analysis of the samples taken during juice production, thawed purple pitaya pur e was diluted with McIlvaine e buffer (pH 6.5) until a maximum absorption of 1.00 0.05 was reached. The obtained dilution was subsequently maintained for all processing samples (Fig. 1). For examination of stored samples, the same dilution as used for the juice sample stored for 1 day at the respective conditions (light and dark, with and without ascorbic acid

652

addition, respectively) to reach a maximum absorption of 1.00 0.5 was applied. Visible spectra (380780 nm) were recorded in 1 cm pathlength disposable cuvettes. Using illuminant D65 and 10 observer angle, chroma (C ) and hue angle (h ) were calculated from CIE a and b values as follows: [C = (a2 + b2 )1/2 ] and [h = arctan(b /a )]. Total colour difference ( E ) of pitaya juice samples stored from 1 to 6 months (t) as compared to the respective sample stored for 1 day (0) was estimated according to the equation [16]: [ E = ( L 2 + C 2 + H 2 )1/2 ] with [ L = (L L )],[ C = (Ct C0 )], t 0 1/2 and [ H = (2 (sin(h t h 0 )/2) (Ct C0 ) ]. All determinations were performed in triplicate. Betacyanin quantication Maintaining the dilutions employed for colour analyses, betanin equivalents (Bc) were assessed applying the equation Bc [mg/l] = [(A F MW 1000/ l)] where A is the absorption value at max (537 nm) corrected by the absorption at 600 nm, F is the dilution factor, MW is the molecular weight of betanin (MW = 550 g/mol), is the molar extinction coefcient of betanin ( = 60,000 l/mol cm), and l is the pathlength of the cuvette [10, 17, 18]. All determinations were performed in triplicate. HPLC analyses As described previously [15, 19], HPLC analysis of purple pitaya betalains was carried out with a Merck Hitachi LaChrom Elite HPLC system (Merck, Darmstadt, Germany) consisting of a pump L-2130, an autosampler L-2200, a JetStream column oven and a diode array detector L-2450. An analytical scale Atlantis dC18 -column (250 mm 4.6 mm i.d.) with a particle size of 5 m (Waters, Wexford, Ireland) tted with a C18 ODS security guard column (4 mm 3.0 mm i.d.; Phenomenex, Torrance, CA, USA) was operated at 30 C. Solvents were 0.2% (v/v) formic acid in water (A) and MeCN (B). At a ow rate of 1 ml/min, the following gradient program was applied: 100% A isocratic for 4 min, 100% A to 93% A in 3 min, 93% A to 90% A in 17 min, 90% A isocratic for 8 min, 90% A to 85% A in 15 min, 85% A to 83% A in 15 min, and 83% A to 0% A in 8 min. Simultaneous monitoring was performed at 535 nm (betacyanins and red betacyanin degradation products) and 470 nm (betaxanthins and yellow betacyanin degradation products), respectively. Results and discussion Purple pitaya juice production at pilot-plant scale Production of purple pitaya juice at pilot-plant scale was accomplished according to a process previously developed

Table 2 Colour characteristics of purple pitaya samples at successive steps of juice processing Processing step (sample) Thawing (1) Separation (2) Enzymation (3) Enzyme inactivation (4) Filtration (5) Pasteurisation (6) HTST system (A) Tubular heat exchanger (B) Processing vessel (C) L 66.2 1.6 61.3 0.9 66.2 0.3 66.0 0.7 74.5 0.3 C 49.7 1.7 51.5 0.7 48.5 0.2 47.2 1.1 51.5 0.7 h 336.5 0.5 338.7 0.2 337.7 0.2 337.2 0.2 331.9 0.1

