International Journal of Pharma Medicine and Biological Sciences Vol. 6, No.

1, January 2017

Degree of Deacetylation of Chitosan Extracted

from White Snapper (Lates sp.) Scales Waste
Noverita D. Takarina, Aldila A. Nasrul, and Alinda Nurmarina
Department of Biology, Universitas Indonesia, Depok, Indonesia
Email: noverita.dian@sci.ui.ac.id, {aamininasrul, alindamarina96}@gmail.com

Abstract—Chitosan is natural biopolymer which can be absorbent materials. Other than that, scales can also be
obtained from natural resources even from wastes such fish used not only in separation technology but also in
scales. Indonesia is one of countries that having high fish catalysis and biomedical applications.
production. White snapper (Lates sp.) is seawater fish that Further research is needed to determine the method
has an economical value since it is preferred by many
and characteristic of chitosan extracted from seawater
consumers. New challenge to this condition is how to
manage the waste from fish processing into useable fish scales, for example white snapper. Local name of
materials. This study was aimed to extract chitosan from white snapper is Barramundi. Barramundi (Lates sp) has
white snapper (Lates sp.) scales and to determine the degree a body covered with scales. The scales derived from the
of deacetylation of the chitosan based on the treatment skin layer called the dermis.
differences in temperature, heating duration, and the NaOH Degree of deacetylation is a quality parameter that
concentration. Chitosan from white snapper scales were indicates an acetyl group which can be removed from the
extracted through three stages, deprotenization, chitosan. The higher the degree of deacetylation of
demineralization and deacetylation. Different treatment on chitosan, the lower the acetyl group contained in the
deacetylation stages based on variation of NaOH
chitosan (Knoor, 1983) [6]. Degree of deacetylation is
concentration, temperature and duration of heating. Degree
of deacetylation were measured using FTIR also determined as a percentage that indicates an acetyl
spectrophotometry based on infrared light absorbance. missing or replaced with amines. Analysis Fourier-
Result showed that chitosan produced were fulfilled Transform Infra-Red (FTIR) is used to determine the
minimum requirement for good quality of chitosan. Highest functional groups present in chitosan. In addition, this
deacetylation degree was 84.05 %, resulted from treatment method can be used also to determine the degree of
number 15 with NaOH concentration of 80%, temperature deacetylation. The working principle of FTIR is infrared
of 120oC and duration of heating for 4 hours. radiation will pass through the sample and then the
radiation will be absorbed. The ones that are not absorbed
Index Terms—chitosan, degree of deacetylation, fish scale, will be forwarded passing the transmittances resulting in
white snapper, FTIR
a spectrum of absorption in the infrared region therefore
cause the vibration of the bond in the molecules. This
I. INTRODUCTION study was aimed to extract chitosan from white snapper
fish (Lates sp.) scales and to determine the degree of
Chitosan is a natural product which is a derivative of deacetylation of the chitosan based on the treatment
the polysaccharide chitin. Chitosan has the chemical differences in temperature, heating duration, and the
name Poly D-glucosamine (beta (1-4) 2-amino-2-deoxy- NaOH concentration.
D-glucose. Chitosan can be used in various fields of
biochemistry, medical or pharmaceutical, food and II. METHODOLOGY
nutrition, agriculture, microbiology, paper industries,
textile membrane or film, cosmetics, and wastewater A. Preparation of Fish Scale
treatment. 15 kg of fish scales were collected from Bening Food
Waste from seafood processing for example Companies at Parung, Bogor. Fish scales then washed
crustaceans shell are abundantly found and are the most using running water to remove dirt and other odd
common source of chitosan[1]. Chitosan also can be particles. After that, samples were air-dried. Dry fish
isolated from freshwater fishes such as carp (Cyprinus scales ware stored in room temperature before the
carpio L.) scales. In addition, Uawonggul et al. (2002) [2] extraction process started.
and Weeraphan (2011) [3], also managed to extract chitin
and chitosan from fish scales of tilapia (Tilapia nilotica). B. Extraction of Chitosan from Fish Scale
Kumaria & Rath (2014) [4] stated that chitin and chitosan Chitosan were extracted based on [3]. The extraction
can be extracted from fish scales of Labeo rohita, too. consists of three stages, deproteinization,
There are other potential uses of fish scales according to demineralization, and deacetylation. Deproteinization
Ikoma et al. (2003) [5], which is as an inorganic was done using a solution of 4.2% NaOH and the ratio
between the fish scales with a solution of NaOH was 1: 6.
Manuscript received November 30, 2016; revised January 23, 2017.

