6 Okky.pmd


Volume 3, Number 3, December 2009
p 139 - 145

ISSN 1978-3477

*Corresponding author: Phone: +62-251-8323848,
Fax: +62-251-8326851, E-mail: okky_sd@yahoo.com

The Quality of Physic Nut (Jatropha curcas) Seeds Affected by
Water Activity and Duration of Storage

OKKY SETYAWATI DHARMAPUTRA1,2*, RANTJE LILLY WORANG1,3,  RIZAL SYARIEF4 AND MIFTAHUDIN1

1Department of Biology, Faculty of Mathematics and Natural Sciences, Institut Pertanian Bogor,
Darmaga Campus, Bogor16680, Indonesia; 2Seameo Biotrop, Jalan Raya Tajur Km. 6, PO Box 116,
Bogor16134, Indonesia; 3Faculty of Mathematics and Natural Sciences, Universitas Manado State,

Tondano 95615, Sulawesi Utara, Indonesia; 4Department of Food Science and Technology, Faculty of Agricultural Technology,
Institut Pertanian Bogor, Bogor16680, Indonesia

The quality of physic nut (Jatropha curcas) seeds should be maintained during storage, either as seeds for seedlings or oil
production to be used for biodiesel. The effects of water activity and duration of storage on the quality, i.e. fungal population, lipid,
fatty acid and free fatty acid contents, and viability of physic nut seeds were investigated. The results showed that the moisture
content of seeds and total fungal population decreased at low water activities, and increased at high water activities (a

w
). At a

w
 0.64,

at the beginning of storage and after 20 weeks of storage, total fungal populations were 5.4 x 103 and 1.8 x 102 cfu g-1 dry basis (db),
respectively. At a

w
 0.93, at the beginning of storage and after 20 weeks of storage, total fungal populations were 5.0 x 103 and 3.3

x 106 cfu g-1 db, respectively. At the beginning of storage fungi infecting seeds were field fungi, i.e. Cladosporium sp., Colletotrichum
sp., Fusarium semitectum, and F. verticillioides. The population of field fungi decreased with the increase of storage duration.
They were replaced by postharvest fungi, i.e. Aspergillus restrictus, A. penicillioides, Eurotium chevalieri, E. rubrum, Penicillium
citrinum, P. implicatum and P. oxalicum. Lipid content and viability of seeds decreased with the increase of water activities and
seed moisture contents, while free fatty acid contents increased with the increase of water activities and seed moisture contents
during storage. Fatty acids of lipid were dominated by unsaturated fatty acids, i.e. oleic and linoleic acids. Physic nut seeds could
be stored at a

w
 0.64-0.75 up to eight weeks when the seeds will be used for seedling, or up to 16 weeks when they will be used for

producing oil.

Key words: Jatropha curcas, physic nut, water activity

Physic nut (Jatropha curcas) seeds are good sources of
biodiesel. The oil contents in the seeds and in the kernels are
25- 30% and 50- 60%, respectively. The oil contains 21%
saturated fatty acids and 79% unsaturated fatty acids (Gubitz
et al. 1999). As the content of unsaturated fatty acids in the
oil is high, it is possible that the content of free fatty acids
will also increase during storage. Their increase could be
minimized by using good packaging and appropriate storage
condition. Water activity (a

w
) or relative humidity and the

temperature of storage were the most important physical
factors affecting seed quality, because they determine the
moisture content of substrate. Safe appropriate content can
prevent fungal infection and the hydrolysis of lipid,
consequently the quality of seeds or grains could be
maintained for longer period. According to Sirisomboon et
al. (2007), the moisture content of  physic nut hulls (fruits)
var. Kanlueang was the highest, compared to its seeds (nuts)
and kernels. Wanita and Hartono (2007) reported that seeds
originated from physic nut fruits having yellow up to blackish
yellow shell colour produce seeds with highest oil content,
i.e. 28- 30%.