76.2 0.1 45.7 0.2 333.8 0.0 76.4 0.1 46.2 0.2 332.9 0.0 78.9 0.4 37.5 0.8 337.7 0.1

for cactus pear (Fig. 1) [7]. Since purple pitayas exhibit a higher content of mucilaginous material, slight modications were necessary. Application of various enzyme preparations individually and in combination was tested (data not shown) to achieve optimum degradation of the pectin-like material, thus sufciently reducing viscosity to allow juice ltration. Best results were achieved when combining two enzyme preparations at high dosages (1000 ppm Rohapect B1L and 1000 ppm Fructozym BE). However, substantial ltering residues, mainly consisting of undegraded mucilage, were retained. It is assumed that replacing decanter centrifugation by a solid ejecting disk centrifuge prior to enzymatic juice treatment would afford improved separation of mucilaginous material, thus allowing lower enzyme dosage and/or enhanced degradation of the remaining pectin-like material. Nonetheless, a clear purple pitaya juice could be obtained with the present process. To get an insight into betalain susceptibility to different heat treatments, three heating systems, i.e. an HTST system, a tubular heat exchanger and a vapour heated double jacketed processing vessel, respectively, were applied for juice pasteurisation. Colour development Chromatic changes of purple pitaya juice during the production process were assessed by monitoring L C h values (Table 2). Basically, juice processing was reected by lightness (L ) increase, indicating betacyanin degradation [15, 19]. Solely decanter separation resulted in decreasing L , which may be explained by the separation of whitish pectin-like material from the juice, affording a darker juice colour. Chiey, the three pasteurisation techniques applied increased L to a different extent. Whereas the HTST system (Sample 6A) and the tubular heat exchanger system (Sample 6B) yielded an L value of about 76, pasteurisation in a processing vessel (Sample 6C) with a preheating time exceeding 1 h caused a slightly increased lightness value of about 79 (Table 2), pointing to greater betacyanin destruction because of more extensive heat exposure. Additionally, chroma (C ) showed characteristic changes during juice processing: Increasing values upon separation (Sample 2) and ltration (Sample 3) may be explained by

653 Table 3 Betacyanin retention (%) in purple pitaya juice at successive steps of juice processing monitored at max by spectrophotometry and by HPLC-DAD HPLC-DAD Peak area sum Betanin 100.0 92.9 85.5 85.2 75.7 66.1 64.9 55.1 100.0 94.1 79.8 82.7 78.8 67.8 67.6 57.9

Processing step (sample)

Spectrophotometer

Phyllocactin 100.0 92.9 86.4 84.2 71.2 53.2 52.4 31.6

Thawing (1) 100.0 Separation (2) 113.6 Enzymation (3) 99.9 Enzyme inactivation (4) 97.4 Filtration (5) 87.0 Pasteurisation (6) HTST system (A) 77.3 Tubular heat exchanger (B) 76.9 Processing vessel (C) 63.1

removal of particles and mucilaginous material [7]. On the other hand, thermal treatment (Samples 4 and 6AC) resulted in a decrease of C , thus indicating the formation of betacyanin degradation products [15, 19]. This conclusion is supported by the observation of a pronounced chroma decrease due to the more rigorous heating in a processing vessel (Sample 6C) as compared to gentle pasteurisation using the HTST system (Sample 6A) or the tubular heat exchanger (Sample 6B), respectively. Interestingly, hue angle (h ) of purple pitaya juice increased slightly upon separation (Sample 2) by about 2 to reach 339 (Table 2), while enzymation (Sample 3) and even thermal enzyme inactivation (Sample 4) did not affect h . In contrast, juice ltration (Sample 5) had a great impact on juice colour, causing a decrease in hue angle by about 5 . It is for this reason that gently pasteurised purple pitaya juices (Samples 6A and B) exhibited smaller hue angle values than the pitaya pur e immediately after thawe ing (Sample 1; Table 2). Even in purple pitaya juice heated under rigorous conditions (Sample 6C), h value was only slightly increased to about 338 . As the hue angle of purple pitaya juice was reported to increase upon betacyanin degradation due to formation of yellow degradation products [19], marginal pigment decomposition during juice pasteurisation may be assumed. Betacyanin retention Betacyanin retention during processing of purple pitaya fruits was monitored by spectrophotometry and HPLCDAD (Table 3). Interestingly, betacyanin retention calculated as betanin (Betanidin 5-O--glucoside; Fig. 2a) from spectrophotometric data was higher than obtained from HPLC analysis of the total peak area at 535 nm throughout the production process. This may be ascribed to coabsorbing juice constituents determined in the spectrophotometric measurements, which are separated from the betacyanins by HPLC before detection. Nevertheless, using both methods a decrease of the betacyanin content upon processing could be revealed, juice ltration (Sample 5) and pasteurisation (Samples 6AC) being most adverse to pigment retention. Again, the HTST system (Sample 6A) and the tubular heat exchanger (Sample 6B) were the most favourable techniques for juice pasteurisation, resulting in