©2017 Int. J. Pharm. Med. Biol. Sci. 16

doi: 10.18178/ijpmbs.6.1.16-19
 International Journal of Pharma Medicine and Biological Sciences Vol. 6, No. 1, January 2017

The process of making 4.2% NaOH solution was by (1)

dissolving 504 grams solid NaOH using distilled water up
to 1000 ml volume in the beaker glass. The solution then Chitosan A1655 = absorbance at a wavelength of 1655 cm
-1
was added by 11 L of distilled water. 2 kg of fish scales -1
Chitosan A3450 = absorbance at a wavelength of 3450 cm
were added to 2 L of 4.2% NaOH solution, mixed, then
heated using hotplate with a constant temperature of TABLE I. DEACETYLATION TREATMENT
60 °C, and stirred using spatula periodically for 6 hours.
Deproteinization residue was filtered and neutralized Parameter
using distilled water. The residue that has reached a pH of Treatment
7 was dried using an oven with a temperature of 60 °C for number NaOH Temperature Time
(%) (°C) (hours)
12 hours. The dried residue was then prepared for
1 60 110 4
demineralization process. 2 60 110 6
Demineralization was carried out using 2 N 4.2% HCl 3 60 120 4
solution and the ratio between deproteinization residue 4 60 120 6
with a solution of HCl was 1: 6. Demineralization began 5 60 130 4
by adding the 4.2% HCl solution in a pyrex glass 6 60 130 6
7 70 110 4
containing deproteinization residue. The solution is then 8 70 110 6
stirred periodically in room temperature for 5 hours. 9 70 120 4
Demineralization residue then filtered and neutralized 10 70 120 6
using distilled water until its pH reached 7. The residue 11 70 130 4
was dried using an oven with a temperature of 60 °C for 12 70 130 6
13 80 110 4
12 hours. Final product of demineralization is chitin.
14 80 110 6
Samples resulted from demineralization process were 15 80 120 4
tested using complete random design with variation on 16 80 120 6
NaOH concentration, temperature, and duration of 17 80 130 4
heating. 18 80 130 6
Table I showed parameters used which were: NaOH
concentrations (60%, 70%, 80%), temperature (110, III. RESULT AND DISCUSSION
120,130oC) and duration of heating (4, 6 hours). The Chitosan is produced through three processes, which
steps of deacetylation of white snapper fish scales divided are deproteinization, demineralization and deacetylation.
into 18 treatments (number 1-18). Deproteinization is protein removal process using
C. Measurement of Degree of Deacetylation on alkaline solution, to produce deproteinization residue.
Chitosan Meanwhile, the demineralization process is removal of
Degree of deacetylation was characterized using FTIR the metal content of deproteinization residue, generating
spectrophotometry with computation by providing residual demineralized form of chitin. Chitosan is
infrared light on a sample of chitosan and then the produced from chitin by a deacetylation process.
infrared absorbance was recorded. Hydroxyl groups Deacetylation is the removal of acetyl groups (-COCH3)
-1 on chitin and turn it into an amine group (NH2) using an
calculated at a wavelength of 3450 cm , whereas the alkaline solution. Degree of deacetylation on chitosan
-1
amide group is at a wavelength of 1655 cm . extracted from white snapper fish scales is showed at
Deacetylation degree of chitosan can be calculated using Table II.
this following equation as in (1) [7]:

TABLE II. DEGREE OF DEACETYLATION ON CHITOSAN EXTRACTED FROM WHITE SNAPPER FISH SCALE
Treatment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
number

