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Technical Paper Quantitation of Carotenoids in Raw and Processed Food Sci. Technol. Res., ++ (+), +-ῌ+2, ,**/ Technical paper Quantitation of Carotenoids in Raw and Processed Fruits in Japan +ῌ + + + + Masamichi YANO , Masaya KATO , Yoshinori IKOMA , Akemi KAWASAKI , Yoshino FUKAZAWA , + + , - . Minoru SUGIURA , Hikaru MATSUMOTO , Yumiko OOHARA , Akihiko NAGAO and Kazunori OGAWA + Department of Citrus Research Okitsu, National Institute of Fruit Tree Science, .2/ῌ0 Okitsunakacho, Shimizu-ku, Shizuoka-shi, Shizuoka .,.ῌ*,3,, Japan , Saga Prefectural Fruit Tree Research Station, 3+ Teraura, Ogi-cho, Ogi-gun, Saga 2./ῌ**.+, Japan - National Food Research Institute, ,ῌ+ῌ+, Kannondai, Tsukuba-shi, Ibaraki -*/ῌ20.,, Japan . Okinawa Subtropical Station, Japan International Research Center for Agricultural Sciences, +*3+ῌ+ Maezato, Kawarabaru, Ishigaki-shi, Okinawa 3*1ῌ***,, Japan. (Present address, Department of Citrus Research Okitsu, National Institute of Fruit Tree Science, .2/ῌ0 Okitsunakacho, Shimizu-ku, Shizuoka-shi, Shizuoka .,.ῌ*,3,, Japan) Received June ,+, ,**.; Accepted October ,1, ,**. To obtain the quantitative and qualitative data available for estimating the intake of carotenoids from fruits in Japan, carotenoids were analyzed with reversed phase high-performance liquid chromatography (HPLC). Ten carotenoids were examined in 1/ raw fruits and +/ processed fruits, all of which were harvested or purchased in Japan. Phytoene was detected in /2 of 3* fruit samples; z-carotene, in /* of 3* ; lycopene, in +- of 3*; a-carotene, in +2 of 3*;lutein,in/0 of 3*; b-carotene, in 2* of 3*; b-cryptoxanthin, in 02 of 3*; zeaxanthin, in /2 of 3*; all-trans-violaxanthin, in // of 3*;and 3-cis-violaxanthin, in .1 of 3* samples. Citrus fruits of the mandarin type (Satsuma mandarin and its hybrids, such as tangor) were rich in b-cryptoxanthin, b-carotene, all-trans-violaxanthin, and 3-cis-violaxanthin; ‘Star ruby’ grapefruit in lycopene; loquat, Japanese persimmon, and peach in b-cryptoxanthin, b-carotene, all-trans-violaxanthin, and 3-cis-violaxanthin; mango in b-carotene, all-trans-violaxanthin, and 3-cis-violaxanthin; acerolas in phytoene; passion fruits in z-carotene. Carotenoid levels in common fruits, such as apple, grape, lemon, pear, strawberry, kiwifruit, cherry, pineapple, and banana, were low. Keywords: carotenoid, fruit, phytoene, lycopene, b-carotene, b-cryptoxanthin, violaxanthin Introduction ,**.a; Sugiura et al., ,**.b). The USDA-NCC Carotenoid Carotenoids are derived from phytoene after steps of Database is a valuable resource for this research. How- dehydrogenation, cyclization, hydroxylation, oxidation, ever, there may be some di#erences in the carotenoid and epoxidation (Fig. +). They have several health bene- composition of fruits and vegetables grown in Japan and fits, such as provitamin A activity, scavenging of free those used for the establishment of the USDA-NCC radicals, enhancement of gap junctions, immunomodula- Carotenoid Database. For example, the characteristics of tion, and modification of enzyme activity involved in cultivars, climate, and conditions for growing fruit in health and disease (Faure et al., +333). Japan may produce carotenoid profiles that are di#erent Therefore, a large number of epidemiological studies from those in the USDA-NCC Carotenoid Database. Ac- have been conducted on the relationship between caro- curate carotenoid data is important when analyzing the tenoid intake or serum-carotenoid levels and the risk of relationship between carotenoid intake from fruit grown various chronic diseases (Ford et al., +333; Yuan et al., ,**+ in Japan and the risk of various chronic diseases. Thus, ; Zeagers et al., ,**+). These studies require that the com- the objective of this study was to characterize the position of the foods being tested for individual carotenoid content in various raw and processed fruits carotenoids are known. The USDA-NCC Carotenoid Data- consumed in Japan. A total of 1/ fresh fruit and +/ base (Holden et al. +333) provides the most valuable re- processed fruit samples were collected in Japan. A total source for this purpose worldwide. We have been inter- of +* carotenoids, such as phytoene, z-carotene, lycopene, ested in the e#ects of carotenoids, particularly in domes- a-carotene, lutein, b-carotene, b-cryptoxanthin, zeaxanthin, tic fruits, on the risk of various chronic diseases in Japan all-trans-violaxanthin, and 3-cis-violaxanthin were deter- (Sugiura et al., ,**,a; Sugiura et al., ,**,b; Sugiura et al., mined by means of reversed phase HPLC analysis. * To whom correspondence should be addressed. Materials and Methods E-mail: ym0*2,@a#rc.go.jp Carotenoids Carotenoids used in this study are as 14 M. YANO et al. tracts by adding NaCl-saturated water. The pigments repartitioned into the diethyl-ether phase were recovered and dried. Subsequently, the residue was re-dissolved in / mL of a methyl tert-butyl ether (MTBE): methanol (+: + v/v ) solution. Percent recoveries of carotenoids under the condition of this sample preparation procedure were 32.,ῌ for b-car, +*/./ῌ for b-cry and 23.2ῌ for z-car. An aliquot (,* mL) was separated by a reverse-phase HPLC system (Jasco) fitted with a YMC Carotenoid S-/ column of ,/*῍..0 mm (i.d) (Waters, Milford, MA) at a flow rate of + mL minῌ+. The eluent was monitored by a photodiode array detector (MD-3+*, Jasco). The sample was analyzed with three di#erent gradient elution sched- ules. To assay t-vio, c-vio, lut, b-cry, a-car, and lyc the gradient elution schedule consisted of an initial -* min of 3/ῌ (v/v) methanol, +ῌ (v/v) MTBE, and .