overall pigment retentions of 77% (calculated from spectrophotometric data) and 6566% (calculated from HPLC data), respectively, as compared to the betacyanin content of purple pitaya pur e immediately after thawing (Table 3). e Even after rigorous heating in a processing vessel (Sample 6C), which took 85 min to reach the nal temperature of 92 C, more than 50% of the initial pigment content were retained, i.e. 63 and 55%, respectively (Table 3). Closer inspection of the two predominant betacyanins in purple pitaya, betanin and phyllocactin (Betanidin 5-O(6 -O-malonyl)--glucoside; Fig. 2b), revealed signicant differences between individual pigments. Phyllocactin being least stable throughout the process can be explained by its previously described tendency towards deacylation resulting in betanin generation [19, 20], which also accounts for the apparently higher betanin stability as compared to the overall betacyanin content (total peak area at 535 nm). Alteration of betalain peak area ratios Different stability of the single betacyanins during purple pitaya juice processing was monitored by comparing peak areas of the respective pigments throughout the process (Table 4). The isomerisation index of betanin, i.e. the betanin/isobetanin peak area ratio, was described previously to decline upon thermal treatment of red beet juice [21, 22], but to increase in the case of purple pitaya juice and pigment preparations thereof [15]. In this study, the isomerisation index was not altered upon separation (Sample 2) and enzymatic treatment (Sample 3), but showed a slight increase upon enzyme inactivation (Sample 4) and ltration (Sample 5). Thermal exposure during juice pasteurisation induced further increments of the betanin/isobetanin ratio (Samples 6AC), which may in part be due to betanin replenishment by phyllocactin deacylation [19, 20]. Interestingly, the isomerisation index of the juice pasteurised in the processing vessel (Sample 8), i.e. the sample with the most intensive heat exposure, was slightly lower as compared to the respective indices of the more gently heated samples. The peak area ratio of betanin and phyllocactin has recently been described to reach an equilibrium of about 1 when heated at 85 C for 1 h [15]. Starting from an initial

654 Fig. 2 Chemical structures of betanin a, phyllocactin b, and indicaxanthin c

HO HO H2C HC CH OH O O OH CH HO
5

CH HC

COO-

a
15

HOOC

N H

COOH

O HOOC CH2 C HC HO HO CH O CH2 O O OH CH HO

5

CH HC

COO

b
15

HOOC

N H

COOH

COOH

c
11

HOOC

N H

COOH

betanin/phyllocactin ratio of 0.5 (Table 4), in this study a slight decrease of this ratio was monitored upon enzymatic treatment (Sample 3) and enzyme inactivation (Sample 4), possibly due to -glucosidase side activities. As expected, the betanin/phyllocactin ratio increased upon pasteurisation depending on the intensity of thermal treatment: Pasteurisation in the HTST system (Sample 6A) and in the tubular heat exchanger (Sample 6B) resulted in a slight increase of the ratio to reach 0.6, while heat treatment in the processing vessel (Sample 6C) increased the betanin/phyllocactin ratio to 0.8. Hence, thermal load of purple pitaya juice can be easily deduced from calculating the peak area ratio of

betanin and phyllocactin, which increments with prolonged heat exposure. By comparing the total peak area in the red (535 nm) and yellow (470 nm) absorption domain, colour shifts of purple pitaya juice can be monitored [19]. Whereas the processing steps prior to pasteurisation (Samples 15) did not alter the initial ratio of the total peak area (535 nm/470 nm) of 3.5, pasteurisation resulted in a decline of the index, tantamount to a colour shift towards yellow. Again, heating in the processing vessel (Sample 6C) caused the greatest chromatic difference (Table 4). The colour shift can be explained by the generation of yellow betacyanin degradation products

