Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in varying sodium hydroxide concentration

Article abstract of DOI:10.1016/j.ijbiomac.2017.11.138

Large amount of sodium hydroxide (NaOH) is consumed to remove the protein content in chitin biomass during deproteinization. However, excessive NaOH concentration used might lead to the reduction of cost effectiveness during chitin extraction. Hence, the

Full text of DOI:10.1016/j.ijbiomac.2017.11.138

Accepted Manuscript
Title: Extraction and physicochemical characterization of
chitin and chitosan from Zophobas morio larvae in varying
sodium hydroxide concentration
Authors: Chu Yong Soon, Yee Bond Tee, Choon Hui Tan,
Abdul Talib Rosnita, Abdan Khalina
PII:
S0141-8130(17)32763-0
DOI:
Reference:
https://doi.org/10.1016/j.ijbiomac.2017.11.138
BIOMAC 8618
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
27-7-2017
6-11-2017
21-11-2017
Please cite this article as: Chu Yong Soon, Yee Bond Tee, Choon Hui
Tan, Abdul Talib Rosnita, Abdan Khalina, Extraction and physicochemical
characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration, International Journal of Biological
Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.11.138
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Manuscript
Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio
larvae in varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, Abdul Talib Rosnitab, Abdan Khalinac
a
Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University, 56000
Cheras, Kuala Lumpur, Malaysia
b
Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
c
Department of Biological and Agricultural Engineering, Faculty of Engineering, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia
Full name
Given name
Chu Yong
Yee Bond
Choon Hui
Rosnita
Family name
Soon
Tee
Tan
Abdul Talib
Abdan
Parentheses
C.Y. Soon
Y. B. Tee
C. H. Tan
A. T. Rosnita
A. Khalina
Chu Yong Soon
Yee Bond Tee
Choon Hui Tan
Abdul Talib Rosnita
Abdan Khalina
Khalina
*Corresponding author
Address: Department of Food Science and Nutrition, Faculty of Applied Sciences, UCSI University,
56000 Cheras, Kuala Lumpur, Malaysia
Tel.: +603 9101 8880
Fax: +603 9102 3606
E-mail address: teeyb@ucsiuniversity.edu.my
E-mail address of each author
Chu Yong Soon
*Yee Bond Tee
Choon Hui Tan
: 1001645059@student.ucsiuniversity.edu.my
: teeyb@ucsiuniversity.edu.my
: tanch@ucsiuniversity.edu.my
1

A. Talib Rosnita
Abdan Khalina
: rosnita@upm.edu.my
: khalina@upm.edu.my
ABSTRACT
Large amount of sodium hydroxide (NaOH) is consumed to remove the protein content in chitin
biomass during deproteinization. However, excessive NaOH concentration used might lead to the
reduction of cost effectiveness during chitin extraction. Hence, the present study aimed to extract and
evaluate the physicochemical properties of chitin and chitosan isolated from superworm (Zophobas
morio) larvae using 0.5 M to 2.0 M of NaOH. The extracted chitin and chitosan were subjected to
Fourier Transform Infrared Spectroscopy (FT-IR), elemental analysis, Scanning Electron Microscope
(SEM), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and X-ray
Diffraction (XRD). The 0.5 M NaOH treatment resulted in the highest yield of chitin (5.43%), but
produced the lowest yield (65.84%) of chitosan. The extracted chitin samples had relatively high degree
of acetylation (DA) (82.39%-101.39%). Both chitin and chitosan showed smooth surface with tiny
pores. The extracted chitin samples were confirmed as α-chitin based on the FT-IR and TGA. The chitin
samples were amorphous with low degree of crystallinity. From TGA, the Chitosan 3 extracted was
partially deacetylated. Both DPPH radical scavenging and ferric-chelating assay showed positive
correlation with DD of chitosan isolates. However, the chitosan isolates were not fully dissolved,
resulting in lower radical scavenging and ferric-chelating ability compared to commercial chitosan.
Keywords: Chitin; Chitosan; Zophobas morio; physicochemical properties
1. Introduction
Chitin is a linear biopolymer with monomer β-(1→4)-N-acetyl-D-glucosamine unit. It is
biocompatible, biodegradable, non-toxic and highly insoluble in most of the polar and non-polar solvent
[1]. Due to these preferable properties, chitin has been extensively used in the food, cosmetics,
composite material, wastewater treatment, and biomedical industry [2–5]. The chitin can be further
2

