Fabrication of Native and Enzymatically Modified Durian Seed (Durio
zibethinus Murr.) Starch
Susi Afrianti Rahayu
1
, Nasrul Wathoni
2,3*
, Sriwidodo
2
, Lisa Sophianingsih
2
1. Academy of Pharmacy, Bumi Siliwangi, Bandung, Indonesia
2. Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Universitas
Padjadjaran, Jatinangor 45363, Indonesia
3. Scientific Consortium of Drug Discovery and Development, Universitas Padjadjaran, Jatinangor
45363, Indonesia
Received : 13 Nov 2018/Revised: 2 Feb 2019/Accepted : 5 May 2019/ Published 13 June 2019
ABSTRACT
Durian seed (Durio zibethinus Murr.) has a high starch content (46.2%) and thus can be used as a new
source of starch for the raw materials of pharmaceutical and food industries. In this study, we fabricated
native and enzymatically modified durian seed starch using a rough enzyme extract from Saccharomycopsis
fibuligera. Wet grinding method was used for starch production. Physicochemical characterization of the
starches was investigated by organoleptic, acidity-basicity, loss on drying, flow capability, compressibility,
ash content and microbial limit. In addition, viscoamylograph had been done to clarify the viscosity
properties of the starches. The result of starch production showed that the durian seed had a starch yield of
17.68%. Physicochemical characterization of the starch showed that the results of quality testing had
fulfilled the Indonesian Pharmacopoeia 4
th
edition standards requirements, such as description,
identification, acidity-basicity, loss on drying, ash content and microbial limit. In addition,
viscoamylograph study showed that the enzymatically modified durian seed starch had a higher viscosity
than the native durian seed starch. Interestingly, modification of the durian seed starch using a rough
enzyme extract improved its flow capability and compressibility. These results suggest that the modified
durian seed starch experienced an increase in viscosity, compressibility and flow capability compared to
native durian starch.
Keywords: durian seed, starch, enzymatic modification
1. Introduction
Starch, a flour-like substance of carbohydrate
with a glucose polymer, is one of the most useful
biopolymer in the food and pharmaceutical
industries as thickeners, gelling agents, texture
modifiers and stabilizers [1, 2]. There are two types
of starch commonly used in the pharmaceutical
industry, namely, native starch and modified starch.
Native starch is a starch without any changes in
physical and chemical properties [3]. On the other
hand, modified starch has a different characteristics
compared to the original one, which can then be
adjusted for certain purposes [4]. The desired
characteristics of starch are a stable viscosity, a
high durability, as well as a thickening capability
towards mechanical and temperature changes [5].
In general, the modification methods of starch
are divided into chemical, physical or enzymatic
methods. Several enzymes are used in starch
hydrolysis which subjected to break down its
structure and to obtain the desired functional
characteristics [6]. Enzymes hydrolyze the (14)
or (16) bond between the α-D-glucopyranose
unit in starch. The most commonly used enzymes
to modify starch are α-amylase, β-amylase,
glucoamylase, pullulanase, and isoamylase. These
enzymes are isolated from mushrooms, yeast,
bacteria, and plants [7].
Durian (Durio zibethinus Murr.), a tropical fruit
origined from Southeast Asia with common name
“king of fruit”, is one of the unique fruits in
Southeast Asia due to its strong aroma and
exclusive taste [8, 9]. In addition, 30-35% of the
*Corresponding author, https://doi.org/10.24198/idjp.v1i2.19287
e-mail : nasrul@unpad.ac.id (N. Wathoni) 2019 Rahayu et al
Vol 1, Issue 2, 2019 (40-45)
http://journal.unpad.ac.id/idjp
S.A. Rahayu et al / Indo J Pharm 2 (2019) 40-45
1-6
41
durian is edible, whereas one fourth of the seeds
and the shell are usually as wastes [10].
Interestingly, durian seed has high starch content
and potential as an alternative starch sources in
pharmaceutical industry [11, 12]. In this study, to
obtain modified durian seed with optimum
functional characteristics, we fabricated and
characterized enzymatically modified durian seed
starch using a rough enzyme extract from
Saccharomycopsis fibuligera.
2. Method
2.1. Materials
Durians were obtained from durian garden,
Lampung, Indonesia. Yeast extract and sago starch
were procured by Leiber, Jakarta, Indonesia. 70%
ethanol, 80% ethanol, phenophthalein 0.1%,
glucose anhydrate, sodium hydroxide 0.1 N were
bought from Brataco Chemical, Jakarta, Indonesia.
All other reagents and chemicals were of analytical
grade and used without any further purification.
2.2. Yeast production
The durian seed starch was produced using a wet
grinding method [13]. 7.403 grams of durian seed
were cleaned from its outer coat and epidermis,
followed by cut into small pieces and then crushed
using a blender with distilled water. The material
was strained using a flannel cloth into a container
until the residue no longer produced any liquid.