ῌ (v/v) water followed by a linear gradient of 0ῌ (v/v) methanol, 3*ῌ (v/v) MTBE, and .ῌ (v/v) water for 0* min (method A). To assay z-car, and b-car, the initial solvent composition consisted of /*ῌ (v/v) methanol, .0ῌ (v/v) MTBE, and .ῌ 0ῌ Fig. +. Carotenoid biosynthetic pathway. (v/v) water followed by a linear gradient of (v/v) methanol, 3*ῌ (v/v) MTBE, and .ῌ (v/v) water for 0* min (method B). Zea was assayed by the gradient elution follows: a-carotene (a-car), lutein (lut), and all-trans- schedule of Rouse# and Raley (+330). The initial compo- violaxanthin (t-vio) were obtained from DHI Water and sition consisted of 3*ῌ (v/v) methanol, /ῌ (v/v) MTBE, Environment (Horshholm, Denmark), lycopene (lyc), b- and /ῌ (v/v) water followed by a linear gradient of 3/ῌ carotene (b-car), and zeaxanthin (zea) were obtained from (v/v) methanol, /ῌ (v/v) MTBE for +, min, 20ῌ (v/v) Extrasynthese (Genay, France), b-cryptoxanthin (b-cry) methanol, and +.ῌ MTBE for 2 min, 1/ῌ (v/v) methanol was obtained from Sokenkagaku (Tokyo). Phytoene and ,/ῌ (v/v) MTBE for +* min, and /*ῌ (v/v) methanol (phy) and z-carotene (z-car) were isolated from phy- and /*ῌ (v/v) MTBE for ,* min (method C). producing Escherichia coli or z-car-producing E. coli (Kato The peaks were identified by comparing their specific et al, ,**.). Nine-cis-violaxanthin (c-vio) was prepared retention times and absorption spectra with the authentic from the flavedo of Satsuma mandarin (Kato et al., ,**.). standards. Standard curves for the carotenoid quan- Sampling and Sample Preparation Ripe samples of tification were prepared with those of the authentic raw fruits were harvested from trees at the National standards at ,20 nm for phy, .** nm for z-car, ./, nm for Institute of Fruit Tree Science (Okitsu), Shimizu-Okitsu t-vio, c-vio, lut, b-cry, a-car, lyc, and zea, and ./- nm for Shizuoka-shi Shizuoka, the National Institute of Fruit b-car. The carotenoid concentration was estimated by Tree Science, Tsukuba-shi Ibaraki, Saga Prefectural Fruit the standard curves and expressed as milligrams per +** Tree Research Station, Ogi-cho Saga, and the Okinawa g fresh weight. Subtropical Station, Japan International Research Center for Agricultural Sciences, Ishigaki-shi, Okinawa, all of Results which are in Japan. The rest of the fruit samples were The carotenoid content of ninety fruit samples was purchased at local Japanese markets. For each sample, - determined. Qualitative and quantitative data of these to / fruits of average size were selected. Each fruit was fruit samples are presented in Table +. Phytoene was homogenized and / g samples were removed for analysis. detected in /3 of 3* fruit samples. The high phy levels Carotenoid analysis was performed in triplicate. were found in acerolas (++..12 mg/+** g fresh wt.), dried Carotenoid quantification For carotenoid analysis, apricots (,.2+. mg/+** g fresh wt.) and tangor cv. ‘Youko’ samples were homogenized in .*ῌ (v/v) methanol con- (,./-1 mg/+** g fresh wt.). Zeta-carotene was detected in taining +*ῌ (w/v) magnesium carbonate basic. Pigments /* of 3* fruit samples. High z-car levels were found in were extracted from the residues using an acetone: meth- raw passion fruit (,..13 mg/+** g fresh wt.) and passion anol (1: - v/v ) solution containing *.+ῌ (w/v) ,,0-di-tert- fruit juice (+.+*- mg/+** g fresh wt.). Lycopene was butyl-.-methylphenol and partitioned into diethyl ether detected in only +- samples. High lyc levels were found by using NaCl-saturated water. The diethyl ether layer in watermelon (0.+2. mg/+** g fresh wt.), guava (..-2- mg/ was mixed with ,*ῌ (w/v) methanolic KOH for saponific- +** g fresh wt.), papaya cv. ‘Fruit tower’ (,..2+ mg/+** g ation. The sample was shaken and wrapped in alumi- fresh wt.), and grapefruit cv. ‘Star ruby’ (+.203 mg/+** g num foil to protect it from light. Prior to capping, the fresh wt.). Alfa-carotene was detected in only +2 sample was gently blanketed with nitrogen, before being samples, but no samples showed high levels (῎+ mg/+** g closed and placed in the dark overnight at room tempera- fresh wt.). Similarly, although lut was detected in /2 of ture. Water-soluble extracts were removed from the ex- 3* samples, no samples showed high levels of lutein (῎+ Quantitation of Carotenoids in Raw and Processed Fruits in Japan 15 Carotenoid contents of raw and processed fruits.
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