Characteristic Odorants of Wasabi (Wasabia japonica matum

Chapter 6

Downloaded by NANYANG TECH UNIV LIB on June 14, 2014 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch006

Characteristic Odorants of Wasabi (Wasabia japonica matum), Japanese Horseradish, in Comparison with Those of Horseradish (Armoracia rusticana) Hideki Masuda, Yasuhiro Harada, Kunio Tanaka, Masahiro Nakajima, and Hideki Tabeta Okayama Laboratory, Ogawa & Company, Limited, 1-2, Taiheidai, Shoo-cho, Katsuta-gun, Okayama-ken, 709-43, Japan The volatile components of both wasabi and horseradish formed by the hydrolysis of thioglucosides with myrosinase are known to possess a strong pungency. The difference between wasabi and horseradish is a green odor. The ratio of the concentration to the odor threshold, C/T, is called the aroma value. Sensory response is logarithmically proportional to the amount of stimulus. In this study, the log of the aroma values, log(C/T), for 14 isothiocyanates have been used instead of their conventional aroma values. ω-Alkenyl isothiocyanates, which possessed higher log(C/T) values than those of horseradish, contributed to the green odor of wasabi. Furthermore, the yields and the values of log(C/T) of the isothiocyanates catalyzed by myrosinase were affected by pH and temperature. Myrosinase (thioglucoside glucohydrolase) is present in plants belonging to the Cruciferae family (7,2). The thioglucosides involved in wasabi, Japanese horseradish, and horseradish are hydrolyzed by myrosinase to form the volatile compounds, including isothiocyanates, thiocyanates, nitriles, etc. (3). Among these compounds, the isothiocyanates have been recognized as the characteristic flavor compounds because of their pungency. Many investigators have reported the amounts of volatile isothiocyanates in wasabi and horseradish (4-6). AUyl isothiocyanate, the main component of wasabi and horseradish, exhibits the most pungent odor (7,8). As for horseradish, the major component after allyl isothiocyanate, phenethyl isothiocyanate, is known to be sufficiently characteristic (9, 10). The remarkable difference between wasabi and horseradish is the green odor. The volatile compounds of wasabi have a more greenish note than those of horseradish. Recently, it has been suggested that w-methylthioalkyl isothiocyanates contribute to the green odor of wasabi (11,12). Furthermore, the odor threshold 0097-6156/%/0637-0067$15.00/0 © 19% American Chemical Society

In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS

value and odor description of allyl isothiocyanate and three kinds of w-alkenyl isothiocyanates, the major components next to allyl isothiocyanate in wasabi, have been reported (13). However, a detailed study on the contribution of odor of each isothiocyanate in wasabi compared to that in horseradish has not yet been made. The properties of myrosinase purified from mustard seed (14, 15) and wasabi (16) were studied. The pH and temperature activity, and the pH and temperature stability of wasabi myrosinase were examined in detail. However, the effects of pH and temperature on the yields of the isothiocyanates have not yet been reported. This study focuses on the determination of the characteristic odorants of wasabi in comparison with those of horseradish by calculation of the logarithmic ratio between the concentration of isothiocyanate and its odor threshold value. The relationship between the yields of the isothiocyanates formed by the action of wasabi myrosinase and the conditions of hydrolysis are presented. Experimental Stems of wasabi (1 kg) and horseradish (1 kg) collected in Japan and New Zealand, respectively, in January 1995. They were crushed, allowed to stand for 1 h at 25 °C and extracted with 5 x 5 L of dichloromethane. Each solution was concentrated to 4.9 g and 6.7 g, respectively, using a rotary evaporator (35°C/300 mmHg). In the study of the influences of pH and temperature, stems of wasabi (1.26 kg) collected in Japan in June 1995, were freeze-dried at 0.1 mmHg for 72 h. The freeze-dried stems (206 g) were crushed to form wasabi powder. The powder (10 g) was hydrolyzed in the phosphate buffer under different pHs (4, 7 and 9) and at different temperatures (3°C and 25°C). Each suspension was extracted with 3 x 50 ml of dichloromethane and concentrated using a rotary evaporator (35°C/300 mmHg). The concentrations of 14 isothiocyanates were determined by GC. A Hitachi G5000 fitted with an FID was used. A DB-1 (30 m x 0.25 mm i . d.) fused-silica capillary column was employed. Operating conditions were as follows: initial oven temperature, 60°C, then to 250°C at 3°C/min and held for 30 min; injector temperature, 250°C; carrier gas, 0.5 ml/min N . Peak areas were obtained with a Hitachi D-2500 Chromato-Integrator. To estimate the concentrations of the components, an internal standard, phenyl isothiocyanate, was used. The oj-alkenyl isothiocyanates, except for allyl isothiocyanate, were prepared by the isomerization of the corresponding w-alkenyl thiocyanates (17). The w-alkenyl isothiocyanates were converted to the corresponding oj-methylthioalkyl isothiocyanates (18). The other isothiocyanates were purchased from commercial sources and purified by vacuum distillation. The odor threshold values in a water solution of the odorants were estimated by the 2/5 test (19). The ten panelists picked both of the flasks containing the odorous solution. Each dilution step was iVlO the concentration of the previous one. The flavor dilution (FD) factors, 2 , were obtained using the aroma extract dilution method developed by Grosch and co-workers (20, 21). The extract was diluted stepwise with dichloromethane in the volume ratio of 1:1 until odorous compounds were no longer detected by GC sniffing. 2