DD (%) 78,35 80,51 77,09 79,25 81,72 82,02 80,19 80,59 82,30 81,45 75,00 79,79 77,49 79,22 84,05 76,09 77,41 75,58

Formation of chitosan from chitin occurs through an obtained from treatment number 15 (NaOH concentration
amide hydrolysis reaction using a base. Chitin plays role of 80%, temperature of 120oC, duration of heating for 4
as an amides and NaOH as a base. The process begins hours), while the treatment number 11 (NaOH
with an additional reaction, namely the inclusion of the - concentration of 70%, temperature of 130oC, duration of
OH group into the group NHCOCH3. Then chitosan heating for 4 hours) showed lowest degree of
produced through the elimination of cluster CH3COO- deacetylation which was 75.00%. The quality standard of
[8]. The degree of deacetylation of the chitosan obtained chitosan according to SNI 7949: 2013 is when the degree
from white snapper fish scales ranged from 75.00% to of deacetylation of chitosan reaches a minimum value of
84.05%. Degree of deacetylation was calculated based on 75%. It showed that chitosan resulted from white snapper
FTIR using equation Dosmzy & Robert (1984). The fish scales fulfilled the standard.
highest degree of deacetylation was 84.05% which was

©2017 Int. J. Pharm. Med. Biol. Sci. 17

 International Journal of Pharma Medicine and Biological Sciences Vol. 6, No. 1, January 2017

Fig. 1 showed the FTIR graph which performed which were 1655 and 3450 wavelength. The distances
chitosan with higher degree of deacetylation by then were measured based on there the highest and lowest
calculating the distance between two absorbance values point.

Figure 1. FTIR graph of treatment showing chitosan with DD higher than 80% (Treatment 2, 5, 6, 7, 8, 9, 10, 15)

©2017 Int. J. Pharm. Med. Biol. Sci. 18

 International Journal of Pharma Medicine and Biological Sciences Vol. 6, No. 1, January 2017

The amount of percent degree of deacetylation can be [9] B. Srijanto, “Kajian pengembangan teknologi proses produksi
kitin dan kitosan secara kimiawi,” in Proc. Seminar Nasional
used to determine the quality of chitosan. Base line
Teknik Kimia Indonesia, 2003, vol. 1, pp. F01-1 –F01-5.
method proposed by [7] used to calculate the percentage
of the degree of deacetylation of the chitosan scales white Dr. Noverita Dian Takarina, M.Sc was born
snapper. Chitosan is said to have been deacetylated in Surakarta, Central of Java on 16th
November 1965. She received Bachelor
perfectly if DD> 90% [9].
degree in 1990 at Faculty of Biology,
Universitas Gadjah Mada, and she received
IV. CONCLUSION her M.Sc degree in 1996 at McMaster
University, Hamilton, Ontario, Canada in
Deacetylation degree of 84,05% was resulted from cooperation with Six Universities
treatment number 15 with temperature of 120 °C, NaOH Development and Rehabilitation-Asian
concentration of 80%, and duration of heating for 4 hours. Development Bank (SUDR-ADB) Scholarship.
She received her Doctoral degree at Institut Pertanian Bogor (IPB) and
funded by Universitas Indonesia scholarship.
ACKNOWLEDGMENT She wrote four articles published by international journal in 2013 and
four articles published by accredited national journal in 2010, 2011, and
We acknowledge Ministry of Higher Education, 2013. While undergoing Doctoral degree, she wrote international
Research, and Technology and Directorate of Research scientific paper, titled Geochemical Fractionation of Copper (Cu), Lead
and Community Engagement, Universitas Indonesia (Pb), and Zinc (Zn) in Sedimen and their Correlations with
Concentration in Bivalve Mollusc Anadara indica from Coastal Area of
through PITTA UI grants Fiscal Year 2016 with contract Banten Province, Indonesia. That paper published by International
number 2047/UN2.R12/HKP.05.00/2016. Journal of Marine Science in 2013. She also wrote national scientific
paper, titled Bioconcentration Factor of Copper (Cu), Lead (Pb), and
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©2017 Int. J. Pharm. Med. Biol. Sci. 19