Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium Edule Seeds

日本調理科学会誌 Vol.J. Cookery Sci. 47，No.Jpn. Vol. 4，202～213（2014） 47 No. 4（2014）〔Article〕

Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium edule Seeds

Martha Santoso* Tomoko Yamaguchi** Teruyoshi Matoba*** Hitoshi Takamura*§

Pangium edule Reinw. is a tropical plant from Southeast Asia and southern Pacific islands whose seeds are commonly consumed by the native population after being treated to remove its high content of cyanogens. It can be consumed directly as vegetable or exposed to pit fermentation that changes the appearance and adds specific savory flavor, and consumed as spice. The fermented feed is called kluwak. It has been reported that the seeds contain high level of antioxidant activity and high amount of its components. Here we report the distribution of the antioxidant activity and component and the effects of pit fermentation and thermal cooking process on the antioxidant components of Pangium edule seed, measured using 1,1-diphenyl-2-picrylhydrazyl（DPPH） and oxygen radical absorbance capacity（ORAC）, total phenolic, ascorbic acid and vitamin E contents, and fatty acid composition assays. In addition, the changes in physicochemical properties of the seeds during pit fermentation were also quantified. The antioxidant components of the seed is mainly distributed in the non-oil fraction when measured using DPPH radical- scavenging activity and almost equally distributed in non-oil and oil fractions when measured using ORAC. The antioxidant components in the non-oil and oil fractions consist mostly of phenolic compounds and γ-tocotrienol, respectively. Pit fermentation significantly increased the antioxidant activity of both fractions, most likely caused by the formation of Maillard reaction products. Similarly, thermal cooking process increased the antioxidant activity of both fractions.

Keyword：kluwak, pit fermentation, antioxidant activity, Pangium edule

tannins, and tocols （Sun, 1990; Di Carlo et al., 1999; INTRODUCTION Rice-Evans et al., 1996; Calucci et al., 2003）. Spices, in par- Antioxidants, especially those from food sources, have ticular, are excellent sources of antioxidants, especially received increased attention by nutritionists and medical phenolic compounds that have been reported to show researchers because they are believed to play important good antioxidant activity（Di Carlo et al., 1999）. roles in the prevention and treatment of a variety of Pangium edule Reinw. is a tropical tree that grows in chronic diseases, such as premature aging, age-related Southeast Asia and Southern Pacific islands that was pre- decline in immune system, cardiovascular disease and can- viously included in the family Flacourtiaceae （Burkill, cer, most of which are mediated by oxidative stress（Sun, 1935） and is currently part of the family Gnetaceae（Kato et 1990）. The proposed mechanisms by which antioxidants al., 1995）. In Indonesia, it is called “picung” or “kepayang” protect cells from oxidative stress is by scavenging free and it grows in many Indonesian islands, especially in radicals and halting the chain reaction of lipid peroxida- Java. All parts of P. edule tree contain relatively high tion, which can cause DNA damaged（Di Carlo et al., amount of cyanogens and therefore are poisonous 1999）. The main source of antioxidants for human comes （Deshpande et al., 2000）. The main product of this plant is from food, and that is the reason why the consumption of the endosperm of its fruit seed, which is usually consumed foods with high antioxidant content is strongly recom- as a spice, following cyanogens removal and pit fermenta- mended （Sun, 1990; Di Carlo et al., 1999）. Numerous tion. The cyanogens were removed by soaking and boiling researches have been done to identify foods that are rich the seed in water, which is followed by pit fermentation in antioxidants. Vegetables, fruits, tea, wine, as well as for approximately 40 days. During fermentation the herbs and spices are known to contain very effective natu- endosperm undergoes dramatic changes in appearance, ral antioxidants such as flavonoids, lignans, phenolic acids, the color changes from milky white to dark brown （Andarwulan et al., 1999a）. The endosperm of the fer- * Nara Women’s University ** Niigata University mented seed is locally known as “kluwak”, a spice that is *** Kansai University of Welfare Science commonly used in Indonesian traditional cuisine, which § Inquiry Faculty of Human Life and Environment, Nara Women’s can give a specific savory flavor and a dark brown color University. Kitauoya-nishimachi，Nara 630-8506, Japan TEL 0742-20-3454 FAX 0742-20-3447 to the food. Kluwak has been previously reported to have