Physic nut seeds can also be used as seeds for seedlings.
According to Adikadarsih and Hartono (2006), the use of
physic nut seeds for seedlings should be derived from the
fruits, which skin is yellow up to blackish yellow in color,
because they have high percentages of viability and vigority,
i.e. 89 and 81%, respectively. To obtain good quality of physic
nut seeds after storage, some factors should be taken into
consideration, i.e. the degree of fruit maturity, safe seed

moisture content, appropriate container and storage
condition, duration of storage, good viability and vigor of
seeds. During storage seeds or grains could be infected by
fungi which cause a decrease in viability, discolouration, loss
in weight, chemical and nutritional changes, heating, caking
and mycotoxin contamination. Chelkowski (1991) reported
that in many cases, fungi infecting seeds are seed-borne
pathogens. They play an important role in the transmission
of numerous pathogenic fungal species to seedlings as well
as to the soil.

The objective of this study was  to analyze the effects of
water activity and duration of seed storage on fungal
population; lipid, fatty acid and free fatty acid contents, and
viability  of physic nut seeds. The population of each fungal
species infecting physic nut seeds in various hydratation
levels was also determined.

MATERIALS AND METHODS

Seed Materials. Physic nut seeds were obtained
from fresh harvested fruits having yellow up to blackish yellow
shell colour. The fruits were obtained from plants (Lampung
accession) cultivated in Loyang Village, Cikedung Subdistrict,
Indramayu Regency, West Java in May 2007. The fruit shell
were peeled using a knife, then the seeds were air-dried on
the selves in a shaded place up to moisture contents of about
8%.

Preparation of Saturated Salt Solution. Selected water
activities (a

w
) were 0.64, 0.75, 0.84 and 0.93 using various

saturated salt solution, i.e. NaNO
2
, NaCl, KCl and KNO

3
,

respectively,
 
in sorption containers. The preparation of each

salt solution was conducted by placing the salt of certain
weight in a sorption container, then distilled water of certain



Microbiol Indones140   DHARMAPUTRA ET AL.

volume was added, consequently water activitiy  selected
was obtained.   During storage the salt solutions were  still
saturated. After being checked using a thermohygrograph at
every sampling, the relative humidity inside each sorption
container were still the same with that at the beginning of
storage.

Packaging and Storing of Physic Nut Seeds. As much as
100 g of physic nut seeds (moisture content ±8%) were placed
in a sac made from plastic net. The sack containing the seeds
was then hung in a sorption container (10 sacks per
container). The seeds were stored in sorption containers with
different water activities. Three replicates (= 3 sorption
containers) were used for each water activity. Sorption
containers were placed on a wooden table under room
conditions. The duration of storages were 0, 1, 2, 3, 4, 6, 8, 12,
16 and 20 weeks. The ambient temperature and relative
humidity of the storage room were recorded using a tinytag
data logger.

Sampling Method and to Obtain Working Samples.
Samples of physic nut seeds were collected from each sack.
The seeds derived from each sack in three sorption containers
were collected for each storage duration. Each sample derived
from each sack was then mixed homogenously, manually
divided into five parts to obtain working samples for the
determinations of moisture content, fungal population, lipid,
fatty acid and free fatty acid contents, and viability of seeds,
and reserved sample.

Determination of Seed Moisture Content and Fungal
Population. Moisture contents of seeds (based on wet basis)
were determined based on oven method. Two replicates were
used for each sample. Fungi from each sample were isolated
and enumerated using serial dilution method followed by
pour plate method on Dichloran 18% Glycerol Agar (DG18)
(Hocking and Pitt 1980). Fungal identification was conducted
based on Klich and Pitt (1988), Pitt (1988), Burgess et al.
(1994), Barnett and Hunter (1998).