655 Table 4 Peak area ratios of characteristic betalains in purple pitaya juice at successive steps of juice processing monitored by HPLC-DAD at 535 nm (betanin, isobetanin, phyllocactin) and 470 nm (indicaxanthin, isoindicaxanthin), respectively Processing step (sample) Thawing (1) Separation (2) Enzymation (3) Enzyme inactivation (4) Filtration (5) Pasteurisation (6) HTST system (A) Tubular heat exchanger (B) Processing vessel (C)
a

Betanin/Isobetanin 4.2 4.2 4.2 4.3 4.4 4.7 4.7 4.6

Betanin/Phyllocactin 0.5 0.5 0.4 0.4 0.5 0.6 0.6 0.8

Total peak area 535 nm/470 nm 3.5 3.5 3.5 3.5 3.5 3.2 3.4 2.8

Betanin/ Indicaxanthin 1666.9 1636.3 1420.0 1475.4 1405.4 1090.9 1136.8 604.5

Isobetanin/ Isoindicaxanthin a a 235.2 166.8 141.0 36.5 37.6 7.3

Isoindicaxanthin not detectable

[19, 22] as well as by betaxanthin formation via condensation of free amino acids in the juice with betalamic acid generated by betacyanin hydrolysis [15]. Interestingly, this fact may not be assessed by mere spectrophotometric analysis, since the hue angle of the pasteurised samples (6AC) is smaller or only slightly higher than that of pitaya pur e e (Sample 1; Table 2). In a previous study [15], thermal treatment of purple pitaya juice was found to induce generation of indicaxanthin (proline-betaxanthin; Fig. 2c) and its C11 -epimer isoindicaxanthin by condensation of betalamic and isobetalamic acids released from betacyanin cleavage with proline, which is abundant in genuine purple pitaya juice. Analogously, indicaxanthin formation at the expense of betanin observable by a decreasing betanin/indicaxanthin peak area ratio was monitored in the course of pitaya juice processing (Table 4). Also the isobetanin/isoindicaxanthin peak area ratio declined, reecting isoindicaxanthin formation by condensation of isobetalamic acid generated from isobetanin cleavage and proline. Indicaxanthin formation during juice processing corroborated the assumption of indicaxanthin generation upon straining, centrifuging and freezing of purple pitaya fruits [15]. Furthermore, the absence of indicaxanthin in manually squeezed juice [23] is plausible. Thus, indicaxanthin may not be regarded a genuine constituent of purple pitaya, but rather an artefact which is formed during processing or storage. Storage of purple pitaya juice To evaluate the storage stability of betacyanins in purple pitaya, juice was processed by straining and centrifuging for complete removal of mucilaginous material, thus eliminating the potential betacyanin stabilising matrix effect. Although ascorbic acid was previously reported to be absent in purple pitaya juice [10], this antioxidant was recently proven to stabilise betacyanins during thermal treatment of purple pitaya juice [15]. Therefore, samples were stored with and without addition of 1% ascorbic acid. Since light was earlier reported to deteriorate betacyanin stability [1214], the effect of illumination was investigated and compared to dark storage.