Then, the suspension was precipitated for 24-48
hours until a clear supernatant obtained. The
supernatant was then decanted. The starch
precipitate was dried in a drying rack at a
temperature of 35-40ºC for 24 h. The powders were
sifted using a 100 mesh sifter.
2.3. Physicochemical properties of native durian
seed starch
Examination of the durian seed starch including:
organoleptic, solubility, acidity-basicity, loss on
drying, microbial limit, pH, real density and bulk
density, pure density, flow capability,
gelatinization temperature and viscosity, amylose
and amylopectin content, degree of whiteness, and
proximate analysis [14].
2.4. Production of rough enzyme extracted from
Saccharomycopsis fibuligera
S. fibuligera was grown on a slanted agar
medium composed of 6% sucrose and 1.5% bacto-
agar in 10% bean sprout extract, continued with an
inoculum containing 1% sago starch and 1% yeast
extract for 48 hours. Afterwards, the inoculum was
moved into a production media which has the same
composition as the inoculum, it formed 10% of the
production medium. This fermentation was done at
a shaking rate of 180 rpm for 72 hours. The enzyme
was then strained using straining paper in cold
conditions. The result was a rough enzyme extract
(containing α-amylase and glucoamylase). The
rough enzyme extract was added by ammonium
sulfate salt at concentration of 25% (v/v) [15].
2.5. Hydrolysis of raw durian seed starch
Optimization of the starch hydrolysis used
various enzyme concentrations in selected times at
optimum enzyme temperature (50
o
C). A 5% of
starch suspension was produced in a reaction
mixture containing 10 ml of enzymes with various
concentration (100, 200, and 300 ppm). The
mixture was hydrolyzed at 2, 4, and 6 h. A sample
of the hydrolyzed result was then centrifuged at
10,000 rpm for 15 minutes and its supernatant were
withdrawn. The absorbance was measured at a
wavelength of 420 nm [16].
2.6. Determination of the total amount of reducing
sugars
The reducing sugar for the supernatant was
determined using the Schales method. Briefly, as
much as 200 µL of the hydrolysis solution was
placed in a closed test tube, followed by addition of
800 µL K
3
Fe(CN)
6
, then incubated for 10 minutes.
A total measurement of the reducing sugar was
also done towards the dilution of the hydrolysis
result samples for 5, 8, and 10 times. A standard
glucose curve was made from the measurement of
glucose absorbance with different concentrations
S.A. Rahayu et al / Indo J Pharm 2 (2019) 40-45
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Table 1. Physicochemical properties of durian seed starch
Examination Literature data Durian starch seed
Organoleptic A very soft, white powder Soft powder, brownish-white.
Solubility Does not dissolve at all in cold
water or ethanol.
Does not dissolve at all in cold
water or ethanol.
Identification:
A. Heating until boiling point for 1
minute a suspension of 1 g of
starch in 50 ml of water, then
cooling.
B. Mixing 1 ml of the starch solution
obtained from identification A with
0.05 ml iodium 0.005 M
A dilute starch solution is
formed.
A blue color appears which
disappears when heated and
reappears when cooled.
A dilute starch solution was
formed.
A blue color appeared which
disappeared when heated and
reappeared when cooled.
Acidity Not more than 2.0 ml is needed 1.7 ml was needed
Loss on drying Not more than 15.0% 13.04 %
Ash content Not more than 0.6% 0.17 %
Microbial impurities limit no Escherichia coli no Escherichia coli
Table 2. Characterization of durian seed starch
Characteristics Durian Seed Starch
pH ± SD 4.26 ± 0.02
Pure density ± SD 2.325 ± 0.57
Water content 87.98%
Ash content 0.17%
Protein content 3.75%
Fat content 17.51%
Carbohydrate content (rough estimate) 78.57%
Rough fiber content 11.62%
(100, 200, 300, 400, and 500 ppm), which was
given the same treatment as the determination of
the total reducing sugar [10, 17].
3. Results and Discussion
3.1. Starch Production
The starch yield obtained was 17.68% with a
brownish-white color. During washing and drying,
some starch was decreased.
3.2. Physicochemical properties of native durian
seed starch
Examination on the starch quality according to
the monograph in the Indonesian Pharmacopoeia
4th edition had been done including solubility,
acidity, loss on drying, ash content, and microbial
impurities limit compared to that of cassava starch.
The results of the examination of starch quality can
be seen in Table 1. The test results showed that the
durian seed starch fulfilled the requirements as
stated in the Indonesian Pharmacopoeia 4
th
edition.
Other quality examinations such as pH, pure
density, water content, protein content, and fat
content were also conducted and the results can be
seen on Table 2.
3.3. Production of rough enzyme extracted from
Saccharomycopsis fibuligera
The enzyme produced from 5 g of yeast extract
and 5 g of starch in a 500 ml aquadest solution was
351 ml (70.2 %). By testing the enzyme activity
with the Goyal et.al method, it was obtained that the
activity of the extracted enzyme was 312.33 U/ml
[18].
