Process Biochemistry 35 (1999) 197204

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 (46.0750.95% db) although the
dominant fatty acid composition did not change. The dominant fatty acids were oleic acid (C
18:1
n-9) and linoleic acid (C
18:2
n-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 (69.8123.3 mg g
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 bre (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
www.elsevier.com/locate/procbio
1. Introduction
Pangium edule Reinw. is a tropical tree that grows in
Micronesia, Melanesia, and Southeast Asia, including
Indonesia. The seeds of this tree are poisonous, mostly
because of the presence of cyanogenic glucosides [1]. In
Indonesia, seed kernels are edible following treatment
and the removal of cyanogenic glucoside. Dage is a
product from boiled seeds after removal of kernels and
water soaking for 23 days. Dage is utilized in West
Java as a vegetable. Another product is a fermented
seed material, called keluwak, which has been used a
spice for soup in Java and South Sulawesi. Keluwak is
also a raw material for another product, kecap pangi
(ketchup, soy sauce like product) and has been used as
a spice in Saparua. An edible oil is also produced from
the seed kernels.
Keluwak is fermented in a specic way. The fruits are
harvested and placed in the eld for 10 days until the
fruit is tainted. The seeds are then removed, washed,
and boiled for 3 h. The seeds are then cooled, placed in
a hole in the ground (indoors) and covered by ash.
After 40 days, the fermented seeds are cleaned and can
be used as spices. Previous research on fermented seeds
indicated that the methanol extract of keluwak had
antioxidant activity [2] and another investigation found
that keluwak oil did not contain cyclopentenyl fatty
acids, common cyclic fatty acids in the Flacourtiaceae
family, while g-tocotrienol is a predominant tocol [3].
* Corresponding author. Tel.: +1-413-5451022; fax: +1-413-
5451262.
E-mail address: kalidas@foodsci.umass.edu (K. Shetty)
0032-9592/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved.
PII: S0032- 9592( 99) 00051- 5
N. Andarwulan et al. / Process Biochemistry 35 (1999) 197204 198
Recently, we have also shown that the dominant fatty
acids in seeds during germination were oleic and
linoleic acid, and the antioxidant g-tocotrienol in germi-
nated seed was dominant in early stage and changed to
be a tocotrienol during germination [4].
Prior to this study, it was not known whether fer-
mentation or post-harvest ripening occurred during
keluwak processing. We suspected that fermentation
was the process because of the possibility growth of
microorganism inside and on the surface of the seed.
The physical properties of seeds such as texture, colour
and avour changed during post-harvest processing.
The potentially fermented seeds during post-harvest
processing have soft texture and dark colour (dark red
to dark brown). These changes are thought to be due to
biochemical reactions linked to the enzyme activity of
microorganisms. The objective of this research was
primarily to investigate the mobilization of lipids,
protein, carbohydrate, free phenolics and the antioxi-
dant activity associated with seeds during post-harvest
processing and natural fermentation. A secondary ob-
jective was to determine the activity of key enzymes,
b-glucosidase, which is an enzyme for breakdown of
glycosidic bond of conjugated phenolics and guaiacol
peroxidase, which may be potentially involved in conju-
gation of phenolic aglycones.
2. Materials and methods
2.1. Plant material and fermentation process
Pangium edule Reinw. seeds were obtained from
Bogor, Indonesia. The fruits were harvested in Novem-
ber 30, 1997 and placed in the eld for 10 days until the
fruit was tainted. The seeds were removed, washed, and
boiled for 3 h. Following this, the seeds were cooled,
placed in a hole in the ground (indoor) and covered by
ash. The post-harvest fermentation process began on
December 4, 1997 and proceeded until January 14, 1998
(40 days). Three groups of fermented seeds were used in
this study according to the time of fermentation: 0 day
refers to the boiled seed following cooling, 20 and 40
days refer to fermented seeds following boiling and
cooling. The fermented seeds after removal of kernels
were freeze-dried and ground. The freeze-dried fer-
mented seeds were stored at 20C until analysed.
2.2. Lipid content and fatty acids composition
Lipid or oil content was measured gravimetrically
after the freeze-dried fermented seed powder was defat-
ted using a Soxhlet extraction method for 6 h using
hexane as the solvent. Oil was obtained after the sol-
vent was evaporated under reduced pressure using the
AOAC ofcial method 963.15.