Determination of Lipid, Fatty Acid and Free Fatty Acid
Contents and Viability.  Lipid, fatty acid and free fatty acid
contents were determined based on Soxhlet (AOAC 1999),
gas chromatography (AOAC 1984) and titration methods,
respectively. Fatty acid contents were determined  at the
beginning of storage, subsequently after 12 and 20 weeks of
storage.

Viability (percentage of germination) of seeds from each
sample was determined by growing 25 seeds in  a rectangular
plastic container (36 x 27.5 x 5 cm) containing sand (5 kg/
container) seven and 10 days after planting under green
house conditions.. Normal seedlings were observed 7 days
after planting, while normal, abnormal and death seedlings
were observed on 10 days after planting.

Statistical Analysis. Split plot design with two factors
was used. The first factor as the main plot was a

w
 with four

levels, i.e. 0.64, 0.75, 0.84 and 0.93. The second factor as the
subplot was the duration of storage with ten levels, i.e. 0, 1, 2,
3, 4, 6, 8, 12, 16 and 20 weeks with three replications. The
observed data were analyzed using anova, followed by
Duncan’s multiple range test at the 5% probability level.

RESULTS

Moisture Contents. Seed moisture content is the most
important environmental factor that influence fungal growth
in stored grains. Moisture content is always in equilibrium
with the relative humidity of the storage. The range and the
mean of ambient temperature and relative humidity of the
storage is presented in Table 1. At the beginning of storage
the average moisture contents of physic nut seeds were
between 8.17-8.36% wet basis. Water activity, duration of
storage and their interaction gave very significant difference
on the moisture contents (Table 2).

At the beginning of storage the moisture contents at a
w

0.64 (8.36%) was higher and significantly different from those
at a

w
 0.75 (8.17%), but they were not significantly different

from those at a
w
 0.84 (8.28%) and a

w
 0.93 (8.31%). With the

increase  of storage duration each a
w
 gave different effect on

the moisture contents. After one week of storage, the moisture
contents at a

w
 0.64 (8.37%) was not significantly different

with those at a
w 

0.75 (8.37%), but they  were very significantly
different from those at a

w
 0.84 (9.39%) and a

w
 0.93 (10.48%).

At a
w 

0.64, the moisture contents decreased with the increase
of storage duration after two weeks of storage, while at a

w 
>

0.64 the moisture contents increased (Table 2).
Fungal Population. Twenty one fungal species and three

fungal isolates were isolated at various a
w
 during twenty

weeks of storage. They were Aspergillus candidus, A. flavus,
A. niger, A. penicillioides, A. restrictus, A. tamarii,
Aspergillus sp. B, Aspergillus sp. D, Cladosporium sp., C.
cladosporioides, Colletotrichum sp., Eurotium chevalieri,
E. rubrum, Fusarium semitectum, F. verticillioides,
Lasiodiplodia sp., Libertella sp., Penicillium citrinum, P.
implicatum, P. oxalicum, Wallemia sebi, and three
unidentified fungal isolates, i.e. isolates D, F, and I.
Cladosporium cladosporioides and P. citrinum were always
isolated at various a

w 
and duration of storage. Water activity,

duration of storage and their interaction gave very significant
difference on total fungal population. The effect of interaction
between a

w
 and duration of storage on total fungal population

is presented in Table 3. From the beginning of storage until
20 weeks of storage total fungal population were not different
at a

w
 0.64, 0.75 and 0.84, but they were significantly different

from those at a
w 

0.93 after 16 and 20 weeks of storage. Water
activity of 0.64 and 0.75 are favorable to be used for storing
physic nut seeds, because fungal population decreased
during storage. Although at a

w
 0.84 after 20 weeks of storage

Table 1  The range and mean of temperature and relative humidity
of storage room during storage

Duration of

storage
(week)

Temperature
o
C)

Relative humidity
(%)

