Stability of Carotenoids in Vegetables, Fruits, Functional Foods, and

storage, especially heat treatment, light exposure, and the presence of ... selected carotenoids are shown in Figure 1. ... mainly due to the developm...
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Stability of Carotenoids in Vegetables, Fruits, Functional Foods, and Dietary Supplements with Particular Reference to trans-cis-Isomerization Andreas Schieber and Reinhold Carle Institute of Food Technology, Chair Plant Foodstuff Technology, Hohenheim University, August-von-Hartmann-Strasse 3, D-70599 Stuttgart, Germany

Carotenoids are widespread plant pigments occurring predominantly in their all-trans configuration. Processing and storage, especially heat treatment, light exposure, and the presence of photosensitizers may lead to the formation of carotenoid cis-isomers which exhibit different physical, chemical and biological properties. Despite a large number of studies carried out during the past two decades, the physiological relevance of cis-isomers has not completely been understood. This review summarizes our recent investigations on the effects of processing on carotenoid stability in vegetables, fruits, functional foods, and dietary supplements. Particular attention was given to methods for the determination of carotenoid stereoisomers and to the role of the physical state of carotenoids and the plant matrix for their stability.

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© 2008 American Chemical Society

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141 Carotenoids are widespread pigments which are biosynthesized by higher plants (/), algae, fungi, and bacteria (2, 3, 4). Their presence in animal tissues, e.g. egg yolk, bird feathers, and the exoskeleton of invertebrates is attributed to ingestion via the food chain followed by accumulation in these tissues. In plants, carotenoids are accumulated and sequestered in chloroplasts and chromoplasts (5, 6). Carotenoids are broadly classified into carotenes, which are strict hydrocarbons, and the more polar xanthophylls (or oxycarotenoids), which contain one or more oxygen atoms. Prominent members of the carotenes are ctcarotene, P-carotene and lycopene, whereas the most important xanthophylls are zeaxanthin, lutein, P-cryptoxanthin, astaxanthin, and canthaxanthin. Carotenoids containing a hydroxyl group may be esterified with fatty acids. The structures of selected carotenoids are shown in Figure 1. Among the 600 carotenoids known so far, only approximately 50 are active as provitamin A precursors, the most important and most efficient being P-carotene with its two P-ionone rings. Carotenoids predominantly occur in their dXVtrans configuration. However, mainly due to the development of novel C stationary phases and advances in hyphenation techniques, in particular LC-MS and LC-NMR spectroscopy, there is abundant proof for the presence of c/s-isomers in fruits (7), vegetables and physiological samples (8, 9, 10). Apart from naturally occurring in foods, heat treatment, light exposure, and the presence of triplet sensitizers and electrophilic compounds are the main reasons for the formation of c/s-isomers. Exposure to light predominantly leads to the formation of 9-ds-P-carotene, whereas 13-c/s-Pcarotene is mainly formed by thermal treatment. Their structures are shown in Figure 2. The c/s-isomers possess different physicochemical properties, such as lower melting points and increased solubility in non-polar solvents compared to their d\\-trans counterparts. Furthermore, a new maximum often referred to as 'c/s-peak' is observed in the UV spectrum. Since trans-cis-\somQnzdX\on is accompanied by a hypsochromic shift in the ^ and smaller extinction coefficients, this transformation may also lead to a decreased color intensity. It is also well known that isomerization results in a decreased provitamin A activity of carotenoids containing a P-ionone ring. However, the physiological relevance of the presence of c/s-isomers in the diet is not completely understood. For a more comprehensive treatise also including basic principles and consequences of trans-cis-isomerization, we refer to a recent review by Schieber and Carle (11). The objective of this contribution is to summarize our studies on carotenoid stereoisomer analysis and the effects of processing on the stability of carotenoids. 30

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Carotene Stereoisomers in Fortified Drinks Fortified drinks containing ascorbic acid (vitamin C), tocopherols (vitamin E), and p-carotene (provitamin A), hereinafter referred to as ATBC drinks, have

In Color Quality of Fresh and Processed Foods; Culver, Catherine A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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9-C/5-P-carotene