The Soxhlet oil extracts of all freeze-dried fermented
seeds were analyzed in duplicate for fatty acid proles.
Methyl ester derivatives were prepared according to
AOCS standard method No. Ce 2-66 and IUPAC
standard method No. 2.301 with modication. Approx-
imately 1 mg of sample oil (diluted using hexane as the
solvent) was placed in a small vial with a teon cap. A
0.5 to 0.7 ml of 2 N NaOH (in methanol) was added to
each sample. After homogenization, the vial was placed
in a heating block at 80C for 10 min. The vial was
removed and 1 ml of BF
3
-methanol reagent (Sigma, St.
Louis, MO) was added. Subsequently, following ho-
mogenization, the vial was placed in a heating block at
80C for 10 min and homogenized every 3 min. Half a
millilitre of hexane was added to the reaction mixture
after it cooled. Following homogenization, saturated
NaCl solution was added to the mixture and this which
was followed by centrifugation at 3000 rpm for 12
min. The upper phase (hexane phase) was removed and
placed in a vial, which contained anhydrous Na
2
SO
4
.
The hexane phase, which contained fatty acid deriva-
tives, was stored at 4C in amber crimp vials wrapped
in aluminum foil and was analyzed within a week.
Standards of methyl ester derivatives were obtained
from Sigma.
Fatty acid derivatives were analyzed with a Varian
Model 3700 gas chromatograph with ame-ionization
detector and equipped with an integrator (SP 4270).
The column was a Supelco10 fused silica capillary
column with dimension of 30 m0.20 mm and 20 mm
lm thickness. The initial column temperature of 150C
was increased at a rate of 3C min
1
to a nal temper-
ature of 240C which was held for 10 min. The injector
and detector temperatures were 250 and 300C, respec-
tively. The fatty acid content was expressed as percent-
age of total fatty acids. The total fatty acid content was
calculated using margaric acid (C
17:0
) as internal stan-
dard and expressed as mg fatty acid g
1
lipid. The acid
value assay was described in AOAC methods [5].
2.3. Tocol analysis
The tocopherols (T) and tocotrienols (T3) were ex-
tracted in duplicate from the freeze-dried fermented
seed powder in minimal light [6,7]. 1 g seed was homog-
enized for 1 min in HPLC grade methanol (20 ml) and
ltered through Whatman c42 media. The superna-
tant was removed and placed in a 25 ml glass vial and
evaporated under nitrogen. The residue was resus-
pended in 15 ml HPLC grade methanol and the homog-
enization and ltration steps were repeated. The
supernatant was removed and added to the rst extract
and dried under nitrogen. The dried extract was dis-
solved in 2 ml of HPLC grade hexane, mixed briey in
a vortex mixer and centrifuged at 13 000 rpm for 5 min,
placed in a 2 ml amber crimp vial and immediately
analyzed.
N. Andarwulan et al. / Process Biochemistry 35 (1999) 197204 199
Extracts were analyzed directly by high perfor-
mance liquid chromatography (HPLC) using a
Hewlett Packard model HP1090 equipped with a No-
vapak C18 column (3.9 mm150 mm) with guard
cartridge and diode array detector at 298 nm. The
eluent was HPLC grade acetonitrilemethanol (85:15)
at a owrate of 0.8 ml min
1
(Table 1). A sample
volume of 10 ml was used. Identication of to-
cotrienols (a-, and g-tocotrienols) were compared with
the retention time and derivative spectrum of each
compound peak [8]. Tocopherol standards (a-, g-, and
d-tocopherols) were obtained from Sigma (Sigma).
The tocols content was expressed in mg g
1
freeze-
dried seed using d-tocopherol as external standard.
2.4. Protein content and amino acids composition
Total crude protein was performed using the micro
Kjeldahl procedure. Non-soluble protein was the
protein that was precipitated in TCA solution and
was also determined using the micro Kjeldahl proce-
dure. Prior to the nitrogen assay for non-soluble
protein, the freeze-dried fermented seed was extracted
using water and then centrifuged. 10% trichloro acetic
acid (TCA) was added to the supernatant and after
centrifugation, protein in the residue (non-soluble
protein) was determined. The soluble protein was de-
termined by the difference (Total crude protein %
non-soluble protein %).