8 （202） Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium edule Seeds

antioxidant activity（Andarwulan et al., 1999b）. However ence standards were purchased from Eisai （Tokyo, there are little data about the major antioxidant compo- Japan）. Fluorescein （sodium salt） and 6-hydroxy- nents and their distribution in kluwak, and there has been 2,5,7,8-tetramethylchroman-2-carboxylic acid（Trolox） no data on the changes on antioxidant activity and its standard were purchased from Sigma-Aldrich（St. Louis, components in kluwak during thermal cooking process. MO, USA）. Randomly methylated β-cyclodextrin Furthermore, the cause for the physicochemical changes （RMCD）（Trappsol） was purchased from Cyclodextrin that occur in kluwak during pit fermentation has never Technologies Development Inc.（High Springs, FL, USA）. been addressed so far. It is also unknown whether the Bio-Rad protein assay dye reagent concentrate was pur- antioxidant activity of kluwak is present prior to fermen- chased from Bio-Rad Laboratories, Inc.（Hercules, CA, tation, or is generated during fermentation. USA）. The water used for HPLC was purified with Milli- The main objective of this research was to analyze the Q Labo equipment（Millipore Japan, Tokyo, Japan）. changes of antioxidant components and activity of P. edule 2. Sample preparation seed during fermentation and thermal cooking process. Sample preparation for pit fermentation was done as Several parameters examined were the antioxidant activ- previously described（Andarwulan et al., 1999b）. In short, ity, measured using 1,1-diphenyl-2-picrylhydrazyl seeds were taken from the ripe fruits of P. edule and then （DPPH） and oxygen radical absorbance capacity（ORAC） were washed with clean water. The seeds were boiled for assays, total phenolic, ascorbic acid and vitamin E con- 2－3 hours, and then left to cool at room temperature for tents, and fatty acid composition. The thermal cooking several hours. The boiled seeds were used as the sample process was limited to boiling, because it was the most for 0-day fermentation（unfermented）. Fermentation pro- common way to prepare foods with kluwak. Additionally, cess was done in Osaka, Japan at ambient temperature the color changes and molecular weight distribution of the during summer. Several 30 cm-deep pits of the same antioxidant component was also addressed, and the possi- diameter were dug. The seeds were covered with wood bility of reactions that occurred during pit fermentation of ash and buried in the pit for 20- and 40- days. During fer- kluwak was discussed. mentation, the humidity of the soil covering the pits was maintained by sprinkling with tap water once per day. MATERIALS and METHODS Following 20- and 40- day of fermentation, the seeds 1. Sample and materials were taken out, washed from the ash, air-dried and stored For the analysis of the effects of pit fermentation, the at －40℃. fruits of P. edule were obtained from a traditional village Prior to analysis, the seed shells were broken down to in Bogor, West Java, Indonesia. Ripened fruits were picked get the soft, meaty endosperm. For the analysis of the from the tree and then the seeds were taken from the effects of pit fermentation, the endosperm was lyophilized fruits. The seeds were then washed and boiled as for 72 hrs（VD-400F, Taitec Co., Tokyo, Japan）. For the explained below. For the analysis of the effects of cooking, analysis of the effects of cooking, the endosperm was fermented P. edule seed（kluwak） were purchased from a mixed with distilled water with ratio of 1 : 5（w/v） and traditional market in Bandung, West Java Indonesia. Both homogenized with a Polytron homogenizer（PT MR2100, the boiled and fermented seeds were brought to Japan at Kinematica AG, Switzerland）.The homogenized mixture ambient temperature and directly treated and analyzed as was then kept inside several sealed tubes, immersed in explained below. boiling water（97－100℃） for 10, 20, 30, 60, 90, and 120 Gallic acid standard, Folin-Ciocalteu reagent, phenolic min and cooled immediately in cool water. An unboiled acid standard, buffer components, DPPH, 2,4-dinitro- homogenized mixture was used as a control. The samples phenylhydrazine, sulfuric acid, phosphoric acid, and were then lyophilized for 72 hrs. 1,4-dioxane were purchased from Nacalai Tesque（Kyoto, 3. Separation of the oil and non-oil fractions Japan）. Acetic acid, 2,6-dichloroindophenol, 2,2′-azobis The lyophilized samples were extracted with 5 times （2-aminidopropane） dihydrochloride（AAPH）, stannous volume of hexane for 30 min in the dark with a KM chloride, metaphosphoric acid, trimethylamine, HPLC shaker（Iwaki Sangyo, Tokyo, Japan）. The suspensions grade of methanol, acetonitrile, hexane and other chemi- were centrifuged at 1,400×g at 4℃ for 20 min. The cals of analytic grade were obtained from Wako Pure extraction was repeated twice. The resulting supernatants Chemical Industries, Ltd.（Osaka, Japan）. Vitamin E refer- were combined and filtered using filter paper and evapo-

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rated by rotary evaporator. The oil fractions obtained 7.4（800 μL） and 0.5 mM DPPH in ethyl acetate（1 mL） were sealed under argon gas and stored at －40℃ until and vigorously shaken. The absorbance for both of oil and analysis. The resulting residue （non-oil fraction） was non-oil fraction reactions was measured by a UV-2100 PC dried under vacuum to remove hexane, and were ground UV-VIS spectrophotometer（Shimadzu, Kyoto, Japan） at into fine powder and stored at －40℃ until analysis. 517 nm after incubated for 20 min at room temperature in 4. Extraction the dark. The DPPH radical-scavenging activity was cal- The non-oil fractions（50－100 mg each） were extracted culated from the difference in absorbance between blank with 2 mL of 90％ methanol containing 0.5％ acetic acid. and samples with Trolox as standard. The DPPH radical- The solutions were stirred for 1 min, and supernatants scavenging activity was expressed as μmol trolox equiva- were recovered by centrifugation at 1,400×g for 10 min. lent per 100 g dry weight. The extraction was repeated twice and the extracts were 8. ORAC assay dried under nitrogen flow. The residues were dissolved in ORAC assay for oil and non-oil fractions was evaluated 90％ methanol, filtered, and used for total phenolic content, according to the lipophilic and hydrophilic ORAC methods DPPH radical-scavenging activity with proper dilution. of Prior et al.（2003）, respectively. Sixty-three milimolar For total ascorbic acid analyses the freeze dried non-oil AAPH and 14 μM fluorescein were dissolved in 75 mM fractions were extracted with 5％ metaphosphoric acid phosphate buffer（pH 7.0） and was incubated at 37℃ solution and incubated at 10℃ for 30 min. The extracts before used. The oil fraction（0.05 g） was dissolved in 250 were filtered and directly used for ascorbic acid analyses. μL acetone and then diluted with 7％ RMCD solution The oil fraction was used directly without further extrac- （50％ acetone / 50％ water, v/v） into proper dilution. The tion. 20 μL oil fraction solution was added to 96-well black 5. Color measurement polystyrene microplate（Costar No. 3915） and added with Color was evaluated at room temperature by using a 200 μL fluorescein solution followed by 75 μL of AAPH. tristimulus color-meter NE2000 （Nippon Denshoku Seven percent of RMCD solution was used as blank and Industries,Tokyo, Japan）. Lyophillized samples were dissolved Trolox was used as standard. For the non-oil reconstituted with water and spotted onto a glass Petri fraction extract, further dilution was made with phosphate dish as previously described（Morales and van Boekel, buffer. A 20-μL portion of the diluted sample was added 1998）. The color values were expressed in terms of the to a well in a 96-well microplate. The 200 μL fluorescein CIE L*a*b* uniform color space, where L* represents solution and the 37.5 μL of AAPH were added in the lightness, a* represents chromaticity on a green（－） to same manner as that for the oil fraction assay except that red（＋） axis and b* represents chromaticity on a blue phosphate buffer was used as blank and to dissolve （－） to yellow（＋） axis. Trolox. The fluorescence microplate reader Gemini XPS 6. Browning measurement （Molecular Devices, Sunnyvale, CA, USA） was set with Browning index of the sample was recorded by their an excitation wavelength of 485 nm and an emission absorbance at 420 nm on a UV-2100 PC UV-VIS spectro- wavelength of 520 nm from the top of the plate and pro- photometer （Shimadzu, Kyoto, Japan）, as previously grammed to read every min for 60 min at 37℃. Plates described（Morales and van Boeckel, 1998）. were shaken for 8 sec following each reading. Data were 7. DPPH radical-scavenging activity expressed as μmol Trolox equivalent per 100 g dry DPPH radical-scavenging activity for the oil fraction weight. was evaluated according to the method of Espin et al. 9. Total phenolic content （2000） with a minor modification. Trolox, oil fraction The total phenolic content for non-oil fraction was samples, and DPPH were dissolved in ethyl acetate. An determined by the Folin-Ciocalteu method（Singleton and aliquot of sample solution（144 μL） was mixed with 0.27 Rossi, 1965）. The extract of non-oil fraction（200 μL） was mM DPPH in ethanol（1800 μL） and vigorously shaken to reacted with 800 μL 7.5％ sodium carbonate in water and adjust the final concentration of DPPH to 0.25 mM. DPPH 1 mL Folin-Ciocalteu reagent for 30 min in room temper- radical-scavenging activity for non-oil fraction was evalu- ature. Gallic acid was used as standard. The absorbance ated as previously described（Blois, 1958）. Trolox and values of the samples and standard were measured at 765 DPPH were dissolved in ethanol. Each of extract solutions nm. Total phenolic content was expressed as μmol gallic （200 μL） was mixed with 100 mM Tris-HCl buffer pH acid equivalent per 100 g dry weight.