treated with concentrated NaOH in extreme conditions to produce chitosan via deacetylation [6–8].
Chitosan is a linear co-polymer obtained by partial deacetylation of chitin derivative which consists of
N-acetyl glucosamine units and D-glucosamine unit blocks. It is less amphiphilic and soluble in mild
acidic solvent [9,10]. Chitosan can be used as the filler in packaging material development due to its
antimicrobial properties to broad spectrum of microorganisms [11]. The chitosan with high degree of
deacetylation (DD) was crucial as biomaterial in tissue engineering due to its polycationic nature that
able to interact with negative-charged cell membrane [12]. Besides, the chitosan is the raw material to
synthesis the O-carboxymethyl chitosan Schiff bases supported metal ion complex heterogeneous
catalyst, which exhibits excellent catalytic activity and is reusable [13–15].
Recently, there is an interest in discovery of potential chitin and chitosan source from insects.
Chitin fibre can be found abundantly in the cuticle of insects and is covered with protein matrix that
contains catechol and minerals [16]. The current source for massive production of chitin is from marine
crustaceans, such as shrimp, lobster, and crab [7,8,17–20]. Other than insect and marine source, chitin
can also be found in fungus [19,21,22]. Nevertheless, the extraction of chitin from all the biosources
mentioned utilizes the same facile chemical treatment procedure, namely demineralization,
deproteinization and decolourization. In this study, superworm (Zophobas morio) was studied for the
extraction of the chitin and chitosan.
Superworm is found abundantly in tropical countries and is widely used as aquaculture feed. A
normal superworm has approximately 57% of water content. The dried superworm contains low amount
of ash content (about 3.5%), closed to 6.9% of total nitrogen and relatively high amount of crude fat
(around 40.8%) [23]. Interestingly, superworm was found to be able to produce antimicrobial peptides
(AMPs) which showed relatively good antimicrobial activity against bacteria [24]. Considering its low
land and space utilization per culture breeding, low requirement of culture maintenance, high adaptation
to tropical climates, and large number of offspring per reproduction, Z. morio could have the potential
as an alternative source of chitin and chitosan production [25,26].
3

The chitin biosources have the significant amount of protein content as mentioned. Therefore,
deproteinization using alkaline treatment should be carried out in order to ensure the high purity of the
chitin. The deproteinization parameters includes the concentration of NaOH, temperature and time of
reaction [6,27–32]. From the study of the chitin extraction from silkworm chrysalides, it was concluded
that the yield of the chitin decreased as the time of basic reaction or the temperature of reaction increased
[6]. However, the relationship of the yield of chitin and its corresponding physicochemical properties
was not yet revealed. Nonetheless, at least to the authors’ knowledge, there was no research done in the
investigation of the NaOH concentration during deproteinization to the physicochemical properties of
chitin from insect source. Hence, the inspiration to conduct the present research was obtained from the
finding of different researchers that utilized varying deproteinization conditions to purify chitin.
In the present study, the chitin and chitosan samples were extracted from Z. morio larvae using
different NaOH concentrations during deproteinization. The physicochemical properties of the chitin
and chitosan samples were evaluated and the effects of the different basicity treatment were studied.
The findings of this present study could provide a new insight of the physicochemical changes of chitin
and chitosan at different extraction conditions and hence, promote the reduction of NaOH chemical
concentration used during chitin extraction. Besides, it also served as an exploration to the potential of
Z. morio as an alternative raw material for chitin and chitosan production, in par with the increasing
demand worldwide.
2. Materials and methods
2.1 Materials
Processed (dried) Z. morio larvae was purchased from Macau Pet Trading Sdn. Bhd., Selangor,
Malaysia. The chemicals used for chitin extraction were hydrochloric acid (HCl), sodium hydroxide
(NaOH), and acetone. Acetic acid was used to dissolve the chitosan. All reagents used were of analytical
4

