Limonoid Glucosides in Fruit, Juice and Processing by-products of Satsuma Mandarin (Chus unshiu Marcov.) YOSHIHIKO OZAKI, SHIGERU AYANO, NOBUYA INABA, MASAKI MIYAKE, MARK A. BERHOW, and SHIN HASEGAWA ABSTRACT In a study on improving utilization of waste from processing of Satsuma mandarin (Citrus unshiu Marcov.) the limonoid glucosides of fruit, juice, and by-products were measuredusing HPLC and TLC. All materials had the 17-P-D-glucopyranoside derivatives of limonoids reported in other commercial citrus fruit. Citrus molasseswas a good source for industrial scale extraction of limonoid glucosides. An extraction system using pol- ystyrene divinylbenzene resins, was developed which could be expanded to industrial scale. Key Words: mandarin, limonoid glucosides, citrus molasses INTRODUCTION THE UTILIZATION OF CITRUS PROCESSING WASTE is increasingly important. New technologies, such as supercritical fluid extrac- tion and preparative chromatography applied to food processing have improved extraction and isolation of chemical constituents of food (Braddock and Cadwallader, 1992). Limonoids are one group of the two bitter principles in Citrus. Among them, limonin is the major cause of limonoid bitterness in a variety of citrus juices. Limonoid research has been focused toward determining ways to remove bitter limonoids from citrus products (Hasegawa and Herman, 1992; Hasegawa et al., 1992). The commercial demand for limonoids has increased consider- ably since they were found to exhibit chemopreventive activity against cancer in laboratory animals (Lam et al., 1994; Miller et al., 1994) and antifeedant activity against certain insects and termites (Klocke and Kubo, 1982; Serit et al., 1991). Limonoids also occur as glucoside derivatives in mature citrus fruit tissues and seeds. Twenty-one limonoid glucosides have been isolated and characterized. All of those were monogluco- sides in which one limonoid molecule is linked with one D- glucose molecule at the 17-position of the open limonoid D-ring by a P-glucosidic linkage as in, for example, limonin 17-P-D- glucopyranoside (LG). Limonin glucoside was shown to exhibit similar chemopreventive activity against cancer in laboratory an- imals (Miller et al., 1994). Limonoid glucosides are almost taste- less and are water soluble, whereas some limonoid aglycones are extremely bitter and have very low solubility in water. Thus, limonoid glucosides would be ideal functional food additives, and it may be advantageous to recover such compounds from citrus processing wastes for other uses (Hasegawa et al., 1994). Satsuma mandarin (Citrus unshiu Marcov.) cultivation origi- nated in Japan. Today they are grown in South Korea, China, Mediterranean costal countries and Spain as well as the U.S. Satsuma mandarins are the most popular fresh citrus fruits and processed juice products consumed in Japan. The annual pro- duction was 1.5 million metric tons in 1993. Concentrations of limonoid aglycones in the juices from mature Satsumamandarin Authors Ozaki, Ayano, Inaba, and Miyake are with the Wakayama Agricultural and Biological Research Institute, 396- 1, Tsukatsuki, Momoyama-cho, Naga-gun, Wakayama 649-61 Japan. Authors Berhow and Hasegawa are with the Fruit & Vegetable Chemistry Laboratory, USDA, ARS, 263 S. Chester Ave., Pasadena, CA 91106. are generally low, thus the limonoid bitterness problem occurs infrequently (Hashinaga et al., 1977; Izumi et al., 1981). Our objectives were to follow the changes in LG, the pre- dominant limonoid glucoside in citrus fruit tissues, during Sat- suma mandarin fiuit development. We then analyzed the limonoid glucosides of processedjuice products and processing by-products of Satsuma mandarin, to evaluate suitability as sources for limonoid glucosides recovery. Commercially debit- tered and deacidified juices of Hassaku (Citrus hassaku) were also analyzed. An extraction experiment was conducted with resins commonly used for commercial debittering of citrus juices as a basis for developing an industrial scale system to recover limonoid glucosides for other uses. MATERIALS & METHODS Materials Frozen concentrated juice sampleswere obtained from the Momoyama processing plant of Wakayama Prefectural Agricultural Cooperatives from October 1990 through February 1991. Juices were processed with an FMC In-Line juice extractor. Processed juices and by-product sam- ples, including single strength juice, concentrated juice, finisher pulp, centrifugal pulp, intact peel residue, pressedpeel residue, dried peel res- idue and citrus molasses, were obtained from the Momoyama plant dur- ing the 1992 processing season. Commercially debittered and native Hassaku juices were also obtained from the Momoyama plant. Fruit samples were obtained 11 times between July 20 and December 23, 1992, from two trees grown at a commercial orchard in Shimotsu, Wakayama, Japan. For each sample date, 10 fruits were picked from each tree. All reported values are expressedas averagesfrom the lots of fruits. A C,, reverse phase analytical HPLC column, TSK-gel ODS120T, was purchased from Tosoh Corp. (Tokyo, Japan). Silica gel TLC plates, SIL G-25, was purchased from Macherey-Nagel (Germany). SEP-PAK@ plus C,, Environmental Cartridge was purchased from Millipore Corp. (Milford, MA). Industrial resins, HP-20 and WA-30 were obtained from Nihon Rensui Co. Ltd. (Tokyo, Japan). All other chemicals were pur- chased from Nacalai Tesque (Kyoto, Japan). Preparation and extraction of fruit samples Flesh and peel of sample fruit were separatedby hand. The contents of limonoid glucosides of the peel and flesh were analyzed by the method of Fong et al. (1992). A portion of each sample was blended in methanol with a Waring blender (New Hartford, CT). For analysis, 5 g of slurry was further homogenized with a Polytron tissue homogenizer (Kinematica, Switzerland) with 20 mL niethanol. The homogenate was centrifuged at 9,000 g for 10 min. The residue was extracted twice with 20 mL of 70% methanol. The extraction procedure was repeated three times. The combined extract was evaporatedto dryness and dissolved in minimal volume of H,O. The extract was then passedthrough a C,, SEP- PAK. The SEP-PAK was activated with 3 mL of methanol and rinsed with 10 mL of H,O, before sample loading. The cartridge was then washed with 10 mL of H,O and limonoid glucosides were eluted with 6 mL of methanol. The eluate was evaporated and the residue was dis- solved in 1 mL of methanol. Preparation and extraction of liquid samples Limonoid glucosides of all liquid samples, including single strength juice, concentrated juice, finisher pulp and citrus molasses, were ex- tracted by the modified method of Fong et al. (1990) as follows. Con- 186-JOURNAL OF FOOD SCIENCE-Volume 60, No. 1, 1995