Range Mean Range Mean

0 - 1 25.1 - 28.3 26.5 61.7 - 80.7 72.6

1 - 2 24.7 - 27.9 26.1 63.9 82.4 75.3

2 - 3 24.4 - 27.9 26.0 65.6 82.8 75.8

3 - 4 25.1 27.9 26.3 60.0 80.7 71.2

4 - 6 25.1 28.7 26.8 58.7 80.7 71.4

6 - 8 25.1 28.3 26.3 48.2 78.1 67.8

8 - 12 25.1 29.1 26.5 55.2 78.5 69.5

12 - 16 25.1 28.3 26.7 46.0 80.7 68.2

16 - 20 25.1 28.7 26.8 55.6 82.4 72.8

-

-

-
-

-

-

-

-
-

-

-
-

-

-

(



Microbiol Indones   141Volume 3, 2009

the total fungal population increased (4.3 x 105 cfu g-1 dry
basis/db), they did not give any significant difference on the
population at beginning of storage up to  16 weeks of storage.
At a

w 
0.84 and 0.93 total fungal population was straight

proportional with the increase of storage duration. At
the beginning of storage the range of total fungal population
was range between 5.0 x 103 – 5.4 x 103 cfu g-1 db at various a

w
.

After 20 weeks of storage the lowest total fungal population
was found at a

w
 0.64 (1.79 x 102 cfu g-1 db), while the highest

was found at a
w
 0.93 (3.25 x 106 cfu g-1 db). At the beginning

of storage the most often fungi isolated were categorized as
field fungi, i.e. Colletotrichum sp., Cladosporium spp. and
Fusarium spp. The population of these fungi decreased with
the increase of storage duration. At low a

w
 (0.64 and 0.75)

total fungal population decreased with the increase of storage
duration, while at high a

w
 (0.84 and 0.93) total fungal

population increased with the increase of storage duration.
Lipid Contents. Physic nut seeds contain high lipid. At

the beginning of storage and after 20 weeks of storage, the
range of lipid contents were between 39.61- 40.85% db and
29.59- 32.46% db, respectively. Water activity, duration of
storage and their interaction gave very significant difference
on lipid content.  The effect of a

w
 and duration of storage on

lipid content is presented in Fig 1.
At the beginning of storage lipid contents of each a

w

were not significantly different. Lipid contents of each a
w

decreased with the increase of storage duration. At a
w
 0.64,

lipid contents decreased during storage, especially after 12
weeks of storage. After 20 weeks of storage lipid contents

were significantly different from those at the beginning of
storage up to 16 weeks of storage. At a

w
 0.75, lipid contents

decreased during storage, especially after 16 weeks of
storage. After 16 and 20 weeks of storage lipid contents were
not significantly different, but they were significantly different
from those at the beginning of storage up to 12 weeks of
storage. At the beginning of storage, at a

w
 0.84, lipid contents

was significantly different from those after one week of
storage. Lipid contents decreased continuously up to 20
weeks of storage. After 20 weeks of storage lipid contents
was  significantly different from those after two up to 16
weeks of storage. At a

w 
0.93 lipid, contents decreased

significantly during storage, especially after two weeks of
storage. After two weeks of storage lipid contents was
significantly different from those at the beginning  and after
one week of storage. Lipid contents decreased continuously
up to 16 weeks of storage and they were significantly different
from those after 20 weeks of storage. Lipid contents at the
four levels of a

w
 decreased with the increase of storage

duration, but they were still in the good ranges for oil
production (about 30%). The highest decrease of lipid
contents  were found at a

w
 0.93 after 20 weeks of storage

(29.59% db).  The decrease of lipid contents was in the form
of negative sigmoid, it looks like S letter upside-down.

Fatty Acid and Free Fatty Acid Contents. Lipid of physic
nut seeds consist of saturated and unsaturated fatty acids.
In this study fatty acid contents of physic nut seeds were
dominated by unsaturated fatty acids, i.e. oleic acid (37.05-
41.29% wb), followed by linoleic acid (26.73- 29.08% wb).

Fig 1  Effect of water activity and duration of storage on lipid content of physic nut seeds.