grade. Commercial chitin (CAS: 1398-61-4) and chitosan (CAS: 9012-76-4) were purchased from
Sigma Aldrich.
2.3 Chitin extraction from Z. morio
The processed larvae was washed with running tap water to remove impurities such as dirt sand
and organic residues. Then, they were dried in convection oven at 70°C overnight. The dried larvae was
then blended in low speed and kept in air-tight plastic seal.
The extraction of chitin was modified in reference to Kaya, Akyuz, et al. (2016). The chitin
extraction process consisted of stages as summarized below:
i.
Demineralization: 4 g of blended worms were mixed with 1.0M of HCl solution (1:20 ratio
of solid to solution) in 35°C water bath at 100 rpm rotation for 30 minutes to remove
mineral content and catechols. The samples were filtered out and washed with distilled
water until neutral pH was achieved in the washing solution.
ii.
Deproteinization: The demineralized samples were mixed with three different
concentration of NaOH solution (0.5 M, 1.0 M and 2.0 M) (1:20 ratio of solid to solution),
which were referred to as Chitin 1, 2 and 3, respectively. Deproteinization was carried out
in 80°C water bath at 100 rpm rotation to promote the reaction for 20 hours. The samples
were filtered out and washed extensively with distilled water until neutral pH.
Decolorization: The partially pure chitin samples were soaked in glacial acetone (1:10 ratio
of solid to solvent) for 30 minutes to remove organic pigment. The chitin was filtered out
from the excess acetone and dried further in fume hood for 30 minutes for volatile acetone
removal. Then, the chitin isolates were dried completely in convection oven at 70°C
overnight.
iii.
2.4 Chitosan preparation
5

The chitin isolates were deacetylated with 50 wt% NaOH in 90°C water bath for 30 hours and
100 rpm rotation. The samples were filtered and washed with distilled water until neutral pH. Chitosan
samples were then dried in convection oven at 70°C overnight. The Chitosan 1, 2 and 3 were the
products of deacetylation from Chitin 1, 2 and 3, respectively.
2.5 Characterization tests
2.5.1 Elemental analysis
Elemental analysis was performed using (LECO CHNS-932, USA). The degree of acetylation
(DA) of the chitin and degree of deacetylation (DD) of chitosan was determined using percentage
composition of carbon (C) and nitrogen (N) as shown in Equation (1) and Equation (2), respectively
[33].
DA(%) =
퐷퐴(%) = [(C/N − 5.14)/1.72]
(1)
(2)
퐷퐷(%) = 100 − [(6.857 − 퐶/푁)/1.7143] × 100
2.5.2 FT-IR analysis
Both chitin isolates and chitosan samples were characterized using FT-IR (Thermo Scientific
Nicolet iS5, USA) with the attenuated total reflectance (ATR) technique to determine the chemical
compositions. The FT-IR spectrum was recorded in the range of wavelength 400 cm-1 to 4000 cm-1 with
4 cm-1 spectral resolution. A total of 64 scans were conducted for each sample.
2.5.3 Scanning electron microscope (SEM)
A scanning electron microscope (JSM-IT100 SEM, Japan) was employed to observe the
microstructure and morphology of all samples. The samples were sputter-coated with a thin gold layer
6

