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 com- monly 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 compo- nents of Pangium edule seed, measured using 1,1-diphenyl-2-picrylhydrazyl（DPPH） and oxygen radical absor- bance 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- （203） 9 J. Cookery Sci. Jpn. Vol. 47 No. 4（2014） 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.