The amino acid composition of fermented seeds
was determined using a high performance liquid chro-
matography (HPLC) method and carried out as fol-
lows: protein in freeze-dried fermented seed powder
were hydrolyzed using 6 N HCl in stoppered vials at
100C for 24 h, and then freeze-dried. Five millitres
of 0.01 N HCl was added to the freeze dried material
and the mixture was ltered through a 0.45 mm lter
(Millipore) followed by addition of 1 M K-borate
buffer, pH 10.4 at a ratio of 1:1. Ten microlitres of
sample solution was placed in a vial and 25 ml OPA
reagent (1:3 dilution of stock solution which con-
tained 50 mg orthophtaldehyde, 4 ml methanol,
0.0025 ml mercaptoethanol, 0.05 ml of 30% Brij-30
and 1 ml of 1 M borate buffer pH 10.4) was added
and the mixture stored for 1 min. The sample was
then ready for HPLC assay. Amino acids were ana-
lyzed by HPLC (Shimadzu) equipped with a Ultra
Techspere ODS 3 column (HPLC Technology) (4.6
mm75 mm), a uorescence detector, and an inte-
grator (Shimadzu C-R6A). The eluent was a gradient
of buffer A (0.025 M, acetate buffer pH 6.5, contain-
ing of 0.025 M Na-acetate pH 6.5, 0.05% Na-EDTA,
9% methanol and 1% THF) and B (95% methanol)
(Table 1) at a ow rate of 1 ml min
1
. Amino acids
standard for calibration and calculation were pur-
chased from Sigma.
2.5. Carbohydrate content
Crude or total carbohydrate was determined by dif-
ference from the proximate analysis (100%water
content%ash content%protein content%lipid
content%). Neutral detergent bre (NDF, as cellulose,
hemicellulose, and lignin) was estimated by the van
Soest method [9]. Reducing sugar (D-glucose) was es-
timated by the dinitrosalicylic acid (DNSA) method
[10]. The starch content was determined after acid
(HC1) hydrolysis and sugar analysis by an anthrone
method.
2.6. i-glucosidase acti6ity assay
The enzyme was extracted from freeze dried fer-
mented seed powder in buffer at a ratio of 1:5 and
5% PVP-4 was added. The extraction buffer consisted
of 150 mM bis-tris propane, 2 mM EDTA, 3 mM
DTT at pH 7.5. The mixture was centrifuged at 4000
rpm, 4C for 30 min and the supernatant utilized for
enzyme and protein assays. The protein content of
each enzyme extract was determined using a Bio-Rad
Protein Assay (Bio-Rad Laboratories, Hercules,
CA) [11].
b-Glucosidase in the fermented seed extract was
quantied in terms of its specic activity. 25 ml 40
mM PNP-b-D-glucopyranoside and 450 ml of 0.1 M
phosphate buffer (pH 6.3) were mixed in a reaction
test tube and 25 ml of supernatant added. After 2 h
incubation at 30C the reaction was stopped by
adding 800 ml of 1 M Na
2
CO
3
. After the mixture was
homogenized, absorption was measured at 400 nm us-
ing water as reference and the blank was directly in-
activated enzyme and substrate. Enzyme activity was
calculated from PNP (p-nitrophenol) produced using
an extinction coefcient (m) of PNP=10 500 l mol
1
per cm. Enzyme activity is reported as mmols min
1
per g protein.
Table 1
Gradient program of eluent for amino acid assay using HPLC
method
Time (min) % B (45% methanol)
0 0
1 0
15 2
5 15
42 13
42 15
20 70
22 100
26 100
28 0
38 0
N. Andarwulan et al. / Process Biochemistry 35 (1999) 197204 200
2.7. Guaiacol peroxidase acti6ity assay
The enzyme was extracted from the freeze dried
fermented seed powder in buffer, under cold conditions.
A sample (50 mg) was homogenized with a glass mortar
in 2.5 ml extraction buffer in an ice bath. The extrac-
tion buffer consisted of 0.1 M potassium phosphate
(Fisher Scientic) buffer, pH 7.5, containing 2 mM
EDTA (Fisher Scientic) and 1% PVP-40 (Fisher Scien-
tic). The homogenate was centrifuged at 13 000 rpm
for 10 min and the supernatant utilized for the enzyme
and protein assays. The protein content of each enzyme
extract was determined using a Bio-Rad Protein As-
say (Bio-Rad Laboratories, Hercules, CA) [11].