Means followed by the same letter are not significantly different according to Duncan’s multiple range test at the 5% level.

Table 2  The effect of water activity and duration of  storage on moisture content (% wet basis) of physic nut seeds

Duration of

storage (week)

Water activity

0.64 0.75 0.84 0.93

0

1

2

3

4

6

8

12

16

20

8.36 rs ± 0.12
8.37 rs ± 0.09
8.26 st ± 0.03
8.19 tu ± 0.01
8.17 tu ± 0.03
8.09 u ± 0.06
8.01 vw ± 0.03
7.92 wx ± 0.05
7.84 x ± 0.06
7.64 y ± 0.07

8.27 st ± 0.09
8.37 rs ± 0.03
8.48 r ± 0.03
8.67 q ± 0.11
8.84 p ± 0.01
8.89 op ± 0.02
8.99 o ± 0.03
9.14 n ± 0.07
9.29 m ± 0.06
9.42 m ± 0.07

8.28 st ± 0.14
9.39 m ± 0.07
9.70 l ± 0.02
9.82 l ± 0.08
9.96 k ± 0.02
10.05 jk ± 0.04
10.13 ij  ± 0.03
10.21 hi ± 0.02
10.29 h ± 0.03
10.44 g ± 0.11

8.31 st ± 0.06
10.48 g ± 0.25
11.11 f ± 0.07
11.27 e ± 0.06
11.38 de ± 0.02
11.42 d ± 0.01
11.49 cd ± 0.04
11.58 c ± 0.03
11.75 b ± 0.05
12.05 a ± 0.11

.
, , ,



The composition of fatty acids of physic nut seeds stored at
a

w 
0.64 and 0.93 at the beginning of storage, subsequently

after 12 and 20 weeks of storage is presented in Table 4.
Free fatty acid contents of physic nut seeds at the

beginning of storage were between 0.13-0.27% db. At various
a

w
 free fatty acid contents increased with the increase of

storage duration. Water activity, duration of storage and their
interaction gave very significant difference on free fatty acid
contents.The effect of  a

w
 and duration of storage on free

fatty acid content is presented in Fig 2. At the beginning of
storage free fatty acid contents at each a

w
 were not

significantly different. Nevertheless, free fatty acid contents
increased with the increase of storage duration. After 12 weeks
of storage free fatty acid contents at a

w
 0.64 was not

significantly different from those at a
w
 0.75, but they were

significantly different from those at a
w
 0.84 and 0.93. Free

fatty acid contents at 1% concentration were obtained at a
w

0.64 and a
w
 0.75 after 16 weeks of storage, at aw 0.84 after 12

weeks of storage, and at a
w
 0.93 after 6 weeks of storage (Fig

2). The requirement of free fatty acid contents of physic nut
oil to be used for biodiesel shold not be less than 1%.

Seed Viability. At the beginning of storage the seed
germination percentages were >90%. Nevertheless, at each
a

w
, the seed germination percentages decreased with the

increase of storage duration. At higher a
w
 the decrease of

seed germination percentages was faster than those at lower
a

w
. Water activity, duration of storage and their interaction

gave very significant difference on seed germination
percentage. The effect of a

w
 and duration of storage on seed

germination percentage is presented in Table 5. At the
beginning of storage up to four weeks of storage at each a

w

the percentages of germination were not significantly
different. Their percentages of germination were  still  good
(> 80%). After six and 12 weeks of storage at a

w
 0.64, 0.75 and

0.84 the percentages of germination were not significantly
different, but they were significantly different from those at
a

w
 0.93. After eight weeks of storage at a

w
 0.64 and 0.75 the

percentages of germination were not significantly different,
but they were significantly different from those at a

w
 0.84 and

0.93.