in liquid nitrogen. The acceleration voltage was fixed at the range of 15-20 kV to ensure high resolution
image.
2.5.4 Thermogravimetric analysis (TGA)
The thermal degradation properties of the Z. morio chitin and chitosan isolates were evaluated
using thermogravimetric analyzer (Perkin Elmer, TGA 7, USA). Approximately 10 mg from each
sample were weighted and placed on the heating pan. The test were conducted in the ramp mode of
temperature range from 30°C to 600°C at constant heating rate of 10°C min-1 under nitrogen atmosphere
(20 mL min-1) to avoid thermo-oxidative degradation.
2.5.5 Differential scanning calorimetry (DSC)
The thermal test was done using differential scanning calorimeter (DSC822e, Mettler Toledo,
Greifensee, Switzerland) in accordance to ASTM D3418 standard. Approximately 10mg of the chitin
samples were heated in a semi hermetic pan from room temperature to 200°C with a heating rate of
10°C min-1 under a nitrogen atmosphere. After 5 min of holding time for the elimination of the thermal
history, the samples were cooled to −30°C, held for 5 min, and heated to 400°C with a heating rate of
10°C min-1.
2.5.6 X-ray diffraction (XRD)
The chitin samples were characterized using X-ray powder diffractometer (XRD; Shimadzu XRD-6000)
employing Cu-Kα (λ=1.5406 Å). The analysis was conducted at 30 kV, 30 mA and 2θ with the scan
angle from 5° to 45°. The degree of crystallinity (Xcr) was obtained from the area ratio of the crystal
phase to the total phase of crystal and amorphous phase in XRD diagram using Equation (3):
푋푐푟(%) = (퐼(110) − 퐼(020))/퐼(110) × 100
(3)
where 퐼( is the maximum/peak intensity at a 2θ angle of crystalline material and 퐼(020) is the
)
110
maximum/peak intensity at a 2θ angle of amorphous material.
7

2.6 Antioxidant test
2.6.1 Materials
DPPH (2, 2-diphenyl-2-picryhydrazyl) free radical, ferrozine iron reagent, iron (II) chloride
tetrahydrate (FeCl2·4H2O) was purchased from Eriendemann Schmidt, Acros Organics (New Jersey)
and Merck (Darmstadt, Germany), respectively. All solvents used were of analytical reagent grade.
2.6.2 (2, 2-diphenyl-2-picryhydrazyl) (DPPH) radical scavenging activity
The free radical scavenging activities of chitosan extracts (62.5, 125, 250, 500, 1000 µg/mL)
were identified. 0.5 mL of chitosan in various concentrations of chitosan extracts were mixed with 2.5
mL of 1 mM methanolic DPPH. The solution was incubated for 30 minutes in the dark room at room
temperature. The absorbance of the solution after incubation was taken at wavelength 517 nm. IC50
(concentration of sample that inhibits 50% of radical activity) was identified. Commercial chitosan was
set as the reference. The inhibitory activity (I) was calculated using Equation (4):
(
)
퐼 % = (퐴0 − 퐴1)/퐴0 × 100
(4)
where 퐴0 is the absorbance of the control group and 퐴1 is the absorbance of the chitosan extract.
2.6.3 Metal chelating ability
Ferrous chelating activity was assessed by the method via reference of Soler-Rivas, Carlos
Espín, & J. Wichers (2000) with some modifications [34]. 200 µL of chitosan extracts (62.5, 125, 250,
500, 1000 µg/mL) were mixed with 100 µL of 2 mM FeCl2 and 400 µL of 5 mM of ferrozine. The
solutions were incubated for 10 minutes before the absorbance measurement in 562 nm. CEC50
(effective concentration of sample to chelate ferrous irons by 50%) was identified. Commercial chitosan
was set as the reference. The chelating effect (C) was calculated using Equation (5):
(
)
퐶 % = (퐴0 − 퐴1)/퐴0 × 100
(5)
8