GPX in the fermented seed extract was quantied in
terms of its specic activity. 360 ml of 0.056 M guaiacol
(Acros Organics, NJ), 40 ml of 50 mM H
2
O
2
(Fisher
Scientic, Fair Lawn, NJ), and 600 ml of the 0.1 M
Phosphate buffer (pH 6.8) were mixed in a reaction test
tube. This gave a 1 ml reaction mixture containing 50
mM potassium phosphate buffer (pH 6.8), 2 mM of
H
2
O
2
and 20 mM of guaiacol. At zero time, 50 ml of
supernatant was transferred to the reaction tube and
mixed. The oxidation of guaiacol by GPX was followed
by monitoring the increase in absorbance (u=470 nm).
The rate of change of absorbance per minute was used
to quantify the enzyme in the mixture using an extinc-
tion coefcient (m) of the oxidized product (tetra guaia-
col) of 26.6 mM
1
cm
1
. Enzyme activity is reported as
mmoles min
1
per g protein [12,13].
2.8. Total phenolic content
Phenolics were extracted from defatted freeze dried
fermented seed powder. Sample (6 g) were homogenized
in 100 ml of ACS grade OmniSolv

methanol (EM
Science, Inc., Gibbstown, NJ) for 3 h using shaker.
After the mixture was placed in a water bath at 70C
for 1 h, it was ltered through Whatman c42 media.
The residue w as rinsed using methanol and the super-
natants combined. The solvent was removed in a rotary
evaporator at 40C under reduced pressure. Total phe-
nolics of the extracts were determined from modied
assay [14], which is similar to the original method [15]
and as used in this laboratory [16]. Approximately 1 ml
of 5 mg of DW extracts ml
1
were taken and placed in
a test tube to which 1 ml of 95% ethanol (ACS grade)
and 5 ml of ltered/deionized water added. Folin-Cio-
calteu reagent (50%, 0.5 ml; Sigma Chemical Co.) was
added to each sample. After 5 min, 1 ml of 5% Na
2
CO
3
(Fisher Scientic) was added mixed with a vortex
mixer, and allowed to stand for 60 min in darkness.
Samples were again homogenized with a vortex mixer
and absorbance was measured at 725 nm. A standard
curve was prepared using gallic acid (Fisher Scientic)
in 95% ethanol. The total phenolic content was ex-
pressed as mg g
1
of DW of phenolic extracts.
Fig. 1. Changes in lipid content of Pangium edule Reinw. seed during
fermentation.
2.9. Antioxidant acti6ity test
The antioxidant activity of the phenolic extract was
evaluated using a modication of the b-carotene
linoleate model system described by Miller [17,18]. A
solution of b-carotene (Sigma) was prepared by dissolv-
ing 2.0 mg of b-carotene in 10 ml of chloroform. One
milliliter of this solution was then pipetted into a
roundbottom ask. After chloroform was removed un-
der vacuum, using a rotary evaporator at 40C, 20 mg
of puried linoleic acid, 200 mg of Tween 40 emulsier
(Aldrich, Milwaukee, WI), and 50 ml of aerated dis-
tilled water were added to the ask with vigorous
shaking. Aliquots (5 ml) of this prepared emulsion were
transferred into a series of tubes containing 2 mg dry
weight of extract. As soon as the emulsion was added
to each tube, the zero time absorbance was read at 470
nm. Subsequent absorbance readings were recorded at
30 min intervals, the samples being incubated in a water
bath at 50C. The protection factor (PF) used to ex-
press antioxidant activity was determined as the ratio of
absorbance of the sample at 30 min to that of the
control.
3. Results and discussion
The lipid content in Pangium edule Reinw. seed in-
creased slightly (46.0750.95% db) (Fig. 1). It was
suspected that more non-polar compounds were re-
leased and/or synthesized during fermentation. The
colour of the seed oil from the fermentation process
was darker following Soxhlet extraction. This dark
colour could be from browning reaction products and/
or lignin degradation products. The browining reaction
or non-enzymic browning, called the Maillard reaction,
might have occurred when seeds were boiled and during
fermentation. The seeds contained sufcient levels of
protein, lipid and carbohydrate, which also contained
reducing sugars. Heat treatment of seeds has generally
N. Andarwulan et al. / Process Biochemistry 35 (1999) 197204 201
Fig. 2. Changes in fatty acid content and acid value of lipid fraction
of Pangium edule Reinw. seed during fermentation.
