Agric. Biol. Chem., 53 (4), 1051 -1055, 1989 1051

Degradation and 0-Methylation of among Volatile Flavor Components of Dried Bonito (Katsuobushi) by Aspergillus Speciest Mikiharu Doi, Masayori Ninomiya, Muneaki Matsui, Yoshihiro Shuto* and Yoshiro Kinoshita* Marutomo Co., Ltd., 1696 Kominato, Iyo, Ehime 799-31, Japan * Department of Agricultural Chemistry, Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790, Japan Received October 28, 1988

Eleven strains of Aspergillus species, isolated from dried bonito (Katsuobushi), were grown in a liquid medium containing the individual phenolic compounds, in order to determine the fate of phenols during the molding process in Katsuobushi production. Of the 10 phenols studied, guaiacol, creosol and 2,6-dimethoxyphenol were 0-methylated by 5 strains of Aspergillus species, and the others were degraded by somestrains studied. Similar changes in the phenols were presumed to occur also during the molding process in Katsuobushi production. It was concluded that the pungent smoky flavor becamemilder mainly through biological degradation or 0-methylation of phenols during the molding process.

Many studies on the volatile flavor com- a/.14) reported the O-methylation of chloro- ponents of "Katsuobushi" have appeared in phenols and chloroguaiacols by bacteria, and the literature.1 ~n) It is knownthat the smoky Wiese and Vargas15) the (9-methylation of 2,5- flavor characteristic of Katsuobushi is due to dichloro-4-methoxyphenol by soil microorgan- wood smoke and that the pungent smoky isms. flavor is moderated during the molding proc- In this paper, wereport the degradation and ess. However, little has been reported con- (9-methylation of phenols by Aspergillus spe- cerning the fate of volatile flavor components cies isolated from Katsuobushi and propose of Katsuobushi during the molding process in that the smoky flavor of Katsuobushi is mod- its production. erated and becomes milder during the molding Kim et al.12) compared the contents of 4- process mainly through biochemical changes alkylguaiacols (4-methyl- and 4-ethyl-) and 4- in phenols. aklylveratroles (4-methyl- and 4-ethyl-) in "Arabushi" (non-molded Katsuobushi) with those in "Honbushi" (molded Katsuobushi). Materials and Methods Onthe basis of the results, they assumed that Isolation of strains. Eleven strains of Aspergillus species most of the veratroles in the latter were derived were isolated from Honbushi produced in Makurazaki, from the corresponding guaiacols through Kagoshima Prefecture. They were tentatively identified16) and classified into three groups: A. repens (5 strains), A. biological (9-methylation. There have been a glaucus (4 strains) and A. candidus (2 strains). These few papers on the O-methylation ofphenols by strains were maintained by periodic transfer to fresh PSA microorganisms. Neilson et al.13) and Allard et medium (2.5% potato flour, 2.0% saccharose and 2.0%

f A part of this paper was presented at the meeting of the Nishinippon Branch of the Agricultural Chemical Society of Japan at Ehime University on Oct. 12, 1988. 1052 M．DoIefα上

