Note Changes in Pungent Components of Two Wasabia

Food Sci. Technol. Res., 9 (3), 288–291, 2003 Note

Changes in Pungent Components of Two Wasabia japonica MATSUM. Cultivars during the Cultivation Period

Masakazu HARA,1 Keiichi MOCHIZUKI,2 Shinichiro KANEKO,2 Toshio IIYAMA,3 Takehiro INA,3 Hideo ETOH1 and Toru KUBOI1

1Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, 422-8529, Japan 2Tamaru-ya Honten Co., LTD., 6-7 Kouyamachi, Shizuoka, 420-0852, Japan 3Shizuoka Agricultural Experiment Station, 2860-25 Yugashima, Amagi-Yugashima, Shizuoka, 410-3206, Japan

Received January 10, 2003; Accepted May 15, 2003

Japanese horseradish, i.e. wasabi (Wasabia japonica MATSUM.), cultivars are classiﬁed as green-stem and red-stem types. It is believed that pungency differs slightly between the types. We analyzed isothiocyanate levels and myrosinase activity in two wasabi cultivars, Maruichi and Mazuma, a green-stem and a red-stem type, respectively. Time-course experiments were performed monthly from May 2001 to October 2002, separating plants into leaves, petioles, and rhizomes. Allyl isothiocyanate production induced by maceration occurred in whole plants in both cultivars, though the major organ producing the isothiocyanate was the rhizome. Levels of myrosinase activity in the rhizome of Maruichi increased in November and December 2001, when allyl isothiocyanate production also increased in Maruichi’s rhizome. Although levels of allyl isothiocyanate did not differ between the two cultivars, Mazuma produced isopropyl isothiocyanate but Maruichi produced little. These results suggest that the difference in pungency between Maruichi and Mazuma may be derived from the isothiocyanate component.

Keywords: isothiocyanate, myrosinase, pungency, wasabi, Wasabia japonica MATSUM.

Japanese horseradish, i.e. wasabi (Wasabia japonica MA- about changes of isothiocyanate-generating activity during culti- TSUM.), is a perennial herb which is traditionally used in Japa- vation in wasabi. There was no difference in allyl isothiocyanate nese dishes. Demand for wasabi is increasing as Japanese foods generation between two wasabi cultivars (Hisatake, 1985), nor spread around the world, and it has been found that the wasabi was there any apparent change observed in allyl isothiocyanate extract has many health benefits, such as radical-scavenging and production during the cultivating period (Ohtsuru & Hanayama, anticarcinogenic activities (Kinae et al., 2001). When wasabi is 1982). Thus, more study is needed on fluctuations during cultiva- grated, it produces a great deal of allyl isothiocyanate which has tion or differences among cultivars in the isothiocyanate-generat- a strong pungency; the allyl isothiocyanate is generated by a ing activity of wasabi. In this paper, we analyzed levels of iso- myrosinase-glucosinolate system (Ohtsuru & Kawatani, 1979; thiocyanates and myrosinase activity during the cultivating pe- Hara et al., 2000). The system, which is common in Brassicaceae riod in two wasabi cultivars, Maruichi and Mazuma. species, consists of two components, the enzyme myrosinase and the substrate glucosinolate. Mechanical disruption of plant tissue Materials and methods provides the enzymatic reaction and glucosinolates are converted Plant materials The two wasabi cultivars, Maruichi and to corresponding isothiocyanates. Allyl isothiocyanate has potent Mazuma, were grown in the same field developed in a mountain antimicrobial and insect-repellent activities (Chew, 1988). Hara stream in Amagi, Shizuoka, Japan. Plantlets that budded from the et al. (2001a) demonstrated that the myrosinase-glucosinolate rhizome, which were approximately the same in size, were trans- system of wasabi existed in the epidermis and vascular cambium planted to the field in the beginning of March 2001. Four plants of the root. The localization suggested that the system may pre- of each cultivar were harvested every month from May 2001 to vent insects and pathogens from invading the root and spreading October 2002. After harvest, fresh weight, vertical length, and through the plant via the vascular cambium (Hara et al., 2001b). maximum diameter of the rhizomes were recorded. The plants, Wasabi cultivars are conveniently classified into green- and which were separated into leaves, petioles, and rhizomes, were red-stem types. The green-stem type is preferred for processed kept at 5˚C and analyzed for isothiocyanate and myrosinase ac- foods because it adds a clear pungency and green color to the tivity within 3 days. foods. In contrast, the red-stem type becomes a smooth paste Isothiocyanate analysis Eight grams of fresh tissue was with fine pungency when grated, so it is usually used fresh. cut into small pieces (ca. 5 mm square). The tissue was im- Although the difference in pungency between the two types has mersed in 40 ml of cold deionized water and homogenized im- been described, the difference in the production of isothiocyanate mediately with a blender (S25KV-18G, IKA Works) in a glass has not yet been elucidated. Moreover, there is little information vial. The vial was capped and incubated at 45˚C for 15 min. Iso- thiocyanate generation reached a plateau with this incubation. E-mail: [email protected] After cooling on ice for 10 min, 5 ml of hexane containing Pungent Components of Two Wasabia japonica 289