Table 3
Tocotrienol content in Pangium edule Reinw. seed during
fermentation
a
Fermentation a-T3 (ug g
1
fw g-T3 (ug g
1
fw
freeze dried sample) (day) freeze dried sample)
0 69.8 Trace
97.0 ND 20
40 123.3 ND
a
Each value is a mean of two independent assays.
The fatty acid composition appeared to be stable indi-
cating that oxidative protection inside the seed was
present during fermentation.
The dominant tocol, g-tocotrienol, doubled (69.78
123.25 mg g
1
freeze-dried seed) during fermentation
(Table 3). The synthesis of g-tocotrienol during fermen-
tation may result from the activities of microorganism.
g-tocotrienol in seed may have antioxidant activity
which protected lipid and/or fatty acid from oxidation.
Rancid or off avour was not present in fermented
seed.
Total protein content and amino acid composition
(Fig. 3 and Table 4) did not change, but soluble protein
decreased while non-soluble protein increased (Fig. 3).
The increasing non-soluble protein could be an indica-
tor of microbial growth [22]. In this case, microorgan-
isms might utilize soluble protein for growth. Glutamic
acid was the dominant amino acid in the fermented
seed, and this was one of avour sources for spices
(Table 2).
Microbial growth could also be monitored through
degradation of carbohydrate [22]. During fermentation,
total crude carbohydrate content and neutral detergent
bre (NDF as cellulose, hemicellulose, and lignin) de-
creased while reducing sugar increased and starch con-
tent did not change (Fig. 4). The increase in reducing
sugar was not equal to the decrease of total carbohy-
drate content or NDF. It may be that total carbohy-
drate, including cellulose was partially degraded and
resulted in colour or avour modication because of
Maillard reaction [19]. The reaction products vary from
low to high molecular weight compounds which in-
cludes compound like melanoidin (dark red to brown
colour [20]). The water content of seeds during fermen-
tation was 62.6565.10%, and it was predicted that
water activity (Aw) supported the Maillard reaction.
Lignin degradation products were phenolic compounds
which also varied, from low to high molecular weight
and several have dark colours [21]. Lignin could be
degraded by microorganisms because of lignocellulolitic
enzyme activity.
Fatty acid content decreased (836.1786.5 mg g
1
lipid) and free fatty acid, which is expressed as acid
value, increased (0.222.68 mg KOH g
1
lipid) (Fig.
2). These results did not correlate with lipid content.
The decrease of fatty acid content in the lipid fraction
and the increase in free fatty acid in seeds indicated that
during fermentation hydrolysis reactions due to lipase
activity of microorganisms may have occurred.
Minor fatty acids in the lipid fraction (Table 2)
slightly decreased, but the dominant fatty acids (oleic
and linoleic acids) did not change during fermentation.
Table 2
Fatty acids composition of Pangium edule Reinw. seed during
fermentation
a
Fermentation (day) Fatty acid (%)
0 20 40
ND ND ND C
14:0
C
16:0
7.58 8.11 8.08
0.93 C
16:1
0.15 0.14
3.52 C
18:0
3.14 3.09
C
18:1
43.71 42.13 42.55
41.15 42.61 42.81 C
18:2
3.15 C
18:3
2.88 2.84
0.22 C
20:0
0.24 0.21
0.40 C
20:1
0.38 0.37
C
22:0
0.17 0.11 0.13
a
Each value is a mean of two independent assays.
Fig. 3. Changes in protein content of Pangium edule Reinw. seed
during fermentation.
N. Andarwulan et al. / Process Biochemistry 35 (1999) 197204 202
Table 4
Amino acids composition of Pangium edule Reinw. seed during
fermentation
a
Fermentation (day) Amino acid (% db)
20 40 0
2.5 Aspartic acid 2.2 2.6
4.4 4.1 4.3 Glutamic acid
1.0 Serime 1.1 1.1
0.3 0.4 0.1 Histidine
0.8 Glysine 0.8 0.9
0.9 0.6 0.8 Threonine
2.6 Arginine 2.4 2.4
0.2 0.2 0.2 Tyrosine
0.1 Methionine 0.1 0.2
0.9 Valine 0.9 0.9
1.4 1.3 1.4 Phenylalanine
0.7 Isoleusine 0.7 0.6
1.5 1.4 1.5 Leucine
0.6 Lysine 0.5 0.5
19.8 19.4 19.3 Total (% db)
a
Each value is a mean of two independent assays.
