Process Biochemistry 35 (1999) 197–204 www.elsevier.com/locate/procbio Mobilization of primary metabolites and phenolics during natural fermentation in seeds of Pangium edule Reinw. Nuri Andarwulan a,b, Srikandi Fardiaz b, Anton Apriyantono b, Purwiyatno Hariyadi b, Kalidas Shetty a,* a Department of Food Science, Uni6ersity of Massachusetts, Chenoweth Laboratory, Box 31410, Amherst, MA 01003, USA b Department of Food Technology and Human Nutrition, Bogor Agricultural Uni6ersity, Bogor, Indonesia Received 15 December 1998; received in revised form 30 March 1999; accepted 10 April 1999 Abstract Fermented seeds of the tropical tree Pangium edule Reinw. are a speciality in Indonesia and have been used as spices. The fermentation process of the seeds is a natural spontaneous process, which occurs 40 days following seed maturity and treatment. This study reports some biochemical changes, especially primary metabolites, and antioxidant activity associated with mobiliza- tion of lipids and phenolics during seed fermentation. The lipid content increased slightly (46.07–50.95% db) although the dominant fatty acid composition did not change. The dominant fatty acids were oleic acid (C18:1n-9) and linoleic acid (C18:2n-6). During fermentation, the decrease in fatty acid content in lipid coincided with the increasing acid value, which indicated that free fatty acids increased in seeds during fermentation. The dominant tocol in the seed, g-tocotrienol, increased (69.8–123.3 mgg−1 freeze-dried seed) during fermentation. In general, overall protein content and amino acid composition did not change but non-soluble protein increased while soluble protein decreased. The changes in carbohydrate fraction showed that total crude carbobydrate, neutral detergent fibre (NDF, as cellulose, hemicellulose, and lignin) decreased, but reducing sugar increased and starch content did not change. Enzyme assays showed that microorganisms may be involved in the fermentation process. b-glucosidase, an enzyme that can cleave glycosidic bonds of conjugated phenolics and guaiacol peroxidase (GPX) activities increased. The total phenolics content in seeds increased substantially corresponding to the increase in b-glucosidase but antioxidant activity of phenolic extracts did not change. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Pangium edule; Fermentation processing; Biochemical changes; Primary metabolises; Phenolics 1. Introduction also a raw material for another product, ‘kecap pangi’ (ketchup, soy sauce like product) and has been used as Pangium edule Reinw. is a tropical tree that grows in a spice in Saparua. An edible oil is also produced from Micronesia, Melanesia, and Southeast Asia, including the seed kernels. Indonesia. The seeds of this tree are poisonous, mostly Keluwak is fermented in a specific way. The fruits are because of the presence of cyanogenic glucosides [1]. In harvested and placed in the field for 10 days until the Indonesia, seed kernels are edible following treatment fruit is tainted. The seeds are then removed, washed, and the removal of cyanogenic glucoside. ‘Dage’ is a and boiled for 3 h. The seeds are then cooled, placed in product from boiled seeds after removal of kernels and a hole in the ground (indoors) and covered by ash. water soaking for 2–3 days. Dage is utilized in West After 40 days, the fermented seeds are cleaned and can Java as a vegetable. Another product is a fermented be used as spices. Previous research on fermented seeds seed material, called ‘keluwak’, which has been used a indicated that the methanol extract of keluwak had spice for soup in Java and South Sulawesi. Keluwak is antioxidant activity [2] and another investigation found that keluwak oil did not contain cyclopentenyl fatty * Corresponding author. Tel.: +1-413-5451022; fax: +1-413- acids, common cyclic fatty acids in the Flacourtiaceae 5451262. g E-mail address: [email protected] (K. Shetty) family, while -tocotrienol is a predominant tocol [3]. 0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S0032-9592(99)00051-5 198 N. Andarwulan et al. / Process Biochemistry 35 (1999) 197–204 Recently, we have also shown that the dominant fatty The Soxhlet oil extracts of all freeze-dried fermented acids in seeds during germination were oleic and seeds were analyzed in duplicate for fatty acid profiles. linoleic acid, and the antioxidant g-tocotrienol in germi- Methyl ester derivatives were prepared according to nated seed was dominant in early stage and changed to AOCS standard method No. Ce 2-66 and IUPAC be a tocotrienol during germination [4]. standard method No. 2.301 with modification. Approx- Prior to this study, it was not known whether fer- imately 1 mg of sample oil (diluted using hexane as the mentation or post-harvest ripening occurred during solvent) was placed in a small vial with a teflon cap. A keluwak processing. We suspected that fermentation 0.5 to 0.7 ml of 2 N NaOH (in methanol) was added to was the process