10 （204） Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium edule Seeds

10. Vitamin E content mm i.d ×25 m, Shinwa Chemical Industries Ltd., Kyoto, Vitamin E（ β-tocopherol and γ-tocotrienol） content in Japan）. The flow rate of argon gas was 0.6 mL/min, and the oil fraction was determined according to the method the column oven temperature was programmed from 160 of Ueda and Igarashi（1990） by HPLC coupled with fluo- to 220℃ at the rate of 2℃/min. The fatty acid composi- rescence detector. The HPLC system consists of a Cosmo- tion was expressed as the w/w percentage of a specific sil 5SL-11 column（4.6×250 mm, Nacalai Tesque Inc., fatty acid to total oil fraction . Kyoto, Japan）, an L-6000 pump and an F-1000 fluores- 13. Identification of phenolic compounds cence detector（Hitachi, Tokyo, Japan）. Excitation and The phenolic compounds were identified according to emission wavelengths were set at 295 nm and 325 nm. the method of Sakakibara et al.（2003）. The non-oil frac- The mobile phase was hexane/1,4-dioxane/2-propanol tion was extracted with 90％ methanol containing 0.5％（98.5/1.0/0.5, by vol.） and flow rate was set at 1.0 mL/ acetic acid. The extracts were dried under nitrogen flow min. The oil fraction was diluted properly in n-hexane and the residues were dissolved in DMSO. The DMSO before analysis. 2,2,5,7,8-Pentamethyl-6-chromanol extracts were injected HPLC （Hitachi HPLC series （PMC） was used as an internal standard. The vitamin E D-7000） equipped with chromatography data station soft- content was expressed as mg of tocopherol or tocotrienol ware, autosampler（D-7200）, column oven（D-7300）, and per 100 g dry weight. As a comparator, sesame seed was diode array detection system（D-7450） to monitor the also used, extracted and prepared with the same method. absorbance at all wavelengths from 200－600 nm. Capcell 11. Ascorbic acid content pak C18 UG120（250×4.6 mm i.d., S-5, 5 μm, Shiseido Ascorbic acid content was determined by HPLC as Co., Ltd., Tokyo, Japan）, joined with guard column（10× described previously（Kishida et al., 1992）. The extract of 40 mm i.d） was used at 35℃. Gradient elution was per- non-oil fraction （100 μL） was mixed with or without formed with solution A, composed of 50 mM sodium phos- 0.2％ 2,6-dichloroindophenol（50 μL）, 1％ stannous chlo- phate（pH 3.3） and 10％ methanol, and solution B, com- ride in 5％ metaphosphoric acid solution（50 μL） and 2％ prising 70％ methanol, delivered at a flow rate of 1.0 mL/ 2,4-dinitrophenyl hydrazine in 4.5 M sulfuric acid solu- min as follows: initially 100％ of solution A; for the next 15 tion（120 μL）. The reaction mixture was incubated in a min, 70％ A; for another 30 min, 65％ A; for another 20 water bath at 37℃ for 3 hrs, followed by the addition of min, 60％ A; for another 5 min, 50％ A; and finally 0％ A ethyl acetate and water（1 mL each）. After mixing and for 25 min. The injection volume for the extract was 10 μL. centrifugation for 5 min（1,400×g, 4℃）, 300 μL of the The detected peaks were compared with respect to reten- ethyl acetate layer was taken out and dried under nitro- tion time and spectra of aglycons of standard chemical. gen. The residue was dissolved in 200 μL of acetonitrile 14. Soluble protein content and applied to HPLC analysis. HPLC analysis was carried The soluble protein content was measured using the out on a Cosmosil 5C18-AR-2 column with dimension of Bradford method（Bradford, 1976）. The soluble protein 4.6×250 mm （Nacalai Tesque, Kyoto, Japan） with a was extracted from the non-oil fraction of the samples as Shimadzu SPD-10AV US-VIS detector set at 505 nm and described previously（Theriault et al., 1999）. Briefly, the a Rheodyn injector fitted with a 20-μL loop. The mobile lyophilized samples were mixed with 0.1 N NaOH and left phase was acetonitrile/water（50/50, v/v） with 0.1％ tri- to extract in room temperature for 30 min. Samples were methylamine, adjusted to pH 3.5 with phosphoric acid, at centrifuged for 5 min at 10,000×g and the supernatant a flow rate 1 mL/min. Ascorbic acid content was calcu- solution was decanted. Aliquots（100 μL） of samples or lated by subtracting the value of sample mixed without 100 μL of NaOH（blank） were each mixed in a test tube 2,6-dichloroindophenol from the value of sample with with 5 mL of 1 : 4 diluted Bradford dye reagent（Bio-Rad 2,6-dichloroindophenol. The data were expressed as μg Laboratories） that has been filtered, stored, and diluted ascorbic acid per 100 g dry weight. according to the manual provided by the manufacturer, 12. Fatty acid composition slightly modified by the addition of 3 mg/mL soluble poly- The fatty acid composition was determined after meth- vinylpyrrolidone（PVP）（approx. 40,000 Da）. After 15 anolysis of oil fraction according to the standard proce- min, an appropriate volume of each replicate was trans- dure（American Oil Chemists’ Society, 1989） using GC ferred to cuvettes. The absorbance at 595 nm was analysis （Shimadzu GC-17A）, with a flame ionization recorded against the dye reagent/NaOH blank using a detector（FID） and the capillary column（HR-SS-10, 0.25 spectrophotometer. Samples were calibrated against a