Fig. 5. b-glucosidase and guaiacol peroxidase activity in Pangium
edule Reinw. seed during fermentation.
correlated with the increase in reducing sugar during
fermentation. Since Pangium edule seed is known to be
toxic because of the presence of cyanogenic glucosides;
this enzyme may also facilitate the degradation of these
toxins in the fermented seed [23]. The enzyme present in
the seed may be from microbial activity. Fungi such as
Aspergillus sydowi and Fusarium equiseti contain lina-
marase for cyanogenic glucoside detoxication [24].
b-glucosidase also has polygalacturonase (pectinase) ac-
tivity [25], and this activity may soften the texture seed
during fermentation.
In addition to the Maillard reaction, the increasing
activity of peroxidase might also be correlated with
darkening of fermented seeds and growth of microor-
ganisms. Guaiacol peroxidase in plant has the potential
to convert free phenolics to polymerized derivatives like
lignans and lignins. This enzyme, from microbial fer-
became soluble but did not completely release all the
reducing sugars. Another possibility was that the reduc-
ing sugars reacted with amino compounds in Maillard
reactions during seed fermentation. Reducing sugars
may also be released from conjugated phenolics.
The specic enzyme activity could also be used for
monitoring microbial growth. The activity of b-glucosi-
dase, an enzyme that breaks glycosidic bond of conju-
gated phenolics, and potential phenolic condensation
enzyme, peroxidase increased during fermentation (Fig.
5). The increase in activity of b-glucosidase may be
Fig. 4. Changes in carbohydrate content of Pangium edule Reinw. seed during fermentation.
N. Andarwulan et al. / Process Biochemistry 35 (1999) 197204 203
mentation, could catalyze the polymerization of pheno-
lic aglycones released by b-glucosidase activity as well
as from lignin degradation products.
The total phenolics in fermented seeds increased sub-
stantially during fermentation but antioxidant activity
of the phenolic extracts did not change (Fig. 6). The
increasing total phenolics due to b-glucosidase activity
(Fig. 5) and the antioxidant activity indicated that
antioxidant protection might not be linked only to
phenolics but also to other non-phenolic metabolises.
Seeds were treated at high temperature by boiling for
3 h prior to processing and therefore an enzymic re-
sponse from these seeds were unlikely. Likewise during
post-harvest processing there was no seed germination
and therefore it is unlikely that the increase in b-glu-
cosidase, total phenolics and guaiacol peroxidase was
due to the endogenous activity from the seeds. Enzymic
activity may therefore be due to natural fermentation
by microorganisms. Preliminary studies in this regard
have identied certain bacteria and fungi that may be
associated with increased enzyme activities and total
phenolics (data not shown).
4. Conclusion
Natural microbial fermentation of Pangium edule
Reinw. seeds following postharvest heat treatment was
suspected based on enhanced activity of selected en-
zymes linked phenolic mobilization. During fermenta-
tion processing metabolites including lipids, protein,
and carbohydrates were mobilized. Among the fatty
acids present in lipids, oleic and linoleic acids did not
change but the concentration of the antioxidant, g-to-
cotrienol increased. Glutamic acid was the major amino
acid that was hydrolyzed from protein, which may
contribute to avour. Among other metabolites, carbo-
hydrates were partially hydrolysed and may have con-
tributed to the fermentation process and microbial
growth. Total phenolics substantially increased without
concurrent increase in antioxidant activity. Phenolics
may contribute partially to oxidation stability, avour
and to some extent antimicrobial activity. This will be
further explored in on-going studies.
Acknowledgements
We would like to thank Indonesian Government
(URGE) exchange program, the Indonesian Cultural
Foundation, and Directorate General of High Educa-
tion, Department of Education and Culture, the Re-
public of Indonesia for grants supporting N.A.
References
[1] Burkill IH. A dictionary of the economic products of the Malay
Peninsula, 1935;2:16531654.
[2] Fardiaz D, Romlah S. Antioxidant activity of picung (Pangium
edule Reinw.) seed. In: Development of Food Science and Tech-
nology in ASEAN. Proceedings of the Fourth ASEAN Food
Conference, February 1721, Jakarta, 1992:814819.
[3] Puspitasari-Nienaber NL, Aitzetmuller K, Werner G. Analytical
investigation on Pangium edule seed oil, Langfassung Pub-
likation Stand 20.12.94 (Poster unter Nr. 98025), 1994.