Colour development Chromatic changes of purple pitaya juice samples stored with and without ascorbic acid supplementation as well as in light and darkness, respectively, were monitored in terms of lightness (L ), chroma (C ) and hue angle (h ), and plotted against the storage period (Fig. 3). The samples containing 1% ascorbic acid exhibited very similar lightness development, L only slightly increasing over 6 months of storage irrespective of light exposure. In contrast, the sample without ascorbic acid supplementation stored in the dark showed considerable lightness increments, while the same juice stored under illumination underwent an L value rise by nearly 20 to yield about 90 units after 6 months, indicating most pronounced betacyanin degradation in this sample [15, 19]. Similar effects were observed for chroma. C slowly decreased in the samples supplemented with ascorbic acid, while samples devoid of ascorbic acid gave rise to accelerated chroma loss in the dark, being stronger upon illuminated storage (Fig. 3). Hence, increased formation of betacyanin degradation products in purple pitaya juice stored without ascorbic acid supplementation may be the reason for the observed chroma decrease [15, 19]. It is worth mentioning that the hue angle of pitaya juice exposed to light for 6 months with added antioxidant was only 2 higher than after 1 day storage (data not shown). Similarly, the non-illuminated sample with ascorbic acid supplementation exhibited a slight h increase, while juice stored without antioxidant underwent a considerable colour shift from red-purple to red within 6 months of dark storage. The illuminated sample even exhibited a hue angle of about 50 after the same storage period (Fig. 3). The changes of the three colour values discussed above were summarised by calculating the total colour difference ( E ) of samples stored from 1 to 6 months compared to the respective sample stored for 1 day (Fig. 3). The latter was chosen as a blank value for maximum betacyanin regeneration after juice pasteurisation for 5 min at 95 C, previously reported to be completed 24 h following thermal treatment [24]. In accordance with the individual colour indices, E of the illuminated juice sample without ascorbic acid supplement was intelligibly greater compared to the

656

dark stored sample (Fig. 3). Interestingly, colour change after 6 months of storage was minimal in the illuminated sample stored with 1% ascorbic acid addition. The reason for this sample being unexpectedly more colour stable than the sample stored in the dark could not be disclosed further. Betacyanin retention Betacyanin retention in purple pitaya juice stored up to 6 months as compared to the respective samples stored for 1 day is shown in Fig. 4. In dark-stored juices without ascorbic acid addition, betacyanin degradation proceeded as far as to about 40% retention after 6 months. Illumination promoted betacyanin degradation, as only one-tenth of the initial pigment content was retained after 6 months storage. Hence, previous reports on betacyanin stability during storage demonstrating accelerated degradation under light exposure were substantiated [12, 13]. However, earlier ndings of betanin degradation being threefold accelerated by illumination of red beet juice at 25 C [12] could not be conrmed for purple pitaya betacyanins, presumably due to their acylated nature. Furthermore, pitaya juice supplemented with ascorbic acid did not exhibit enhanced betacyanin degradation during light-exposure. On the contrary, the samples containing ascorbic acid stored in the light and in the dark showed intelligibly higher betacyanin rentention than the samples without antioxidant. In the illuminated sample containing 1% ascorbic acid, more than 70% of the initial betacyanin content was retained after 6 months. As discussed above for overall colour, also betacyanin retention in the dark-stored samples with additive was observed to be slightly inferior to the respective samples under light exposure. Moreover, superior heat stability of purple pitaya betacyanins as opposed to red beet upon thermal treatment [24] was also observed for storage stability: Betacyanins in red beet juice were reported to be prone to fade, resulting in a yellow tint within 6 days without stabilisation, and only 52% of the initial betacyanin content were retained after 30 days illuminated storage of red beet juice at an optimised antioxidant concentration of 0.1% isoascorbic acid [25]. Alteration of betacyanin peak area ratios As described above for purple pitaya samples taken during juice processing, peak area ratios of characteristic betalains were calculated for pitaya juice stored for 1 day, 3 and 6 months, respectively (Table 5). In contrast to thermally treated purple pitaya juice [15], all stored samples underwent betanin C15 -isomerisation, indicated by decreasing betanin/isobetanin ratios (Table 4). In accordance with juices heated in the presence of ascorbic acid, betanin isomerisation in stored samples with 1% antioxidant was even more pronounced, resulting in a betanin/isobetanin ratio of 1.1 in the samples stored for 3 and 6 months with and without light exposure, respectively.