DISCUSSION

Moisture content is the most important factor which
influences fungal growth in stored seeds or grains. High
moisture content is favorable for fungal growth. Dirjenbun
(2006) determined the safe moisture content for storing
physic nut seeds was 7-9%. In this study the average moisture
contents at the beginning of storage were 8.17-8.36% wet
basis (Table 2). Water activity 0.64 was favorable a

w
 for storing

physic nut seeds up to 20 weeks, because their moisture
contents were 7.64- 8.37%. Physic nut seeds were still good
if stored at a

w
 0.75 up to eight weeks. Water activity of 0.84

and 0.93 were not favorable to store physic nut seeds,
because after one week of storage, their moisture contents
increased to  more than 9%. The moisture contents of seed or
grain is in equilibrium with the relative humidity of the storage.
Seeds can have lost (desorption) or absorb (absorbtion) the
humidity of their environment. Seeds stored in a room with
high relative humidity will absorb the humidity of their
environment. On the other hand, seeds will have lost
their humidity, if they were stored at low relative humidity. At
a

w
 0.64 or relative humidity 64%,  physic nut seeds had lost

their humidity during 20 weeks of storage. Their moisture
contents were  8.36% (at the beginning of storage), and then
they became 7.64% after 20 weeks of storage. At a

w
  e” 0.75

during 20 weeks of storage physic nut seeds absorbed the
humidity of their environment. Moisture content of physic
nut seeds increased at a

w
 0.75, 0.84 and 0.93 during 20 weeks

Table 4  Composition of fatty acids of physic nut seeds stored at
a

w
 0.64 and 0.93 at the beginning of storage, subsequently after 12

and 20 weeks of storage

Table  3  The effect of water activity and duration of storage on total fungal population (cfu g-1 db) of physic nut seeds

Means followed by the same letter are not significantly different according to Duncan’s multiple range test at the 5% level.

Microbiol Indones142   DHARMAPUTRA ET AL.

Duration of

storage

(week)

Water activity

0.64 0.75 0.84 0.93

0

1

2

3

4

6
8

12

16

20

5.3x103 c ± 7.0x10

4.2x103c ± 1.1x103

3.6x103 c ± 1.3x103

2.5x103 c ± 4.4x102

1.4x103c ± 7.1x102

9.9x102 c ± 3.2x102

9.5x102c ± 3.6x102

2.8x102 c ± 3.9x102

2.8x102 c ± 3.1x102

1.8x102 c ± 7.3x10

5.2x103 c ± 6.0x102

5.1x103 c ± 1.6x103

4.2x103c  ± 2.4x103

3.1x103c  ± 1.6x103

1.3x103 c ± 6.7x102

1.2x103 c ± 5.2x102

1.2x103 c ± 6.8 x102

6.2x102 c ± 4.1x102

2.7x102 c ± 291.96

2.7x103 c ± 1.4x102

52x103 c  ± 3.1x103

5.2x103 c ± 1.5x103

5.2x103 c ± 2.2x103

5.7x103 c ± 1.7x103

6.2x103 c ± 2.8x103

2.0x104 c ± 2.2x103

2.1x104 c ± 2.5x103

2.2x104 c ± 1.1x103

2.3x105 c ± 1.1x105

4.3x105 c ± 1.2x105

5.0x103 c ± 1.5x103

6.5x103 c ±  2.3x103

8.5x103 c ± 1.6x103

1.0x104 c ± 3.8x103

3.3x104 c ± 5.5x103

1.8x105 c ± 1.2x105

3.0x105 c ± 2.4x105

308591 c  ± 152907

1.2x106 b ± 2.1x105

3.3x106a  ± 1.3x106



Microbiol Indones   143Volume 3, 2009

of storage, however, a
w 

0.64 can maintain safe moisture
content.

At the beginning of storage the most often fungi isolated
was field fungi, i.e. Colletotrichum sp., Cladosporium sp.,
C. cladosporioides, F. verticillioides and F. semitectum. Their
population decreased with the increase of storage duration,
and then they were replaced by storage fungi.