[4] Andarwulan N, Fardiaz D, Wattimena GA, Shetty K. Antioxi-
dant activity associated with lipid and phenolic mobilization
during seed germination of Pangium edule Reinw. J. Agric. Food
Chem. 1999 (in press).
[5] AOAC Ofcial Methods of Analysis, 11th ed, AOAC, Washing-
ton, DC, 1975.
[6] Budin JT, Breene WM, Putnam DH. Some compositional prop-
erties of camelina (Camelina sati6a L. Crantz) seeds and oils. J
Am Oil Chem Soc 1995;72:30915.
[7] Oomah BD, Kenaschuk EO, Mazza G. Tocopherols in axseed.
J Agric Food Chem 1997;45:207680.
[8] Brzuskiewicz LM, Analysis of tocopherols and tocotrienols using
HPLC with UV-Vis photodiode-array detection. MS Thesis,
Department of Biochemistry at the University of Massachusetts,
Amherst, 1989
[9] Roberstson JB, van Soest PJ, The detergent system of analysis
and its application to human food. In The analysis of Dietary
Fiber in Food. In: James WPT, Theander O. Marcel Dekker,
New York, 1981:12356.
[10] Miller GL. Use of dinitrosalicylic acid reagent for determination
of reducing sugar. Anal Chem 1959;31:4268.
[11] Bradford M. A rapid and sensitive method for quantication of
microgram quantities of protein utilizing the principle of protein-
dye binding. Anal Biochem 1976;72:24854.
[12] Laloue H, Weber-Lofti F, Lucau-Danila A, Guillemat P. Iden-
tication of ascorbate and guaiacol peroxidase in needle chloro-
plasts of spruce trees. Plant Physiol Biochem 1997;35:3416.
[13] George P. Intermediate compound formation with peroxidase
and strong oxidizing agents. J Biol Chem 1953;201:41351.
[14] Chandler SF, Dodds JH. The effect of phosphate, nitrogen, and
sucrose on the production of phenolics, and solasodine in callus
cultures of Solanum laciniatum. Plant Cell Rep 1983;2:1058.
[15] Singleton VL, Rossi JA Jr. Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am J Enol
Vitic 1965;16:14458.
Fig. 6. Changes in total phenolics and antioxidant activity of phenolic
extracts of Pangium edule Reinw. seed during fermentation.
N. Andarwulan et al. / Process Biochemistry 35 (1999) 197204 204
[16] Shetty K, Curtis OF, Levin RE, Witkowsky R, Ang W. Pre-
vention of vitrication associated with in vitro shoot culture of
oregano (Oreganum 6ulgare) by Pseudomonas spp. J Plant
Physiol 1995;147:44751.
[17] Hammerschmidt PA, Pratt DE. Phenolic antioxidants of dried
soybeans. Food Sci 1978;43:5569.
[18] Wanasundara U, Amarowicz R, Shahidi F. Isolation and iden-
tication of an antioxidative component in canola meal. J
Agric Food Chem 1994;42:128590.
[19] De Bry L. Anthropology of the Maillard reaction. In:
Labuza TP, Reineccius GA, Monnier VM, OBrien J, Baynes
JW editors. Maillard Reaction in Chemistry, Food, and
Health, The Royal Society of Chemistry, Cambridge,
1994:2836.
[20] Rizzi G, The Maillard reaction in foods. In: Labuza TP,
Reineccius GA, Monnier VM, OBrien J, Baynes JW, editors.
Maillard Reaction in Chemistry, Food, and Health, The Royal
Society of Chemistry, Cambridge, 1994:1119.
[21] Crawford RL. Lignin Biodegradation and Transformation.
Toronto: John Wiley and Sons, 1991.
[22] Carvalheiro F, Roseiro JC, Collaco MAT. Biological conver-
sion of tomato pomace by pure and mixed fungal cultures.
Process Biochem 1994;29:6015.
[23] Ikediobi CO, Onyike E. The use of linamarase in gari produc-
tion. Process Biochem 1982;17:28.
[24] Ikediobi CO, Ogundu EC, Ukoha AI. Production of linama-
rase by Aspergillus sydowi and Fusarium equiseti. Process
Biochem 1985;20:99102.
[25] Brimer L, Cicalini AR, Federici F, Petruccioli M. Beta-glucosi-
dase as a side activity in commercial pectinase preparations of
fungal origin. The hydrolysis of cyanogenic glycosides. Ital J:
Food Sci 1995;4:38794.
.