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standard of ovalbumin in 0.1 N NaOH. Protein content RESULTS AND DISCUSSION was expressed as g protein per 100 g dry weight. 15. pH measurement 1. Effects of pit fermentation on color Lyophillized samples were reconstituted in distilled During pit fermentation, the endosperm of P. edule water at a 0.1％（w/v） concentration. pH values were seeds experienced drastic changes in color, from light measured using a Mettler Toledo MP220 pH meter brown to black（Fig. 1）. The darkening of the endosperm （Mettler-Toledo, Greifensee, Switzerland）. color was also indicated by the significant decrease of the 16. Molecular weight distribution of the antioxidant lightness （L*） values at 20- and 40-day fermentation components （Table 1）. The a* and b* values also decreased signifi- The 90％ methanolic extract of the non oil fraction of cantly during fermentation, indicating the color transitions unfermented, 20- and 40-day fermented seeds was ultra- from reddish to greenish and yellowish to bluish, respec- filtrated by using Centriplus ultrafiltration membranes tively. The darkening of endosperm color strongly indi- with molecular weight cut-off of 50, 30, 10 and 3 kDa cates the occurrence of browning reaction. Enzymatic （Millipore）. Approximately 2.0 mL of the sample extract browning reaction is unlikely to occur since the seeds was ultrafiltrated and washed twice with the same vol- were treated by extensive boiling prior to fermentation, ume of 90％ methanol. which should inactivate the polyphenol oxidase that may 17. Statistical analysis be present in the seeds. It is highly likely that non-enzy- All the values were presented as means of triplicate matic browning reaction occurred during fermentation, analyses. The data analyses were performed using which is discussed in the latter part of this paper. Microsoft Excel. Two-sample t-test was used to calculate 2. Antioxidant activity statistical significance. The values were reported as The changes of antioxidant activities of the non-oil and means±standard deviation（SD） in all the results tables oil fractions during fermentation were measured using and graphs unless otherwise specified. The p values ＜ DPPH and ORAC methods, and the results are shown in 0.05 were considered as significant. Table 2. The DPPH radical-scavenging activity（RSA） of

Fig. 1. The changes of endosperm of Pangium edule seeds during fermentation.（A） before fermentation, （B） 20-day fermentation, and（C） 40-day fermentation.

Table 1. Color and browning index（A 420nm） measurement

Fermentation time A420nm （day） or boiling time L* value a* value b* value Non-oila Oilb （min） 0 d 46.90±0.08 14.46±0.06 17.88±0.02 0.04±0.02 0.13±0.01 20 d 23.33±0.27 9.69±0.06 11.61±0.09 0.45±0.07 0.20±0.01 40 d 8.24±0.06 4.27±0.05 3.053±0.04 1.45±0.08 0.35±0.02 0 min 8.03±0.04 2.76±0.17 2.22±0.05 1.38±0.02 0.29±0.03 10 min 6.35±0.07 2.91±0.02 2.08±0.02 2.17±0.12 0.53±0.02 20 min 6.37±0.02 2.85±0.03 2.24±0.15 2.26±0.04 0.48±0.10 30 min 6.35±0.05 2.72±0.05 2.30±0.02 2.27±0.03 0.51±0.05 60 min 6.26±0.05 2.82±0.19 2.28±0.34 2.28±0.02 0.48±0.07 90 min 6.01±0.06 2.66±0.31 2.06±0.19 2.28±0.01 0.55±0.08 120 min 6.15±0.04 2.53±0.03 2.00±0.01 2.27±0.02 0.49±0.05 aAbsorbance at 420 nm of 5％ solution of lyophillized sample diluted in 90％ methanol bAbsorbance at 420 nm of undiluted sample

12 （206） Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium edule Seeds

Table 2. Changes in the antioxidant activity indicated as DPPH-RSA and ORAC of the oil and non- oil fractions of Pangium edule seeds during fermentation and thermal cooking process.

a Fermentation time Antioxidant activity （day） or boiling time DPPH-RSA ORAC （min） Oil fraction Non-oil fraction Oil fraction Non-oil fraction 0 d 80.1±5.8 2,718.1±143.0 3,488.1±141.6 5,322.5±215.6 20 d 100.0±5.1 2,964.2±178.2 5,769.2± 80.1 6,058.1±217.5 40 d 108.9±7.3 3,702.7±222.5 6,480.3±270.9 7,260.1±114.1 0 min 121.8±1.2 3,215.5± 27.6 6,440.2± 56.9 7,056.0±131.6 10 min 126.7±0.6 5,253.1±334.1 6,633.8± 16.4 7,882.3±315.8 20 min 124.5±0.8 5,445.5±381.6 6,440.3± 25.6 7,703.4± 41.5 30 min 126.3±1.8 5,740.7±185.4 6,193.0±109.3 8,195.1±524.2 60 min 124.5±3.8 6,435.2± 29.3 6,469.6±145.7 8,821.4±120.9 90 min 125.5±1.2 6,747.9±325.0 6,143.2±320.3 9,149.5±102.5 120 min 125.3±3.9 6,912.0±242.1 6,444.1± 33.3 9,236.1± 10.5 aValues are expressed as μmol trolox equivalent/100 g DW