At a
w
 0.64,  total fungal population decreased with the

increase of storage duration (Table 3). It was assumed that
the decrease of total fungal population was due to the
constant or the decrease of moisture contents during storage.
Moisture content is an important factor affecting fungal
growth. At a

w 
0.75 total fungal population also decreased,

but the decrease was not as fast as at a
w
 0.64. According to

Douglas and Boyle (1996) low moisture contents inhibited
fungal growth, but they did not kill the fungi. Among
microorganisms infecting seeds, fungi was the most tolerant
to low water availability, therefore they had an important role
on seed deterioration. At a

w
 0.84 and 0.93 total fungal

population increased with the increase of storage duration
due to the increase of moisture content. Consequently, fungi
infecting physic nut seeds grew and developed better,
especially the fungi which have abundant spores such as
Aspergillus and Penicillium.The moisture content of seeds
was also affected among others by seed respiration and fungal
activity. Fungi absorbed the nutrition of seeds and they
excreted water as the result of their metabolism.  The growth

of storage fungi was affected by the moisture content of the
substrate, temperature, duration of storage, the level of fungal
infection before storage, foreign materials, insects and mites
activities. These factors were related one with anothers. Seed
coat was the main defense to prevent fungal penetration into
the inner seed tissue.  The crack of seed which occurred
mechanically could give the opportunity of fungi to infect
the inner part of the seeds. Seed-borne fungi can cause the
decrease of germination.

Lipid or oil of physic nut seeds which contained high
unsaturated fatty acid (oleic acid) (37.05-41.29% db.), followed
by linoleic acid (26.73- 29.08% db) (Table 4)  will cause
hydrolyses process. This process caused the decrease of
lipid  and increased free fatty acid contents, either in the
seeds or in the oil itself. Lipid contents of physic nut seeds
stored at low a

w
 decreased with the increase of storage

duration, but the decrease was not as rapid as the seeds
stored at high a

w
 (Fig 1). At high a

w
 the moisture contents of

physic nut seeds were also high, so they affected  the activity
of lipolytic fungi to hydrolyse the lipid of the seeds, apart
from lipase enzyme produced by the tissue inside of the seeds.
Pomeranz (1992) reported that the occurrence of fungi will
accelerate the degradation of lipid during storage. The
decrease of lipid content was probably due to the activity of
lipolytic fungi which grew dominantly and can live for long
period. This condition caused the production of free fatty
acid and rancidity. Lipid of seeds can be degraded  by lipase

Table 5  The effect of water activity and duration of storage on the percentage of physic nut seed germination

Means followed by the same letter are not significantly different according to Duncan’s multiple range test at the 5% level.

Fig 2  Effect of water activity and duration of storage on free fatty acid content of physic nut seeds. , ,

, .

Duration of

storage (week)

Water activity

0.64 0.75 0.84 0.93

0

1

2
3

4

6
8

12

16
20

98.67 a ± 2.31
96.00 abc ± 0
94.67 abc ± 2.31
93.33 abcd ± 2.31
92.00 abcde ± 2.31
86.67 cdef ± 2.31
84.00 defg ± 0
77.33 gh ± 4.62
62.67 ijk ± 6.11
29.33 m ± 12.22

98.67 a ± 2.31
97.33 ab ± 2.31
96.00 abc ± 0
93.33 abcd ± 2.31
90.67 abcdef ± 2.31
88.00 bcdef ± 0
88.00 bcdef ± 0
76.67 gh ± 2.31
70.67 hi ± 12.86
25.33 m ± 14.05

97.33 ab ± 2.31
94.67 abc ± 2.31
92.00 abcde ± 0
88.00 bcdef ± 0
86.67 cdef ± 2.31
82.67 efg ± 4.62
76.00 gh ± 4.00
69.33 hij ± 2.31
56.67 k      ± 11.02
2.67 n ± 4.62