where 퐴0 is the absorbance of the control group and 퐴1 is the absorbance of the chitosan extract.
3. Results and discussion
3.1 Yield
The yield of chitin and chitosan isolates from Z. morio larvae was shown in Table 1. The
extracted Z. morio chitin was in flakes structure and the brown colour intensity reduced from Chitin 1
to Chitin 3. The yield of chitin decreased as the concentration of NaOH increased. Higher concentration
of NaOH showed higher capability to remove more protein contents deposited at the chitin. The yield
of chitin from superworm was relatively low, which was in the range of 4.77% to 5.43%. The chitin
yield was slightly higher than larvae wasp (Vespa crabro) (2.1%) but lower than Colorado potato beetle
(Leptinotarsa decemlineata) (7%). However, the chitin yield might increase by folds after
polymorphism to adult beetle [29,30].
The chitin extracts were further treated with concentrated NaOH to partial deacetylate the acetyl
group in C2 of the pyranose ring [18]. Interestingly, the yield of chitosan increased when the chitin was
pre-treated with higher concentration of NaOH during deproteinization. This might be due to the high
concentration of NaOH which aided in the removal of protein residues. Chitin 1 which showed low DA
value (82.39%) was further deproteinized during the deacetylation in concentrated NaOH. Hence,
Chitin 1 which showed the highest chitin yield when compared to Chitin 2 and 3, had the lowest chitosan
yield in the subsequent chitosan production. The range of chitosan yield from Z. morio chitin extracts
(65.84% to 75.52%) was close to larvae (67%) and adult (72%) of L. decemlineata [29]. The purification
of chitosan by extensive dilution of concentrated NaOH and filtration could lead to partial yield lost.
3.2 Elemental analysis
9

The element compositions of carbon (C), hydrogen (H), and nitrogen (N) in Z. morio chitin and
chitosan were examined using elemental analysis to estimate the DA of chitin and DD of chitosan as
shown in Table 1. The N composition of extracted chitin was slightly lower than the theoretical value
(6.89%) of fully acetylated chitin and a low experimental N composition indicated low protein amount
that was left in the insect chitin [35,36]. The corresponding DA of the extracted chitin was 82.39%,
92.83% and 101.39% within the range of NaOH concentration tested. The DA of Chitin 3 was close to
100% and indicated a relatively high purity chitin extract [32]. The leftover impurities could be mineral
salts and protein residues. The concentration of NaOH was one of the parameter to affect the purity of
the chitin. However, the highest DA of chitin (Chitin 3) had the lowest yield. Meanwhile, the DA of
commercial chitin was recorded as 95.53%, which was close to Chitin 2 at 93.83%.
Beside the concentration of NaOH, the DA of chitin could be affected by other factors such as
the type of biomass, method of reaction, temperature and time of extraction. From the literarature, the
NaOH concentration used ranged from 1M to 4M. The chitin extraction from cockroach (Periplaneta
americana) produced 98.67% chitin when treated with 4M of NaOH in 150°C for 20 hours [27].
However, for wasp, the deproteinization conditions were milder than the previous study, which was
only with 1M of NaOH in 60°C for 16 hours, able to produce the chitin with DA ranging from 108.8%
to 127.5% [30].
Generally, the C composition in chitin were slightly higher than the chitosan extracted from Z.
morio and commercial chitosan. Meanwhile, the H and N compositions in the chitosan were higher than
chitin after the treatment of NaOH. During deacetylation, partial of the acetyl chain in N-acetyl
glucosamine was removed and converted to amine group by forming new N-H bond [18]. The removal
of acetyl chain from chitin caused the reduction of C composition in the chitosan but increased N and
H compositions. The C, H and N contents of Z. morio chitosan were close to that of adult and nymph
grasshopper (Dociostaurus maroccanus) [37]. The chitosan DD value obtained in the present research
was the lowest in Chitosan 2 (64.82%), and the highest in Chitosan 1 (81.06%). From Table 1, the
10