agar). were 250°C. The flow rate of the carrier gas (He) was lOml/min.Theion source temperature of the massspec- Medium.The PSAmediumwas adopted for pre- trometer was 270°C and the ionizing energy 70 eV. cultures. MY20 medium (0.5% peptone, 0.3% malt ex- Collection of volatile flavor components. Non-molded tract, 0.3% yeast extract and 20% glucose) was used for (Arabushi) and molded (Honbushi) Katsuobushi were metabolic experiments. shaved and powdered just before use. One kilogram of each powder was suspended in 5000ml of distilled water Metabolic experiments. One of the compoundsin Table and the mixture was steam-distilled at atmospheric pres- I, whose presence in Katsuobushi had been reported in the sure until 5000ml of distillate had been collected. The literature cited above, was added as follows. Forty-nine ml distillate was saturated with sodium chloride and then of the MY20medium was placed in a 100-ml Erlenmeyer extracted with three 3000-ml portions of . The flask, which was then autoclaved at 120°C for 15min. combined ether extracts were dried over anhydrous so- After cooling, 5mg of the compound in 1 ml of dium sulfate and then concentrated to about 400 mg, and was added aseptically to the flask. A strain on a 2-mm the concentrate of the volatile flavor components was cube of the PSA medium, pre-cultured at 25°C for 7 days, subjected to gas chromatographic analysis. wasinoculated into the flask and subjected to stationary incubation at 25°C for 20 days. Uninoculated medium Reagents. Creosol was synthesized by Clemmensen re- served as a control. All of the experiments were repeated duction of .17) 2,6-Dimethoxyphenol was obtained twice, with essentially identical results. from Aldrich Chemical Co., and the other reagents from Wako Pure Chem. Ind., Ltd. Extraction and analysis of substrates and metabolites. The culture broth was filtered, and the filtrate was satu- rated with sodium chloride and then extracted with three Results and Discussion 50-ml portions of diethyl ether. The combined extracts were dried over anhydrous sodium sulfate and then con- Degradation and O-methylation ofphenols by centrated to about 3 ml under reduced pressure. A solution Aspergillus repens MA0197 of hexadecane in diethyl ether (0.6mg/ml) was added to the concentrate as an internal standard and then the Figure 1 shows that a new compound(peak mixture was made up to 5ml and subjected to GC 3) was produced when A. repens MAO197was analysis. incubated at 25°C for 20 days in the presence of creosol (4-methylguaiacol. Since the new GCconditions and quantitative analysis compoundwas not produced on incubation of Analysis of substrates and metabolites. GCwas con- the strain without creosol under the same ducted with a Shimadzu model GC-9A equipped with a flame ionization detector on a glass column (3.2mm i.d.x2m) packed with 0.5% EGA (coated on 60~80 meshChromosorbW). The column temperature was pro- grammed from 90°C to 195°C at the rate of 4°C/min and then held at 195°C. The injector and detector tem- peratures were 180°C. The flow rate of the carrier gas (He) was 25 ml/min. Quantitative analysis was perform- ed with a Shimadzu Chromatopac model C-R2AXwith hexadecane as an internal standard. Analysis of volatile flavor components of Katsuobushi. The GCanalyzer used was the same as above. A fused silica WCOTcapillary column (0.24mm i.d. x 50m) coat- ed with Thermon 3000A was used. The column tempera- ture was programmed from 90°C to 195°C at the rate of 4°C/min and then held at 195°C. The injector and detector temperatures were 180°C. Analysis was carried out ac- cording to the method of Kim et al.12) GC-MSconditions. GC-MSwas carried out with a Fig. 1. Gas Chromatograms of Creosol and Its Me- Shimadzu AUTOGC-MSmodel 9020-DF. GC was per- tabolite. formed on a silica capillary column (HiCap CBP 20; Cultures uninoculated (A) and inoculated with Aspergillus 0.25mmi.d. x 25m). The column temperature was pro- repens MAO197 at 25°C for 20 days (B). Creosol (1), grammed from 100°C to 230°C at the rate of5°C/min and hexadecane (2), as an internal standard, and the metab- then held at 230°C. The injector and detector temperatures olite of creosol (3). Degradation and O-Methylation of Phenols in Dried Bonito 1053

Table I. Degradation and O-Methylation of Phenols by Aspergillus repens MAO197

Recovered (mg) Compound Growth Unchanged Methylated Creosol 3.86 0.24 + + + Guaiacol 2.92 0.59 + + + Fig．2．MassSpectrumofPeak3Material． 2,6-Dimethoxyphenol 1.22 1.29 + + + 2,4-Dimethylphenol 4. 64 0.00 + 3,4-Dimethylphenol 4. 59 0.00 + 3,5-Dimethylphenol 4.28 0.00 + 0- 4. 15 0.00 + + m-Cresol 3.02 0.00 + + /7-Cresol Trace 0.00 + + + Trace 0.00 + + +

Table II. Degradation and 0-Methylation of Guaiacol by Different Aspergillus Species

Fig. 3. Compounds That Undergo 0-Methylation by Recovered (mg) Aspergillus repens MAO1 97. Strain Group Growth Unchanged Methylated MA 0196 B Trace 0.00 4- + + conditions, the compoundin peak 3 was ap- MA 0197 A 2.92 0.59 + ++ parently a metabolite of creosol. The mass MA 0198 A 2.53 0.73 + + + spectrum(Fig. 2: m/z: 152(M+), 137, 122, 109, MA 0199 B Trace 0.00 + + + 91) indicated18) that it was 1,2-dimethoxy-4- MA 0200 B Trace 0.00 + + + methylbenzene (4-methylveratrole), i.e., creo- MA 0201 A 2.32 0.96 + + + MA 0202 A 3.59 0.31 + + + sol was 0-methylated by A. repens MA0197. MA 0209 C 4.98 0.00 + + The O-methylations of guaiacol and 2,6-di- MA 0210 A 4.61 0.33 + methoxyphenol were also confirmed with the MA 0212 B Trace 0.00 + 4- same GC-MSmethod. The structures of the MA 0214 C 4.95 0.00 + + phenols capable of undergoing O-methylation and the methoxyphenols thereby produced are summarized in Fig. 3. The other phenols were studied here also seem to be metabolized degraded without 0-methylation (Table I), but through some similar pathways. no degradation products could be detected. Yeratrole, or O-methylated guaiacol, was de- Degradation and O-methylation ofguaiacol by graded slightly. different Aspergillus species Allard et al.14) proposed that phenols ex- The ability of a variety of Aspergillus species posed to cells were detoxified through O- isolated from Katsuobushi to methylate guai- methylation since all the (9-methylated metab- acol was investigated. Table II shows that 5 olites were less toxic to the microorganisms strains of A. repens (Group A) were found to than their precursors. It is known that some exhibit such ability. Four strains of A. glaucus aromatic compounds are metabolized through (Group B) degraded guaiacol completely, the oxidation of their alkyl side chains, and whereas 2 strains of A. candidus (Group C) hydroxylation of the aromatic ring and/or showed neither O-methylation nor degrada- cleavage of the ring, and some of the metab- tion. olites enter the TCA cycle.19) The phenols 1054 M. Doi etal.