Fig. 3 Colour development of purple pitaya juice stored up to 6 months in light and darkness with and without addition of 1% ascorbic acid

657 Table 5 Peak area ratios of characteristic betalains in purple pitaya juice 1 day after pasteurisation and stored for 3 and 6 months monitored by HPLC-DAD at 535 nm (betanin, isobetanin, phyllocactin) and 470 nm (indicaxanthin, isoindicaxanthin), respectively Storage period Betanin/Isobetanin Betanin/Phyllocactin Total peak area 535 nm/470 nm 3.1 1.9 1.3 3.1 1.5 1.1 3.4 3.1 2.8 3.4 3.2 3.2 Betanin/ Indicaxanthin 13.6 34.0 35.6 13.6 15.9 2.1 18.3 77.7 81.9 18.3 82.2 96.5 Isobetanin/ Isoindicaxanthin 51.0 33.9 34.0 27.2 16.2 1.8 43.9 75.2 78.8 44.3 88.0 94.3

Juice stored in the dark without ascorbic acid addition 1 day 3.8 0.6 3 months 1.6 4.5 6 months 1.3 18.1 Juice stored in the light without ascorbic acid addition 1 day 3.8 0.6 3 months 2.0 3.3 6 months 1.9 a Juice stored in the dark with 1% ascorbic acid addition 1 day 3.0 0.6 3 months 1.1 4.4 6 months 1.1 15.7 Juice stored in the light with 1% ascorbic acid addition 1 day 3.0 0.6 3 months 1.1 2.9 6 months 1.1 8.6
a

Phyllocactin not detectable

100

Fig. 4 Betacyanin retention (%) in purple pitaya juice stored up to 6 months in light and darkness with and without 1% ascorbic acid addition monitored by spectrophotometry at 537 nm
Betacyanin retention [%]

90

80

70

60

50

40

30

20

10

0 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

without ascorbic acid, dark

without ascorbic acid, light

with ascorbic acid, dark

with ascorbic acid, light

Storage period [months]

The betanin/phyllocactin ratio rose signicantly, exceeding 1.0 during storage of all pitaya juice variants, while the light-exposed samples reached lower ratios (Table 5), irrespective of ascorbic acid supplementation. Hence, it may be assumed that betanin is more prone to light-induced degradation than phyllocactin. On the other hand, phyllocactin deacylation to form betanin might be impaired by illumination. The ratio of the total peak area sum monitored at 535 and 470 nm, respectively, was observed to decline during storage of purple pitaya juice, pointing to a colour shift towards yellow (Table 5). As expected, the red/yellow ratio of samples without added ascorbic acid decreased to a greater extent than in supplemented samples. Therefore, enhanced

formation of yellow betacyanin degradation products in unstabilised purple pitaya juice is suggested. Again, pitaya juice with 1% antioxidant exhibited a minor colour shift upon illumination than the respective sample stored in the dark. In all samples under observation, the betanin/indicaxanthin ratio increased during the rst 3 months (Table 5). While illuminated juice without antioxidant showed a notable decrease of this ratio during months 36, the betanin/indicaxanthin ratio of the remaining samples incremented, especially when stabilised with 1% ascorbic acid. From these data it can be deduced that betanin is more stable than indicaxanthin.