0.05% phenyl isothiocyanate as an internal standard was added and isothiocyanates were extracted. The extract was dried over

Na2SO4 and the sample was analyzed by gas chromatography (Varian CP-3800). The column was 0.25 mm i.d. 30 m (DB- WAX, J & W) and the column oven was programmed from 60˚C to 220˚C at 10˚C/min. The carrier gas was He and the detector was FID. Injector and detector temperatures were 250 and 280˚C, respectively. Isopropyl, allyl, 4-pentenyl, and phenethyl isothiocyanates were identiﬁed by GC/MS with authentic samples and data of a reference (Spencer & Daxenbichler, 1980). Myrosinase activity Myrosinase activity was measured by the glucose liberation method (Hara et al., 2000) with slight modiﬁcation. Fresh tissues (5 g) were ground in a mortar with 15 ml of reaction buffer (100 mM potassium phosphate buffer pH 7.0) containing 5 mM dithiothreitol on ice. The homogenate was centrifuged for 15 min at 12000g. The supernatant was adjust- ed to 80% saturation with ammonium sulfate and proteins were collected by centrifugation. The precipitate was dissolved in the reaction buffer (2.5 ml) and applied to a PD-10 column (Amer- sham Pharmacia Biotech) equilibrated with the reaction buffer. The assay mixture (1 ml of total volume) containing 950 l of 100 M allylglucosinolate (Sigma) and 50 l of enzyme extract prepared as above (pH 7.0) was incubated at 37˚C. After incubation for 0, 5, 10, and 30 min, the reaction was terminated by boil- ing for 5 min. The sample was centrifuged at 12000g for 15 min, and the content of glucose in the supernatant was deter- mined with an enzymatic Glucose (GO) Assay Kit (Sigma). One Fig. 1. Changes in fresh weight (A), vertical length (B), and diameter (C) of rhizome during the cultivating period in Maruichi and Mazuma. Open and closed circles indicate Maruichi and Mazuma, respectively. The value and bar show the mean and variation of two samples, respectively.

Fig. 2. Time-course of the production of allyl and isopropyl isothiocyanates during the cultivating period in Maruichi and Mazuma. Leaf (A, D), petiole (B, E), and rhizome (C, F) were separated and isothiocyanates were analyzed. Allyl isothiocyanate (A–C) and isopropyl isothiocyanate (D–F). Open and closed circles indicate Maruichi and Mazuma, respectively. The value and bar show the mean and variation of two samples, respectively. 290 M. HARA et al.