because of the possibility growth of each sample. After homogenization, the vial was placed microorganism inside and on the surface of the seed. in a heating block at 80°C for 10 min. The vial was The physical properties of seeds such as texture, colour removed and 1 ml of BF3-methanol reagent (Sigma, St. and flavour changed during post-harvest processing. Louis, MO) was added. Subsequently, following ho- The potentially fermented seeds during post-harvest mogenization, the vial was placed in a heating block at processing have soft texture and dark colour (dark red 80°C for 10 min and homogenized every 3 min. Half a to dark brown). These changes are thought to be due to millilitre of hexane was added to the reaction mixture biochemical reactions linked to the enzyme activity of after it cooled. Following homogenization, saturated microorganisms. The objective of this research was NaCl solution was added to the mixture and this which primarily to investigate the mobilization of lipids, was followed by centrifugation at 3000 rpm for 1–2 protein, carbohydrate, free phenolics and the antioxi- min. The upper phase (hexane phase) was removed and dant activity associated with seeds during post-harvest placed in a vial, which contained anhydrous Na2SO4. processing and natural fermentation. A secondary ob- The hexane phase, which contained fatty acid deriva- jective was to determine the activity of key enzymes, tives, was stored at 4°C in amber crimp vials wrapped b-glucosidase, which is an enzyme for breakdown of in aluminum foil and was analyzed within a week. glycosidic bond of conjugated phenolics and guaiacol Standards of methyl ester derivatives were obtained peroxidase, which may be potentially involved in conju- from Sigma. gation of phenolic aglycones. Fatty acid derivatives were analyzed with a Varian Model 3700 gas chromatograph with flame-ionization detector and equipped with an integrator (SP 4270). 2. Materials and methods The column was a Supelco™10 fused silica capillary column with dimension of 30 m×0.20 mm and 20 mm 2.1. Plant material and fermentation process film thickness. The initial column temperature of 150°C was increased at a rate of 3°C min−1 to a final temper- Pangium edule Reinw. seeds were obtained from ature of 240°C which was held for 10 min. The injector Bogor, Indonesia. The fruits were harvested in Novem- and detector temperatures were 250 and 300°C, respec- ber 30, 1997 and placed in the field for 10 days until the tively. The fatty acid content was expressed as percent- fruit was tainted. The seeds were removed, washed, and age of total fatty acids. The total fatty acid content was boiled for 3 h. Following this, the seeds were cooled, calculated using margaric acid (C17:0) as internal stan- placed in a hole in the ground (indoor) and covered by dard and expressed as mg fatty acid g−1 lipid. The acid ash. The post-harvest fermentation process began on value assay was described in AOAC methods [5]. December 4, 1997 and proceeded until January 14, 1998 2.3. Tocol analysis (40 days). Three groups of fermented seeds were used in this study according to the time of fermentation: 0 day The tocopherols (T) and tocotrienols (T3) were ex- refers to the boiled seed following cooling, 20 and 40 tracted in duplicate from the freeze-dried fermented days refer to fermented seeds following boiling and seed powder in minimal light [6,7]. 1 g seed was homog- cooling. The fermented seeds after removal of kernels enized for 1 min in HPLC grade methanol (20 ml) and were freeze-dried and ground. The freeze-dried fer- filtered through Whatman c42 media. The superna- mented seeds were stored at −20°C until analysed. tant was removed and placed in a 25 ml glass vial and evaporated under nitrogen. The residue was resus- 2.2. Lipid content and fatty acids composition pended in 15 ml HPLC grade methanol and the homog- enization and filtration steps were repeated. The Lipid or oil content was measured gravimetrically supernatant was removed and added to the first extract after the freeze-dried fermented seed powder was defat- and dried under nitrogen. The dried extract was dis- ted using a Soxhlet extraction method for 6 h using solved in 2 ml of HPLC grade hexane, mixed briefly in hexane as the solvent. Oil was obtained after the sol- a vortex mixer and centrifuged at 13 000 rpm for 5 min, vent was evaporated under reduced pressure using the placed ina2mlamber crimp vial and immediately AOAC official method 963.15. analyzed. N. Andarwulan et al. / Process Biochemistry 35 (1999) 197–204 199 Extracts were analyzed directly by high perfor- reagent (1:3 dilution of stock solution which con- mance liquid chromatography (HPLC) using a tained 50 mg orthophtaldehyde, 4 ml methanol, Hewlett Packard model HP1090 equipped with a No- 0.0025 ml mercaptoethanol, 0.05 ml of 30% Brij-30 vapak C18 column (3.9 mm×150 mm) with guard and 1 ml of 1 M borate buffer pH 10.4) was added cartridge and diode array detector at 298 nm.