13-c/s-P-carotene Figure 2. Structure of 9-cis- and 13-cis-fi-carotene

In Color Quality of Fresh and Processed Foods; Culver, Catherine A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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144 experienced growing popularity. The provitamin A moiety is usually provided as synthetic P-carotene but may also originate from carrot juice as a natural source of carotenes. Prior to the investigation of commercial carrot juices and ATBC drinks, we have established an efficient method for the separation of the most important carotene stereoisomers including a\\-trans-a-, a\\-trans-$~, 9-cz's-P-, and 13-cis-p-carotene. Since lipids co-extracted from carrot juices did not interfere with the compounds of interest, a saponification step could be omitted (12). Later, the analytical system was extended to the simultaneous extraction and determination of carotene stereoisomers and tocopherols. Remarkably, the separation of 8-, y-, p-, and a-tocopherol as well as ot-tocopheryl acetate could be accomplished without compromising the resolution of the carotene stereoisomers (75). a-Carotene could be detected only in those ATBC drinks containing carrot juice as the carotene source. Their relative amounts of c/s-isomers, calculated as percentage of all-frans-p-carotene, ranged from 6.7 % to 13.6 %, with 13-c/s-Pcarotene being the predominant c/s-isomer (1.1-2.5 mg/1). In contrast, extraordinarily high relative amounts (31.8-44.5 %) were found in those samples with added synthetic P-carotene (72). These findings may be explained by the particular technology used for the production of the the ATBC basic material. Usually, commercial P-carotene preparations containing micro-crystalline allfr*ara-P-carotene, together with vitamin E and weighting agents (e.g. sucrose acetate-isobutyrate), are dissolved in a hot mixture of lipophilic solvents. The lipophilic phase is finely dispersed by homogenization in the aqueous phase, which contains a hydrocolloid solution, a syrup or fruit juice concentrate, and antioxidants (14). Since isomerization does not appear with crystalline carotene, hot dissolution of a\\-trans-P-carotene is considered the crucial step leading to the formation of c/s-isomers. Excessive heating may also cause de-esterification of a-tocopheryl acetate, which is comparatively stable towards oxidative degradation and therefore frequently used in supplements (75).

Effects of Processing on /rafis-c/s-Isomerization of P-Carotene in Carrot Juices Whereas ATBC drinks represent a comparatively simple matrix, a variety of parameters affect the chemical stability of carotenes in carrot juices. Our preliminary investigations on the presence of carotene stereoisomers in commercial carrot juices had revealed that the contents of all-rnms-cc-carotene ranged from approximately 20 mg/1 to 50 mg/1 and those of all-fr-a^s-P-carotene from 33 mg/1 to 85 mg/1. With respect to c/s-isomers, up to 4 mg/1 and 10 mg/1 of 9-cis- and 13-c/s-P-carotene were found, with relative amounts ranging from

In Color Quality of Fresh and Processed Foods; Culver, Catherine A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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145 4.0 % to 16.2 %, respectively (72). The ds-isomers of a-carotene were not included in this study. Since ds-isomers could not be detected in raw carrot roots, the effects of processing on /ram'-c/s-isomerization of P-carotene during carrot juice production were investigated in detail. For this purpose, carrot juices were produced on pilot-plant scale according to a standard process (75) which included blanching, comminution, and dejuicing using a decanter. The juices were either sterilized or acidified and pasteurized, and finally packaged aseptically. Time-temperature regimes for blanching and sterilization were varied. Another modification of the basic process consisted in the addition of grape seed oil to the coarse mash to investigate the effects of solubilization on carotene stability (16). Unheated juices produced from carrots blanched at 80°C for 10 min were devoid of c/s-isomers, and heat preservation during the standard process resulted in only weak isomerization (2-5 %). Only extensive blanching at timetemperature regimes not applied during production of carrot juice (80-100 °C for 30-60 min) led to the formation of 13-cw-P-carotene at levels between 1.8 and 10 %. Microscopic investigations revealed that raw and moderately blanched carrot roots contained helical and ribbon-like chromoplasts (Figure 3) (77), while excessive blanching caused the formation of yellow-colored lipid droplets containing soiubiltzed carotenes. In pasteurized juices slightly higher levels of 13-c/s-P-carotene were found compared to unheated juices. Sterilization had an additive effect on isomerization and resulted in an average increase in \3-cis-f>carotene of approximately 5 %. Both extended sterilization at 130 °C and the addition of grape seed oil to the coarse mash followed by heat preservation led to enhanced isomerization and caused formation also of 9-m-p-carotene. UV/Vis spectroscopic investigations of carotene-containing particles obtained by density-gradient centrifiigation of carrot juice demonstrated the presence of crystalline carotene. It is assumed that in carrot juice crystalline carotene is suspended and covered by polar lipids. Hydrocolloids of protein and/or polysaccharide nature may protect carotenes from being dissolved in neutral lipids during thermal preservation. However, it appears that at temperatures exceeding 120 °C a significant destruction of the protective matrix occurred which caused dissolution and isomerization of carotenes (16).