purity of the chitin had no effect to the final DD of the partial deacetylated chitosan. The DD value of
chitosan from larval potato beetle was 76% [29], which was close similar to the Chitosan 3 (74.14%)
under the same NaOH concentration during deacetylation.
3.3 FT-IR analysis
The FT-IR spectra bands of chitin isolated from Z. morio using different NaOH concentration
and commercial chitin were shown in Fig. 1 (a). The bands which denoted the structure of chitin
includes 1150 cm-1 (complex vibrations of NHCO group) and 1650 cm-1 (C-O secondary amide stretch).
Chitin polymer is linked by poly-β-(1→4)-N-acetyl-D-glucosamine unit blocks in three different
polymorphs; α (antiparallel), β (parallel), and γ-chitin (combination α and β-chitin) [38]. From the peaks
of 1550 cm-1 (C-N stretch), 1620 cm-1 (N-H bend) and 1650 cm-1 (C-O secondary amide stretch), it was
hypothesized that the chitin isolated from Z. morio was in α-form because the amide I band was split
into two bands, namely, 1620 cm-1 and 1650 cm-1, respectively [27]. α-chitin was also observed in other
arthropoda sources, such as shell of shrimps [39], lobsters [19], and crab [40]. However, X-ray
diffraction should provide more accurate determination of the polymorph of the chitin.
Fig. 1 (b) showed the FT-IR spectrum of the Chitosan 1, 2, 3 from Z. morio and commercial
chitosan. The structures of both Z. morio and commercial chitosan were closely similar. However, there
were some bands absent in the commercial chitosan sample, which were the complex vibrations of
NHCO group (1260 cm-1), H band, CH3 symmetric deformation (1377 cm-1), N-H band (Amide I) (1620
cm-1), and symmetric N–H stretching (3105 cm-1). Z. morio chitosan showed primary amide unit by
giving two bands of N-H stretching at 3105 cm-1 (symmetric) and 3350 cm-1 (asymmetric) while
commercial chitosan showed secondary amide by having one N-H bands near 3300 cm-1.
11

3.4 Morphology analysis
Fig. 2 showed the surface morphology of Z. morio cuticle before and after extraction. The
surface of Z. morio cuticle before extraction in Fig. 2 (a) was rough and uneven, which could denote
the layer with high protein content and some mineral salts. After treatment of demineralization and
deproteinization at different concentration of NaOH, the surface of the chitins were as showed in Fig.
2 (b), (c) and (d), respectively. There were not much difference in surface morphology when the NaOH
concentration was varying. This result also indicated DA of chitin had no effect to the surface
morphology. From Fig. 2, tiny pores were evenly distributed on the smooth surface of the chitin isolates.
The micro-size pores were appeared on intermitant. Similar surface morphology was reported in pupa
of Vespa crabro (bee), Archispirostreptus gigas (large diplopods) and the dorsal pronotum of Blaberus
giganteus (cockroach) [30,41,42].
The different metamorphosis stages of insects showed different structure of chitin arrangement.
The chitin extracted from black soldier flies larvae (Hermetia illucens) had complex repeating units of
hexagonal honeycomb structure and was different to the adult chitin which was smooth and had parallel
distribution of fibres [43]. The different body parts of the insect also showed the different chitin surface
morphology, as proven in adult honeybee [44]. Hence, in this present research, superworm larvae should
only present one type of chitin surface morphology due to its uniform structure arrangement of shell in
each segments.
The surface morphology of commercial chitosan and Chitosan 3 from Z. morio were shown in
Fig. 3 (a) and 3 (b), respectively. The surface of commercial chitosan appeared as micro-fibrillated
crystalline structure. After partial deacetylation of chitin, the surface morphology of Chitosan 3
remained the same with Chitin 3, that was, tiny pores on the smooth surface with micro-sized pores.
Hence, the deacetylation of Z. morio chitin had no effect to the surface morphology.
12