the unfermented and fermented P. edule seeds is mainly distributed in the non-oil fraction（>95％）. There is a significant discrepancy between the antioxidant activity of the oil fraction when measured using DPPH radical-scavenging activity and ORAC, in which ORAC measurement gave a much higher values（approx. 50 times） than DPPH radical-scavenging activity. As a result, the ORAC of unfermented and fermented P. edule seed is distributed approximately 40－50％ in the oil fraction. It has been reported that compared to DPPH radical-scavenging activity, ORAC method is more suitable for the measurement of antioxidant activity of oil-soluble materials（Prior et al., 2003; Teow et al, 2007）. It may be the reason why Fig. 2. Relationship between the DPPH-RSA and ORAC values（ μ the antioxidant activity of the oil fraction obtained by mol trolox equivalent/100 g DW） of the oil（empty symbols） and non-oil（filled symbols） fractions during fermenta- ORAC has much higher values than that obtained by tion（circles） and cooking process（triangles）, as shown in DPPH measurement. Table 2. Data for the oil fraction during cooking process are The antioxidant activity of the non-oil fraction also has not shown because there are only minor differences among the different cooking times. approximately 2 times higher values when measured using ORAC compared to that measured using DPPH Table 3. pH and soluble protein content radical-scavenging activity（Table 2）. Both methods, how- Fermentation day pHa Protein（g/100 g DW）b ever, gave significant correlations for the oil and non-oil 0 d 5.38±0.08a 35.77±1.34a fractions as shown in Fig. 2. 20 d 3.68±0.04b 12.46±0.88b Pit fermentation caused an increase on the antioxidant 40 d 2.95±0.10c 13.19±0.67b

a activity of the oil and non-oil fractions of P. edule seed pH value of 1％ solution of lyophilized sample diluted in H2O. b （Table 2）. The increase is significant（ p＜0.05） for each Protein content measured by Bradford method using ovalbumin as standard（g protein/100 g DW）. fraction and fermentation day, except for the DPPH radi- For each parameter, values with different letters in the same col- cal-scavenging activity of the non-oil fraction between 0- umn are significantly different （P＜0.05）. and 20-day fermentation（ p＝0.12）. To identify the antioxidant components of P. edule seeds, we extracted and 3. Effects of pit fermentation on antioxidant compo- analysed the antioxidant components in the oil and non-oil nents in the oil fraction fractions using the available methods for the common anti- P. edule seed is an oily seed, which more than half of it oxidants such as vitamin E, vitamin C, and phenolic com- （60％） consists of oil fraction（hexane-soluble fraction）. pounds. The oil fraction was slightly increased to 65％ during fer-

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Table 4. Fatty acid composition of Pangium edule seeds during fermentation and thermal cooking process Fermentationtime Fatty acid（％）（day） or boiling time （min） C16 : 0 C18 : 0 C18 : 1 C18 : 2 C18 : 3 C20 : 0 0 d 7.11 3.53 47.34 43.12 3.41 0.34 20 d 7.01 3.67 45.46 40.61 3.13 0.32 40 d 7.29 3.56 40.27 41.67 3.01 0.34 0 min 8,30 4.29 49.36 36.28 3.22 0.41 10 min 8.15 4.34 49.25 36.09 3.19 0.33 20 min 8.22 4.18 49.74 36.52 3.50 0.39 30 min 7.97 4.26 49.51 35.98 3.42 0.37 60 min 8.21 4.31 49.07 36.04 3.44 0.35 90 min 7.63 4.29 49.29 36.21 3.79 0.33 120 min 7.94 4.18 49.30 36.34 3.00 0.33

mentation. Analysis of the fatty acid composition of the oil Table 5. Vitamin E isoforms content of Pangium edule seeds fraction showed that oleic acid（18 : 1） and linoleic acid during fermentation and thermal cooking process （18 : 2） are the major fatty acids found in P. edule seed Fermentation day β-tocopherol γ-tocotrienol （d）（mg/100 g DW）（mg/100 g DW）（Table 4）. The percentage of unsaturated fatty acid did 0 d 2.2±0.10 13.2±0.3 not change significantly during fermentation, suggesting 20 d 2.1±0.05 12.9±1.0 the presence of antioxidative protection. This data con- 40 d 2.1±0.10 12.0±0.6 firmed the high antioxidant activity（ORAC） of the oil fraction（Fig. 2）. HPLC measurement on the amounts of vitamin E iso- 4. Effects of pit fermentation on antioxidant compo- forms showed that γ-tocotrienol is the most abundant nents in the non-oil fraction vitamin E isoform in the unfermented seed（13.16±0.3 The amount of antioxidant components that commonly mg/100 g dry weight）, followed by β-tocopherol（2.2± localize in the non-oil fraction of plant materials（phenolic 0.1 mg/100 g dry weight）, α- and γ-tocopherol（approx. compounds and vitamin C） was also examined. The unfer- 0.27 mg/100 g dry weight each） and α- and δ-tocotrienol mented seeds contain relatively high total phenolic content （trace amounts）. β-Tocotrienol and δ-tocopherol were that increased significantly （p＜0.05） during fermentation not detected in the oil fraction. Tocotrienol has been （Fig. 3） which may contribute to the increase of DPPH reported to have a higher antioxidant capacity than radical-scavenging of the non-oil fraction during fermen- tocopherol（Theriault et al., 1999）, although with lower bioavailability when taken orally（Packer et al., 2001）. Our results agree with the previous report, in which it was suggested that γ-tocotrienol plays an important role in the antioxidative protection of the unsaturated fatty acid of P. edule seed during fermentation（Andarwulan et al., 1999b）. Phenolic compound was not detected in the oil fraction at all period of fermentation tested（data not shown）. Interestingly, the amounts of γ-tocotrienol and β -tocopherol were reduced, although not significantly p（＝ 0.19） during fermentation（Table 5）. This is in contrast to the increase in antioxidant activity of the oil fraction. Thus, it was necessary to analyze further the antioxidant component of the oil fraction that was formed during fermentation, which is discussed in the latter part of this Fig. 3. The phenolic contents of the non-oil fraction of Pangium edule seeds during fermentation（filled bars） and kluwak paper. during boiling process（empty bars）, expressed as μmol gallic acid equivalent per 100 g DW.

