Fabrication of Native and Enzymatically Modified Durian Seed (Durio

zibethinus Murr.) Starch

Susi Afrianti Rahayu

, Nasrul Wathoni

2,3*

, Sriwidodo

, Lisa Sophianingsih

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

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 (14)

or (16) 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 : nasru[email protected]d (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

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

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

Fe(CN)

, 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

1-6

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

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

(ºC)

Temp (ºC)

Viscosity

(BU)

93ºC (BU)

93ºC/20’

(BU)

50ºC (BU)

50ºC/20’

(BU)

Native

91.5

120

starch

Modified

88.5

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

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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