94.67 abc ± 4.62
90.66 abcdef ± 2.31
86.66 cdef ± 2.31
84.00 defg ± 0
81.33 fg ± 2.31
61.33 jk ± 2.31
48.00 l ± 4.00
6.67 n ± 6.11
1.33 n ± 2.31
0.00 n ± 0



into free fatty acid and glycerol, especially if the moisture
content of seeds was high. Free fatty acid was an index of
deteriorated seeds containing lipid during storage. According
to Gupta and Rai (2003) A. fumigatus, A. niger, Alternaria
sp., C. cladosporioides and P. purpuragenum isolated from
soil of dairy dung, house-based waste and mill soil were the
good producers of intra- and extracellular lipase activities.

Free fatty acid contents increased with the increase of a
w

and storage duration. It was assumed, that the ocurrence of
various fungal species in physic nut seeds can degrade lipid.
According to Pomeranz (1992), Aspergillus, Cladosporium
and Penicillium were capable to degrade lipid compound
and have high lipolytic activity. These fungal species were
often isolated from seeds containing high lipid. At the
beginning fungi hydrolyze lipid enzymatically with lipase,
and then they was changed into glycerol through ß-oxydation.

The requirement of free fatty acid contents of physic nut
oil to be used for biodiesel is 0.8% (Sudradjat et al. 2007).
Free fatty acid contents as much as 1% at a

w
 0.64 and 0.75

were obtained after 16 weeks of storage (Fig 2). If free acid
content was high, they will block the reaction of methyl esther
(biodiesel) formation, i.e. methanol, which should  react with
triglyceride, but methanol formation was prevented by the
reaction of soap formation. In this case methanol
consumption becomes high as much as 40% (more expensive),
consequently the production of  biodiesel decreases as much
as 30%. According to Sauer (1988), the decrease of seed
quality was followed by the increase of free fatty acid  value.
The level of free fatty acid value gave an indication
concerning the decrease of seed quality. The level of free
fatty acid depends on fungal species infecting the seeds. In
this study, free fatty acid produced by lipase of physic nut
seeds was not separated from that produced by  fungi.
Dhingra et al. (2001) reported that the viability of soybean
seeds inoculated with the conidia of Aspergillus ruber
decreased with the increase of free fatty acid and storage
duration.

According to Dirjenbun (2006)  good seed germination
was e” 80%. At the beginning of storage seed germinations
were > 90%, nevertheless they decreased at various a

w 
during

storage (Table 5). The decrease of germination was very rapid
at a

w
 0.93 (< 70%) after 6 weeks of storage with the curve in

the form of negative sigmoid. At a
w 

0.84 the seed germination
percentage was good up to 6 weeks of storage, at a

w
 0.75 it

was good up to 12 weeks of storage, while at a
w
 0.64 it was

good up to 8 weeks of storage.
Decrease of seed quality was determined by the capability

of germination and the vigor at the beginning of storage, the
moisture content of seeds, storage condition, and fungal
infection. Seed germination was affected by two factors, i.e.
internal and external factors. Internal factor consisted of the
level of seed maturity, seed size, dormancy and germination
inhibitor. External factor consisted water content, temperature,
oxygen, light and growth media. Seeds harvested before
reaching the level of physiologic maturity was attained, they
do not have high viability. Adikadarsih and Hartono (2006)
reported that the lowest percentage of physic nut germination
(7%) and vigor (4.3%) were found in physic nut seeds derived

from fruits which fruit skin is green in color, while the highest
percentage of germination (91.67%) and vigor (84.67%) were
found in seeds derived from fruits which fruit skin is yellow
in color.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Theresia Prawitasari
for her suggestions and encoureagement. We would like also
to thank the staff members of Plant Pathology and Service
Laboratories, Seameo Biotrop, who had in one way or another
contributed to this research, and for a BPPS scholarship to
Rantje Lilly Worang.

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Microbiol Indones   145Volume 3, 2009