14 （208） Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium edule Seeds tation. We also identified the main phenolic compounds in 5. Maillard reaction products contribute to the increase the unfermented seeds, which are（＋）-cathecin （53.87 of DPPH radical-scavenging activity during fermen- mg/100g dry weight） and （－）-epicathecin （19.45 tation mg/100g dry weight）, as shown in Fig. 4. In contrast to The color of P. edule seeds darkened significantly dur- the increase of total phenolic content during fermentation, ing fermentation（Fig. 1, Table 1）, and this change may the amounts of （＋）-cathecin and （－）-epicathecin indicate that browning reaction occurred during fermenta- reduced significantly during fermentation. It is possible tion. Because the seeds were treated by boiling prior to that the increase of the amount of phenolic content during fermentation and the dark colour develops slowly by time, fermentation, measured by Folin-Ciocalteu method, is the most likely reaction to occur is Maillard reaction caused by the generation of other antioxidant components （Belitz et al., 2004a）. Maillard reaction produces dark- during fermentation, most likely the Maillard reaction colored, high molecular weight melanoidins, which not products that has been reported to interfere with the only contribute to the aroma and color of the food but also Folin-Ciocalteu measurement（Yilmaz and Toledo, 2005）. have an antioxidant activity （Verzelloni et al., 2007）, In addition, we also measured the vitamin C content of the which may be the reason for the increase of DPPH radi- non-oil fraction of unfermented seeds, which was only cal-scavenging activity and total phenolic compound as present in trace amounts（approx. 0.142 mg per 100 g shown above. dry weight） and thus should not contribute significantly to To examine the contribution of melanoidins to the anti- the DPPH radical-scavenging activity of the non-oil frac- oxidant activity of P. edule seeds, we examined the molec- tion. The amount of vitamin C remains constant through- ular weight distribution of DPPH radical-scavenging aciv- out fermentation（data not shown）. ity and total phenolic content in the non-oil fraction of the P. edule seeds and its changes during fermentation. In this section, we performed DPPH assay instead of ORAC 60 (+)-Catechin 0-day fermentation because it is simpler to be performed and we have shown 50 that DPPH and ORAC assays on the non-oil fraction of

AU ) kluwak correlate significantly（Fig. 2）. The non-oil frac- 40 tion（in 90％ methanol containing 0.5％ acetic acid） of 0-, un it (m 30 20-, and 40-day fermented seeds were subjected to ultra- ce filtration to separate the antioxidant components based on 20 (-)-Epicatechin their molecular weights. Four types of membrane with Absorban 10 different molecular weight cut-offs were used（50, 30, 10 and 3 kDa）, and the DPPH radical-scavenging activity 0 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 and total phenolic content of each fraction were examined. Retention time (min) As shown in Fig. 5A, the DPPH radical-scavengning activity of the non-oil fraction of unfermented P. edule 60 seeds is distributed at various molecular weights, with the 40-day fermentation 50 main activity distributed at < 3 kDa, followed by those

AU ) having molecular weights more than 50 kDa. During fer- 40 mentation, the activity of antioxidant components with

30 MWs between 3-50 kDa increased to a significant level（ p ＜0.05）. Accordingly, the phenolic content of the non-oil 20 fraction of unfermented P. edule seeds is distributed at

ce unitAbsorban (m 10 molecular weights below 3 kDa（Fig. 5B）, and during fermentation the amount of phenolic compounds with higher 0 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 molecular weights also increased, which may correspond Retention time (min) to formation of Maillard reaction products during fermentation（Verzelloni et al., 2007; Yilmaz and Toledo, 2005）. Fig. 4. HPLC profile of the phenolic compounds of the non-oil fraction of Pangium edule seeds on 0 and 40-day fermentation The other parameters of Maillard reaction such as the monitored at 280 nm. browning index, change of pH and soluble protein content

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Melanoidins as the end products of Maillard reaction are polymeric subtances with high molecular weights have been previously reported to exhibit strong antioxidant activity（Chevalier et al., 2001）. In fact, melanoidins were reported to be the main antioxidant component of soy sauce and coffee （Yokotsuka, 1986; Cämmerer, 2006）, fermentation products that also undergo Maillard reaction. The data that are shown here suggested that Maillard reaction may contribute to the antioxidative properties of fermented P. edule seeds. Besides producing antioxidative compounds, Maillard reaction is also known to produce many desirable flavor compounds, which may also contribute to the pleasant taste of the fermented P. edule seeds, kluwak. Unfortunately, we have not been able to isolate and characterize the antioxidative products of Maillard reaction in kluwak, since the compounds in kluwak is very complex. The color of the oil fraction of P. edule seeds also changed during fermentation, as indicated by the absorbance at 420 nm（Table 1）, indicating that there may be oil-soluble Maillard reaction products that were formed during fermentation, which contribute to the increase of antioxidant activity in the oil fraction during fermentation. We did not analyze the molecular weight distribution of Maillard reaction products in the oil fraction because of technical limita- Fig. 5. Molecular weight distribution of DPPH-RSA（A） and total tion, i.e. the ultrafiltration unit that we used is not compat- phenolic content（B） of the non-oil fraction at different fer- ible with strong organic solvent like hexane. The mentation days. contribution of Maillard reaction products to the antioxidant activity in the oil fraction needs to be further anal- were also examined, and the results are shown in Tables ysed. 1 and 3. The pH of 0.1％ solution（in water） of the non- 6. Effects of thermal cooking process on antioxidant oil fraction of P. edule seeds decreased as fermentation activity time increased. It is possible that the carboxylic acids pro- To examine the effects of thermal cooking process on duced during Maillard reaction caused pH decrease（Van the antioxidant activity and components of fermented P. Boekel, 2001）, although the possibility of microbial involve- edule seeds, we used the samples that were available ment cannot be excluded. The change in the content of commercially from a traditional market in Indonesia as soluble protein of P. edule seeds was also assayed using described in the Experimental section. The commercial the Bradford method, and it was found that protein con- sample had slight differences in the parameters examined tent decreased during fermentation. The decrease is sig- from the 40-day fermented sample, most likely because of nificant between 0- and 20-day fermentation but not sig- the sample heterogeneity. nificant between 20- and 40-day fermentation. The As shown in Table. 2, thermal cooking process increased absorbance at 420 nm has also been widely used to iden- the antioxidant activity of both the non-oil and oil frac- tify the brown pigment produced in Maillard reaction tions of kluwak. In the non-oil fraction of kluwak, there is （Verzelloni et al., 2007; Van Boekel, 2001）, and the value a significant increase（63％） of the DPPH radical-scav- increased as fermentation time increased. These data enging activity upon boiling for 10 min, compared to the combined with the darkening of the endosperm color uncooked kluwak. Further boiling up to 30 min did not strongly suggest that Maillard reaction occur during fer- increase the DPPH radical-scavenging activity signifi- mentation. cantly （p＞0.05）. However, there was a significant