Table III. Contents of Volatile Phenols and Methoxyphenols in Katsuobushi before and after the molding process Content (mg/lOO g) Compound "Arabushi" "Honbushi"

Guaiacol 4.69 (5.72) 4.00 (4.76) Veratrole 0.34 (0.41) 0.86 (1.02) Creosol 3.06 (3.73) 2.55 (3.04) l,2-Dimethoxy-4- 0.60 (0.73) 0.72 (0.86) methylbenzene 2,6-Dimethoxyphenol 2.64 (3.22) 2.31 (2.75) 1,2,3-Trimethoxybenzene 0.25 (0.30) 0.32 (0.38) Phenol+o-Cresol 18.33 (22.35) ll.86 (14.12) m-Cresol 5.98 (7.29) 4.42 (5.26) /7-Cresol 4.48 (5.46) 3.31 (3.94) Fig. 4. Time Course of Degradation and (9-Methylation 2,6-Dimethylphenol 0.31 (0.38) 0.25 (0.30) of Guaiacol by Aspergillus repens MAO197at Different 3,4-Dimethylphenol 0.41 (0.50) 0.39 (0.46) Temperature. Values in parentheses are the contents on a dry basis. Guaiacol (O) and methylated guaiacol (veratrole) (#).

Fate of volatile phenols and methoxyphenols in termine the optimumconditions for the O- Katsuobushi during the molding process methylation of phenols from the viewpoint of The contents of volatile flavor components flavor-creation. The degradation and O- in Katsuobushi before and after molding, methylation ofguaiacol by A. repens MA0197 "Arabushi" and "Honbushi," respectively, were examined at different temperatures. were comparedin order to gain an insight into Broth cultured at 20°C, 25°C and 30°C, re- the fates of phenols and methoxyphenols in spectively, was analyzed at one weekintervals Katsuobushi (Table III). for 7 weeks (Fig. 4). The contents of veratrole (the product of O- The degradation of guaiacol was highest at methylation of guaiacol), 1,2-dimethoxy-4- 20°C and lowest at 30°C, while the O- methylbenzene (that of creosol) and 1,2,3- methylation of guaiacol was highest at 25°C. trimethoxybenzene (that of 2,6-dimethoxy- These results demonstrated that the molding phenol) in "Honbushi" were higher than temperatures of 25°C to 28°C used in com- those in "Arabushi"; whereas the contents of mercial Katsuobushi production are well suit- all the phenols were lower in "Honbushi" than ed for the 0-methylation of guaiacol. in "Arabushi." It might be concluded that It is concluded that the (9-methylation and the degradation or O-methylation of phenols degradation of phenols by Aspergillus species amongvolatile flavor components of Katsuo- play an important role in the creation of the bushi actually occurred during the molding characteristic flavor of Katsuobushi. process. References Optimum temperature for O-methylation of guaiacol 1) K. Nishibori, Nippon Suisan Gakkaishi, 31, 41 (1965). It is known12) that the agreeable smoky fla- 2) K. Nishibori, Nippon Suisan Gakkaishi, 31, 47 (1965). vor of Katsuobushi is brought about through 3) K. Nishibori and K. Okamoto, Nippon Suisan molding. Since one of the components re- Gakkaishi, 37, 156 (1971). 4) K. Nishibori and K. Okamoto, Nippon Suisan sponsible for the flavor is 1,2-dimethoxy-4- Gakkaishi, 37, 610 (1971). methylbenzene, the product of O-methylation 5) K. Nishibori and K. Okuyama, Nippon Suisan of creosol, it seemed to be important to de- Gakkaishi, 38, 231 (1972). Degradation and O-Methylation of Phenols in Dried Bonito 1055

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