658

When monitoring the peak area ratios of isobetanin and isoindicaxanthin, different developments with and without ascorbic acid addition were obvious: The ratio declined in samples without supplementation, while redoubling of this ratio was observed in the stabilised samples (Table 5). Hence, it may be concluded that the formation of isoindicaxanthin at the expense of isobetanin was minimised by ascorbic acid by inhibiting isobetanin hydrolysis. Alternatively, enhanced formation of isobetanin by betanin isomerisation may be assumed to be responsible for this development, since isomerisation of indicaxanthin is less notable because of the signicantly lower indicaxanthin content compared to betanin. Conclusions It was shown that purple pitaya juice can be processed at pilot-plant scale by adapting a process previously established for cactus pear. By applying minimal heat-load, two-thirds of the betacyanin content of pitaya pur e were e retained after pasteurisation. However, the high mucilage content of pitaya was disadvantageous for juice clarication by ltration, requiring high enzyme dosages and hindering further concentration of pigments, respectively. On the other hand, the mucilage may contribute to minimise betacyanin degradation upon heating and storage, since unclaried juices were reported to exhibit improved pigment stability compared to those claried by centrifugation [15, 24]. Moreover, the genuine hydrocolloids may be advantageous for the application of purple pitaya juice as a colouring foodstuff, thus rendering thickening agents dispensable. Hence, for several applications such as dairy products, unclaried juices containing the total mucilage of the fruit may be advantageous. Storage stability of purple pitaya betacyanins even in claried juice was shown to be superior to the respective pigments in red beet and to be signicantly enhanced by the addition of 1% ascorbic acid. Additionally, supplementation with the antioxidant was found to inhibit light damage, thus allowing storage in transparent packaging materials. In summary, betacyanins in purple pitaya juice were shown to exhibit a proper heat and storage stability. Particularly when stabilised with ascorbic acid, also claried purple pitaya juices appear to be a promising application, thus rendering purple pitaya a valuable source for a redviolet colouring foodstuff.

Acknowledgements The authors are grateful to Mr. K. Mix for his excellent technical assistance in the production of purple pitaya juice. AB Enzymes (Darmstadt, Germany), Biocatalysts (Cardiff, UK), DSM Specialties (Seclin, France), Erbsl h Geiseno heim Getr nketechnologie (Geisenheim, Germany) and Novozymes a Switzerland (Dittingen, Switzerland) are acknowledged for providing the enzyme preparations.

References
1. Wissgott U, Bortlik K (1996) Trends Food Sci Technol 7:298 302 2. Shelke K (2004) Food Processing, http://www.foodprocessing .com/articles/2004/210.html; assessed November 2005 3. Stintzing FC, Carle R (2004) Trends Food Sci Technol 15:1938 4. Bednar CM, Kies C, Carlson M (1991) Plant Foods Human Nutr 41:261268 5. Lu G, Edwards CG, Fellman JK, Mattinson DS, Navazio J (2003) J Agric Food Chem 51:10261029 6. Stintzing FC, Schieber A, Carle R (2000) Obst Gem Kartoffelver (Fruit Vegetable Potato Process) 85:196204 7. Mohammer MR, Stintzing FC, Carle R (2005) Innov Food Sci Emerg Technol 6:221231 8. S enz C, Sep lveda E, Araya E, Calvo C (1993) Lebensm Wiss a u Technol 26:417421 9. Essa HA, Salama MF (2002) Nahrung/Food 46:245250 10. Stintzing FC, Schieber A, Carle R (2003) Eur Food Res Technol 216:303311 11. Mohammer MR, Stintzing FC, Carle R (2005) Food Res Int 38:975981 12. Attoe EL, von Elbe JH (1981) J Food Sci 46:19341937 13. von Elbe JH, Maing I-Y, Amundson CH (1974) J Food Sci 39:334337 14. Cai Y, Sun M, Corke H (1998) J Agric Food Chem 46:4491 4495 15. Herbach KM, Rohe M, Stintzing FC, Carle R (2006) Food Res Int 39:667677 16. Gonnet J-F (1998) Food Chem 63:409415 17. Cai Y, Corke H (1999) J Food Sci 64:869873 18. Wyler H, Meuer U (1979) Helv Chim Acta 62:13301339 19. Herbach KM, Stintzing FC, Carle R (2006) J Agric Food Chem 54:390398 20. Herbach KM, Stintzing FC, Carle R (2005) Rapid Comm Mass Spectrom 19:26032616 21. von Elbe JH, Schwartz SJ, Hildenbrand BE (1981) J Food Sci 46:17131715 22. Herbach KM, Stintzing FC, Carle R (2004) J Food Sci 69:C491 C498 23. Stintzing FC, Schieber A, Carle R (2002) Food Chem 77:101 106, 517 24. Herbach KM, Stintzing FC, Carle R (2004) Eur Food Res Technol 219:377385 25. Bilyk A, Kolodij MA, Sapers GM (1981) J Food Sci 46:1616 1617