3.4. Hydrolysis of durian seed starch
From the linear regression curve of the
absorbance of the standard glucose solution, the
linear equation y = 0.001x – 0.002 was obtained,
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starch
with R2 = 0.999. The results of the absorbance
measurements of the optimization sample was used
to determine the reducing sugar based on the linear
equation of the standard glucose curve. The
optimization showed that in order to hydrolyze 5%
of durian seed starch, the optimum time and
concentration of enzymes required is 6 hours and
200 U/ml. The yield value of reducing sugars was
similar to the hydrolysis experiment published by
Purnomo et al [16].
3.5. Physicochemical properties of modified durian
seed starch
BU. When the temperature was lowered and with
stirring, the viscosity value of the durian seed starch
increased to 120 BU. The increase in viscosity
when the temperature was lowered was affected by
the retrograding capabilities of the starch. The
viscosity value of the durian seed starch remained
constant by stirring without any changes in
temperature. This shows that the viscosity stability
of durian seed starch was at 50ºC.
Table 3. Density and Compressibility Testing of Native and
Modified Starch
Native starch
Modified
Determination of the total reducing sugars
showed that the native starch had been successfully
modified. The modified durian seed starch had a
total amount of 148.79 μg/ml of reducing sugars.
This value is smaller than that from the
optimization results, which was as much as 578.33
μg/ml.
Average bulk
density (g/ml) ± SD
Average tapped
density (g/ml) ± SD
Average pure
density (g/ml) ± SD
Average
compressibility
0.370 ± 0.008 0.45 ± 0.012
0.500 ± 0.025 0.60 ± 0.021
2.325 ± 0.565 -
26.85 ± 2.45 23.02 ± 4.12
The results of density, compressibility (Table 3),
flow capability, and angle of repose testing (Table
4) showed that there were an increase in the flow
(%) ± SD
Table 4. Flow Properties of Native and Modified Starches
properties of modified starch.
Angle of Repose Flow Capability
Gelation temperature is the temperature at which
the starch granules burst because of the addition of
No.
(degrees) ± SD (g/s) ± SD
Without With Without With
water and heat. From the Brabender
vibration vibration vibration vibration
viscoamylograph testing, it is known that the
gelatinization temperature of native durian seed is
91.5 ºC. Durian seed starch does not have a peak
temperature, thus it also does not have a peak
Native
starch
Modified
starch
- - - -
24.07 ± 23.75 ± 13.14 ± 16.14 ±
1.04 0.76 1.72 2.51
viscosity value. After gelatinization, the starch’s
viscosity usually increases due to the lack of water,
which acts as a lubricant between the expanding
granules. With an increase in temperature and
stirring, the viscosity value of durian seed starch is
10 BU (Brabender Units). After stirring, the
viscosity of the durian seed starch increased to 35
Modified starch has lower gelation temperature
compared to native starch, however, modified
starch has a higher viscosity compared to native
starch. This results showed that modified starch has
more viscous compared to native starch. The
temperature needed to form a gel is low but the
viscosity is high.
Table 5. Flow Properties of Native and Modified Starches
Gel Temp
Peak
Viscosity
Viscosity
Viscosity
Viscosity
(ºC)
Temp (ºC)
93ºC (BU)
93ºC/20’
(BU)
50ºC (BU)
50ºC/20’
(BU)
Native
91.5
-
10
35
120
120
starch
Modified
88.5
-
50
90
220
220
starch
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Table 6. Characterization of durian seed starch
Amylose content
Native starch 38.89
Modified starch 30.78
The results of amylose content testing (Table 6)
showed that modified starch had a lower amylose
content than native starch. The amylose content
affects the viscosity of the gel to be formed. The
lower the amylose content, the viscous the gel.
Table 7. Degree of Whiteness testing
Degree of Whiteness (%)
Native starch 97.93
Modified starch 84.36
The results of the degree of whiteness testing
(Table 7) showed that native starch has a higher
degree of whiteness compared to modified starch.
Native starch has a 97.93% degree of whiteness and
modified starch has an 84.36% degree of whiteness.
The water used in the extraction process, the
duration of precipitation and the temperature of the
drying process affects the degree of whiteness of
native starch. The reduction in the degree of
whiteness of the modified starch can be attributed
to the addition of the enzyme and the heating during
hydrolysis.
4. Conclusion
We successfully fabricated native and
enzymatically modified durian seed starch using a
crude enzyme extracted from Saccharomycopsis
fibuligera. Physicochemical characterization of the
starch showed that the results of quality testing had
fulfilled the Indonesian Pharmacopoeia 4
th
edition
standards requirements, such as description,
identification, acidity, loss on drying, ash content
and microbial limit. It can be concluded that the
modified durian seed starch experienced an
increase in viscosity, compressibility and flow
capability compared to native durian starch.
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