16 （210） Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium edule Seeds

increase between 30 and 60 min boiling time. Similarly, increase of the antioxidant activity in the oil fraction. We boiling caused an increase in the ORAC of the non-oil also observed that the amount of unsaturated fatty acid fraction, with the most notable increase between the 0 （Table 4） and vitamin E isomers（data not shown） of the and 10 min boiling time（16％）. The increase is significant oil fraction of kluwak did not change significantly（ p＞ only up to 10 min boiling time. Further boiling did not 0.05） during thermal cooking process. The vitamin E con- increase the ORAC of the non-oil fraction significantly tent of kluwak is not changed upon cooking because vita- compared to the 10 min boiling time. min E is relatively stable against heat treatment at tem- In the oil fraction, boiling for 10 min caused a 4％ and peratures below 180℃（Belitz et al., 2004b）. 3％ increase in the DPPH radical-scavenging activity and CONCLUSION ORAC, respectively. Both increases are not significant （p＞0.05）, and further boiling did not increase the anti- The antioxidant activities of unfermented and fer- oxidant activity significantly. Taken together, 10 minute mented P. edule seeds are distributed almost equally in boiling caused a significant increase of antioxidant activity the oil and non-oil fraction. The antioxidant components in of the non-oil and oil fractions measured by DPPH radi- the non-oil and oil fractions of unfermented seeds consist cal-scavenging activity and ORAC, and further boiling did mostly of phenolic compounds and vitamin E（ γ-tocotri- not necessarily increase the antioxidant activity of either enol isomer）, respectively. The antioxidant activity of the fraction significantly. both fractions increased significantly during fermentation, 7. Effects of thermal cooking process on antioxidant which may be attributed to the formation of Maillard components in the non-oil and oil fractions reaction products. The change in colour, reduction of pH, Total phenolic content of the non-oil fraction of kluwak decrease of soluble protein content and increase of increased with cooking time（Fig. 3）. Significant increases medium to high MW of antioxidant components confirm were observed until 10 min of boiling time. At this point, the occurrence of Maillard reaction during fermentation. there is a 21.5％ increase in total phenolic content of non- The antioxidant activity of P. edule seeds has been pres- oil fraction of kluwak compared with that without boiling, ent before fermentation, and fermentation caused compo- comparable to the increase of antioxidant activity sitional changes in the antioxidant components in both the observed by ORAC（16％）. Longer boiling time did not oil and non-oil fractions, leading to an increase in the anti- cause significant increase in the total phenolic content. oxidant activity. Similar to the fermented sample, we did not observe any Thermal cooking process caused a significant increase significant amount of （＋）-catechin and （－）-epicatechin in the antioxidant activity of kluwak, which is likely to be or vitamin C in the unboiled or boiled samples（data not contributed by Maillard reaction products. Boiling for 10 shown）, suggesting that phenolic compound did not con- min caused a significant increase in the antioxidant activ- tribute to the increase of antioxidant activity during ther- ity of the oil and non-oil fractions, but further boiling did mal cooking process. It is likely that Maillard reaction not cause any significant change. Overall, we suggest that progressed during thermal cooking process and thus we Maillard reaction products contribute strongly to the anti- measured the changes in color and browning index. As oxidant activity of kluwak during fermentation and less

shown in table 1, the lightness （L*） value and A420nm strongly during thermal cooking process. decreased and increased significantly during 10 min of ACKNOWLEDGMENT boiling time. Further boiling did not significantly decrease

or increase the L* value or A420nm, respectively. These This work was supported by Grant-in-Aid for Scientific data suggest that Maillard reaction products contribute to Research（C） 23500927. the increase of antioxidant activity of kluwak during the first 10 min of boiling process. REFERENCES The antioxidant activity of the oil fraction also increased American Oil Chemists’ Society（1989）, “Of cial and tentative significantly only during the first 10 min of boiling process. methods of the American Oil Chemists’ Society”, American Correlatively, we also observed that the decrease in L* Oil Chemists’ Society, Champaign IL, USA, Method Ce 2-66 Andarwulan, N., Fardiaz, D., Wattimena, G. A. and Shetty, K. value and increase in A are only significant during the 420nm （1999a）, Antioxidant activity associated with lipid and phe- first 10 min of boiling（Table 1）, suggesting that oil-solu- nolic mobilization during seed germination of Pangium ble Maillard reaction products are also responsible in the edule Reinw, J. Agric. Food Chem., 47, 3158-3163