n

In Biotechnology for Improved Foods and Flavors; Takeoka, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

6.

MASUDA ET AL.

Characteristic Odorants of Wasabi

69


Results and Discussion The remarkable difference between wasabi and horseradish was the concentrations of the co-alkenyl isothiocyanates (5-8) (Table I), the aryl isothio cyanates (10) and (11) (Table I), and the co-methylthioalkyl isothiocyanates (12-14) (Table I). The odor threshold values of the co-alkenyl isothiocyanates (5-7) were lower than the other isothiocyanates. In addition, these w-alkenyl isothiocyanates had about half the value of that of allyl isothiocyanate (2) (Table I). The alkyl isothiocyanates (1, 3 and 4) (Table I) possessed a chemical odor but no pungent note. Hence, it seems that those compounds make relatively small contributions to the characteristic odor of wasabi and horseradish. The other isothiocyanates except for( 1, 3 and 4), however, had pungent and/or radish-like odors. The concentration divided by the odor threshold value, C/T is called the aroma value (22). Odorants with high aroma values are important contributors to the characteristic flavors. In general, the estimated feel of the stimulus is logarithmically proportional to the objectively measured strength of the stimulus (25). Accordingly, in this research we have evaluated the log of the aroma value, log(C/T), instead of the conventional aroma value. The values of log (C/T) of the isothiocyanates versus their retention indices (RI) are shown in Figure 1, top. Allyl isothiocyanate (2) had the highest value of log(C/T) in both volatiles (Figure 1, top). As for the w-alkenyl isothiocyanates (5-8), the values of log(C/T) in wasabi were higher than those in horseradish. Above all, (5) and (6) possessed the highest value after (2). On the other hand, the aryl isothiocyanates (10 and 11) in horse radish had higher values than those in wasabi. The isothiocyanate (11) had highest value except for (2). Hence, it seems that the remarkable difference in odor between wasabi and horseradish is due to (5-8), and (10 and 11). In general, the oj-methylthioalkyl isothiocyanates (9 and 12-14) had lower log(C/T) values than those for the other isothiocyanates. The value of log(C/T) for 6-methylthiohexyl isothiocyanate (13) in wasabi was the highest of all the a)-methylthioalkyl isothiocyanates. The last note given out by wasabi was similar to the odor of (13). Consequently, it is suggested that the character impact compound in the last note of wasabi is attributed to be the presence of (13). The flavor dilution (FD) factor, 2 , is the maximum dilution value at which the odor is detected by GC sniffing (24). The odorant with high FD factor is considered to be potent (25). In general, the FD factor is proportional to the aroma value of the compound. Figure 1 (bottom) shows the exponents (n) of the FD factor 2 , versus their retention indices (RI) of the isothiocyanates. The behavior of each compound in Figure 1 (bottom) was similar to that in Figure 1 (top). Therefore, the values of log(C/T) for each isothiocyanate observed in Figure 1 (top) were found to be proportional to the corresponding exponents (n) of the FD factor 2 shown in Figure 1 (bottom). Usually wasabi is prepared by grinding the raw stem at room temperature. The pungency of wasabi decreases rapidly as time passes. The yields of the alkyl isothiocyanates in the different pHs at 3°C are shown in Figure 2 (top). The distinguishing increase in the yields of the alkyl isothiocyanates (1, 3 and 4) took place within 1 minute as a result of the rapid hydrolysis of thioglucoside. The yields of (1, 3 and 4) increased in the order: pH 4

Characteristic Odorants of Wasabi (Wasabia japonica matum

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