(Fig. 2A-C). Although the levels fluctuated in the cultivating period, modes of fluctuation differed little between the cultivars. In rhizome, Maruichi produced more allyl isothiocyanate than Mazuma in most periods (Fig. 2C), but allyl isothiocyanate production in petiole was higher in Mazuma than in Maruichi from April to October 2002 (Fig. 2B). Considering that the rhizome produces more allyl isothiocyanate than other organs, it is possible to conclude that the allyl isothiocyanate-generating activity of Maruichi was stronger than that of Mazuma. In Mazuma, isopropyl isothiocyanate was generated in the entire plant, but the generation was remarkable in the petiole and rhizome (Figs. 2D-F). By contrast, Maruichi produced little isopropyl isothiocyanate except in the petiole in June 2002 and rhizome from June to August 2002 (Figs. 2E, F). On the other hand, 4-pentenyl and phenethyl isothiocyanates were detected in both cultivars at similar levels during cultivation (ca. 2% of allyl isothiocyanate, data not shown). These results suggest that the generation of isopropyl isothiocyanate is a characteristic of Mazuma and the feature may explain the difference in pungency between the two cultivars. Myrosinase activity Myrosinase activity in Maruichi and Mazuma changed significantly during cultivation (Fig. 3). The level of activity was usually higher in Maruichi with some excep- tions (from March to May 2002 and October 2002 in petiole and October 2002 in leaf). A common characteristic among leaf, petiole, and rhizome in Maruichi was that the activity increased in winter from November 2001 to January 2002. It is assumed that Fig. 3. Time-course of myrosinase activity during the cultivating period in Maruichi and Mazuma. Leaf (A), petiole (B), and rhizome (C) were sepa- in Maruichi both allyl isothiocyanate production and myrosinase rated and myrosinase activity was measured. Open and closed circles indi- activity increased in winter of the first cultivation year. However, cate Maruichi and Mazuma, respectively. The value and bar show the mean during the total cultivation period no noticeable relation was and variation of two samples, respectively. observed between allyl isothiocyanate production and myrosinase activity in either cultivar. It is suggested that not myrosinase activity but allyl glucosinolate content may be the rate limiting factor for allyl isothiocyanate production in wasabi. unit (U) of myrosinase activity was defined as the enzymatic This study demonstrated that the isothiocyanate component hydrolysis of 1 nmol glucosinolate min1. differed between Maruichi and Mazuma. This is the first report to show that the pungent components can vary among wasabi Results and Discussion cultivars during the cultivating period, as far as we know. Isopro- Rhizome growth Fresh weight, vertical length, and diame- pyl isothiocyanate was generated in Mazuma but little was deter of rhizome in two wasabi cultivars are shown in Fig. 1. Fresh tected in Maruichi, although all isothiocyanates except isopropyl weight did not increase in the first four months of analysis (from isothiocyanate were detected in both cultivars. Although pure May 2001 to August 2001) (Fig. 1A). The increase started in isopropyl isothiocyanate smells weakly pungent, it possesses a September 2001 and continued until July 2002. Growth in the characteristic flavor. It is possible that Mazuma has a pungency second year, especially from April to July 2002, was remarkable. distinct from Maruichi due to the addition of isopropyl isothiocy- Time-course curves did not differ between Maruichi and Mazu- anate. However, sensory evaluations suggested that if isopropyl ma except that there were some periods when values were slight- isothiocyanate was combined with allyl isothiocyanate at the ly higher for the former cultivar. Similar results were obtained in molar ratio of 1 to 10, which is an approximate rate in Mazuma, rhizome length (Fig. 1B). The increase in diameter occurred in the pungency of the mixture was not clearly different from that the first year of cultivation (from May 2001 to February 2002) of authentic allyl isothiocyanate. Thus it is unlikely that the char- but stopped later (Fig. 1C). Both cultivars showed a similar ten- acteristics of flavor in Mazuma depend solely on the presence of dency in the change of diameter. isopropyl isothiocyanate. Other emissive compounds, such as Isothiocyanate generation Wasabi produces several kinds green notes, may help to alter wasabi pungency. Moreover, the of isothiocyanates when the tissue is grated. Isopropyl, allyl, 4- consistency of wasabi paste may affect the pungency recognized pentenyl, and phenethyl isothiocyanates were analyzed by gas by humans, since paste from Mazuma is stickier than that from chromatography, and allyl isothiocyanate was found to be gener- Maruichi. ated at overwhelmingly higher levels. Figure 2 indicates time- The present study suggests that isopropyl isothiocyanate may courses of the production of allyl and isopropyl isothiocyanates be a key compound to identifying Maruichi and Mazuma. How- in leaf, petiole, and rhizome of the two cultivars. Allyl isothiocy- ever, it is unknown whether all the green-stem types produce anate levels were lower in leaf and petiole but higher in rhizome more isopropyl isothiocyanate than all the red-stem types. An Pungent Components of Two Wasabia japonica 291 analysis of isothiocyanates in many wasabi cultivars which 269 (in Japanese). belong to green- and red-stem types is needed. Hisatake, M. (1985). 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