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Andarwulan, N., Fardiaz, D., Wattimena, G. A. and Shetty, K. Morales, F. J. and van Boekel, M. A. J. S.（1998）, A study on （1999b）, Mobilization of primary metabolites and phenolics advanced Maillard reaction in heated casein/sugar solutions: during pit fermentation in seeds of Pangium edule Reinw, colour formation, Int. Dairy J., 8, 907-915 Process Biochem., 35, 197-204 Packer, L., Weber, S. U. and Rimbach, G.（2001）, Molecular Belitz, H. D., Grosch, W. and Schieberle, P.（2004a）, Carbohy- aspects of α-tocotrienol antioxidant action and cell signal- drates. “Food Chemistry” 3rd ed., Translated by M. M. ling, J. Nutr., 131, 369S-373S Burghagen, Springer-Verlag, Berlin, pp. 245-341 Prior, R. L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, Belitz, H. D., Grosch, W. and Schieberle, P.（2004b）, Vitamins. L., Hampsch-Woodill, M., Huang, D., Ou, B. and Jacob, R. “Food Chemistry” 3rd ed., Translated by M. M. Burghagen, （2003）, Assays for hydrophilic and lipophilic antioxidant Springer-Verlag, Berlin, pp. 409-426 capacity （oxygen radical absorbance capacity （ORAC Blois, M. S.（1958）, Antioxidant determination by the use of （FL））） of plasma and other biological and food samples, J. stable free radical, Nature, 181, 1199-1200 Agric. Food Chem., 51, 3273-3279 Bradford, M. M.（1976）, A rapid and sensitive method for the Rice-Evans, C. A., Miller, N. J. and Paganga, G.（1996）, Struc- estimation of microgram quantities of protein utilizing the ture-antioxidant activity relationships of flavonoids and principle of protein-dye binding, Anal. Biochem., 72, 248- phenolic acids, Free Radic. Biol. Med., 20, 933-996 254 Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H. and Burkill, I. H.（1935） A dictionary of the economic products of Kanazawa, K. （2003）, Simultaneous determination of all the malay peninsula. Crown Agents, London, pp. 1653-1654 polyphenols in vegetables, fruits, and teas, J. Agric. Food Calucci, L., Pinzino, C., Zandomeneghi, M., Capocchi, A., Chem., 51, 571-581 Ghiringhelli, S., Saviozzi, F., Tozzi, S. and Galleschi, L.（2003） Singleton, V. L. and Rossi, J. A., Jr.,（1965）, Colorimetry of total Effect of γ-irradiation on the free radical and antioxidant phenolics with phosphomolybdic-phosphotungstic acid contents in nine aromatic herbs and spices, J. Agric. Food reagent, Am. J. Enol. Vitic., 16, 144-158 Chem., 51, 927-934 Sun, Y.（1990）, Free radicals, antioxidant enzymes, and carci- Cämmerer, B. and Kroh, L. W.（2006）, Antioxidant activity of nogenesis, Free Radic. Biol. Med., 8, 583-599 coffee brews, Eur. Food Res. Technol., 223, 469-474 Teow, C. C., Truong, V-D., McFeeters, R. F., Thompson, R. L., Chevalier, F., Chobert, J. M., Genot, C. and Haertle, T.（2001）, Pecota, K. V. and Yencho, G. C.（2007）, Antioxidant activi- Scavenging of free radicals, antimicrobial, and cytotoxic ties, phenolic and β-carotene contents of sweetpotato geno- activities of the Maillard reaction products of β-lactoglobu- types with varying flesh colors, Food Chem., 103, 829-838 lin glycated with several sugars, J. Agric. Food Chem., 49, Theriault, A., Chao, J.T., Wang, Q., Gapor, A. and Adeli, K. 5031-5038 （1999）, Tocotrienol: a review of its therapeutic potential, Deshpande, S. S., Salunkhe, D. K., Oyewole, O. B., Azam-Ali, S., Clin. Biochem., 32, 309-319 Battcock, M. and Bressani, R. （2000）, Fermented grain Ueda, T. and Igarashi, O.（1990） Determination of vitamin E in legumes, seeds and nuts. A global perspective. Food and biological specimens and foods by HPLC-pretreatment of Agriculture Organization of the United Nations, Rome, pp. samples and extraction of tocopherols, J. Micronutr. Anal., 20-21 7, 79-96 Di Carlo, G., Mascolo, N., Izzo, A. A. and Capasso, F.（1999）, Van Boekel, M. A. J. S.（2001） Kinetic aspects of the Maillard Flavonoids: old and new aspects of a class of natural thera- reactions: a critical review, Food/Nahrung, 45, 150-159 peutic drugs, Life Sci., 65, 337-353 Verzelloni, E., Tagliazucchi, D. and Conte, A.（2007） Relation- Espín, J. C., Soler-Rivas, C. and Wichers, H. J.（2000）, Charac- ship between the antioxidant properties and the phenolic terization of the total free radical scavenger capacity of and flavonoid content in traditional balsamic vinegar,Food vegetable oils and oil fractions using 2,2-diphenyl-1-picryl- Chem., 105, 564-571 hydrazyl radical, J. Agric. Food Chem., 48, 648-656 Yilmaz, Y. and Toledo, R. （2005）, Antioxidant activity of Kato, M., Inoue, T. and Nagamitsu, T.（1995）, Pollination biology water-soluble Maillard reaction products, Food Chem., 93, of Gnetum（Gnetaceae） in a lowland mixed dipterocarp 273-278 forestin Sarawak, Am. J. Bot., 82, 862-868 Yokotsuka, T.（1986）, Soy sauce biochemistry, Adv. Food Res., Kishida, E., Nishimoto, Y. and Kojo, S.（1992）, Specific determi- 30, 195-329 nation of ascorbic acid with chemical derivatization and high-performance liquid chromatography, Anal. Chem., 64, （Received Jan, 8, 2014 Accepted May, 29, 2014） 1505-1507

18 （212） Effects of Pit Fermentation and Thermal Cooking Process on the Antioxidant Activity and Components of Pangium edule Seeds

パンギノキ種子の抗酸化活性および成分に対する穴埋め発酵と加熱調理の効果

サントソ・マルタ* 山口智子** 的場輝佳*** 髙村仁知*§

和文抄録パンギノキ（Pangium edule Reinw.）は，東南アジアおよび南太平洋諸島の熱帯植物であり，現地では，その種子から高含量のシアン化合物を取り除いた後に食される。パンギノキの種子はそのまま植物性食素材として消費するほか，穴埋め発酵により，特徴的なセイボリーなフレーバーを付け加えた香辛料として消費される。発酵した種子はクルワックと呼ばれる。これまでに，種子が高レベルの抗酸化活性および抗酸化成分を有することが報告されている。本研究では，パンギノキ種子における抗酸化活性および抗酸化成分の分布，ならびに穴埋め発酵および加熱調理の影響について，1,1-ジフェニル-2-ピクリルヒドラジル（DPPH）法，酸素ラジカル吸収能（ORAC）法，全フェノール量，アスコルビン酸量，ビタミン E 量，脂肪酸組成を測定した。さらに，穴埋め発酵がパンギノキ種子の物性に及ぼす影響についても解析した。種子に含まれる抗酸化活性および抗酸化成分は，DPPH ラジカル捕捉活性を指標とした場合，主に水系画分に存在したが，ORAC を指標とした場合，水系，油系の両方に存在した。水系，油系の主な活性成分はそれぞれフェノール化合物およびγ－トコトリエノールであった。穴埋め発酵により，水系および油系の抗酸化活性は有意に増加したが，これはメイラード反応生成物によるものと思われる。同様に，加熱調理過程においても両方の画分で抗酸化活性が増加した。

キーワード：クルワック，穴埋め発酵，抗酸化活性，パンギノキ

* 奈良女子大学 ** 新潟大学 *** 関西福祉科学大学 § 連絡先奈良女子大学研究院生活環境科学系〒 630-8506 奈良市北魚屋西町 TEL 0742-20-3454 FAX 0742-20-3447

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