Isomerization of P-Carotene in Mango Fruits Whereas carrots are important sources of provitamin A in countries of the western hemisphere, mango fruits substantially contribute to the P-carotene supply in tropical countries. Although they are mainly consumed as fresh fruits, the economical importance of processed mango products such as canned mango

In Color Quality of Fresh and Processed Foods; Culver, Catherine A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Figure 3. Helical [1] and ribbon-shaped [2] chromoplasts in raw carrot roots (Reproduced with permission from reference 17. Copyright 2004.) (See page 12 of color inserts.)

slices, puree, and nectar has considerably increased. Furthermore, dehydrated products like mango fruit bars and slices represent an efficient means to improve the pro-vitamin A status of the population especially during off season. Two processes for the dehydration of mango fruits were assessed for their effects on /raws-cw-isomerization of P-carotene. The first process was performed using an overflow dryer which was operated for 3-3.5 h at 75 °C and an air velocity of 1 m/s. In the second process, the mangos were dried in a solar tunnel dryer at 60-62 °C for 7-8 h. Surprisingly, cw-isomers of P-carotene could be detected even in fresh mango fruits at relative contents of 19-27 %, which increased to 37 % upon thermal drying. In those samples dried in the solar tunnel dryer relative contents ranging from 51 % to 64 % were found. The profile of c/s-isomers was clearly dependent on the process technology used for mango drying. Whereas 13-cw-P-carotene was the predominant c/s-isomer formed after thermal treatment, solar tunnel drying mainly caused the formation of 9-cis-P-carotene (18).

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Determination of Xanthophyll Stereoisomers in Thermally Processed Vegetables and Dietary Supplements The dihydroxy carotenoids lutein and zeaxanthin have been identified as the major constituents of the macular pigment of the human retina. There is a growing body of evidence that they act as antioxidants in the macular region and display a protective role in the prevention of age-related macular degeneration (19). Therefore, a higher intake especially of lutein through either xanthophyllrich vegetables and fruits or dietary supplements may have beneficial effects on the visual performance of people suffering from age-related eye disorders. While green leafy vegetables such as spinach, lettuce, and kale are the most important dietary sources of lutein, zeaxanthin is mainly ingested with sweet corn and orange paprika. Since only limited information on the effects of processing on the stability of lutein and zeaxanthin was available, spinach and sweet corn were used as model vegetables to assess the influence of blanching and canning on the isomeric profile of these xanthophylls. For this purpose, an HPLC method for the simultaneous determination of lutein and zeaxanthin stereoisomers was developed. The canning process of sweet corn ( T = 121 °C; F= 5) resulted in a decrease of total lutein from 2.3 to 1.7 mg/100 g and of total zeaxanthin from 0.9 to 0.7 mg/100 g. In contrast, the moderate blanching conditions (100 °C; 2 min) led to a relatively large degradation of lutein in spinach (44 vs. 37 mg/100 g). Zeaxanthin could be detected only in trace amounts. Pronounced differences were also observed for the profile of aj-isomers in both vegetables. Whereas heat treatment of sweet corn resulted in a significant increase in the 13-c/sisomers of lutein and zeaxanthin, blanching of spinach mainly decreased lutein c/s-isomers (20). Subequent investigations on the effects of heating and illumination on /rara-c/s-isomerization and degradation of P-carotene and lutein in isolated spinach chloroplasts indeed revealed that carotenoid stability needs to be evaluated for every single pigment in its genuine environment and that stability data obtained from model experiments may not necessarily be portable to complex food matrices (21). In contrast to functional foods, dietary supplements are very similar to pharmaceutical products and frequently marketed as oral dosage formulations like gelatin capsules, dragees, tablets or powders. As a consequence, the applicability of procedures for the extraction of carotenoids from fruits, vegetables and functional foods to dietary supplements is limited. We have therefore developed methods for the determination of carotenoid stereoisomers from the above formulations comprising enzymatic digestion of the gelatin capsule using papain, extraction with acetone-hexane, and separation by HPLC. Whereas all-/Aws-P-carotene could be detected in all samples investigated, all/r 90 %). c/s-Isomers could be detected only in very low quantities (28). It is expected that a similar approach may also be suitable for the isolation of biologically active compounds other than carotenoids, e.g. polyphenols and phytosterols.

Conclusions Mainly due to advances in analytical chemistry, considerable progress has been made to understand the phenomenon of carotenoid isomerization in foods. The physical state of carotenoids and the nature of the matrix have been identified as crucial factors affecting their stability and the extent of isomerization. However, in order to obtain a more comprehensive knowledge, further investigations, e.g. into the role electrophilic compounds such as quinones play in carotenoid isomerization (29), would be desirable. The development of methods for the isolation of carotenoids should also facilitate

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149 studies on their bioavailability, metabolism, distribution, and possible health effects.

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