3.5 TGA and DSC
Fig. 4 (a) showed the Thermogravimetric Analysis (TGA) and Differential Thermogravimetric
(DTG) of the chitin samples. All of the samples had the first mass degradation step within 50°C to
100°C which indicated the evaporation of moisture in the samples. The maximum degradation
temperature (Tm) of Chitin 1, 2, 3 and commercial chitin in were recorded as 390.4°C, 391.7°C, 391.1°C
and 397.0°C, respectively. The TGA analysis in the present study was closely similar to the chitin in
butterfly (Argynnis Pandora) with Tm from 386.9°C to 389.6°C, which showed that the chitin extracted
from Z. morio was α-chitin [28]. At the same time, the Tm of α-chitin was higher than that of β-chitin
(270-350°C) due to its antiparallel alignment with the adjacent polymer chain that able to form more
hydrogen bonds [5].
From Fig. 4 (b), the commercial chitosan showed two degradation steps (66.5°C and 301.0°C).
Meanwhile, the Chitosan 3 showed three degradation steps, which were at 63.1°C, 308.7°C and 396.8°C.
Similar trend was reported with silkworm chrysalides [6]. The second and third mass degradation steps
of Chitosan 3 could be due to the degradation of D-glucosamine and N-acetyl glucosamine, respectively.
This also showed that Chitosan 3 was partially deacetylated. The derivative weight loss of D-
glucosamine (0.15 wt% s-1) was two-folds greater than N-acetyl glucosamine (0.08 wt% s-1) in partial
deacetylated Chitosan 3. The thermal degradation temperature of chitosan was lower than chitin’s due
to its amine group which has lower polarity than chitin’s amide group with comparatively higher
polarity.
Endothermic and exothermic curves were observed from DSC analysis as shown in Table 2.
The chitin samples showed the wide endothermic heating peaks from 30°C to 170°C during the first
heating program, which could be attributed to the evaporation of water content in the chitin samples
[45,46]. There was an obvious positive shift of the heating curve in Chitin 1, where the To, Tp and Tc
were higher than Chitin 2 and 3. The lower DA of Chitin 1 had more protein impurities content which
led to higher water holding ability. Meanwhile, Chitin 3 had the narrowest first heating peak (34°C to
13

153°C), and the lowest exothermic enthalpy peak (-275.11 J g-1), indicated higher purity of chitin with
less water content in the chitin sample.
For the exotherm heating peaks observed during the second heating program, the heating peaks
were from 270°C to 330°C. The increase of NaOH concentration of treatment during deproteinization
resulted slight increase of thermal stability (To) as shown in Chitin 3 (285°C). This finding was in lined
with the TGA result above where the chitin samples degraded thermally around 270°C. The exothermic
peak were around 300°C were close to the findings in the commercial chitin-chitosan (306°C) [46].
3.6 XRD
The X-ray Diffraction (XRD) results of chitin samples were shown in Fig. 5. Two sharp peaks
and four weak peaks were found in all XRD spectra of the chitin samples. The peaks of Chitin 1 were
9.28°, 19.38°, 21.08°, 23.14°, 26.50°, and 27.98°. The peaks observed in Chitin 2 were 9.24°, 19.32°,
21.02°, 23.24°, 26.06°, 27.96° and in Chitin 3, the peaks were 9.28°, 19.40°, 21.02°, 23.16°, 26.32°,
and 28.24°. The two sharp peaks were found to be around 9.26° and 19.36° for all the chitin samples,
which were diffraction planes (020) and (110) of the orthorhombic crystal structure [47]. The chitin
XRD peaks were found close similar to the chitin extracted from insects and even commercial chitin
[29].
The degree of crystallinity (푋푐푟) was calculated based on the intensity of the crystalline region
and amorphous region. The 푋푐푟 of Chitin 1, 2 and 3 were 67.82%, 60.62 and 57.62, respectively. The
푋푐푟 decreased when the chitin were deproteinized in higher concentration of NaOH. Nevertheless, the
푋푐푟 obtained from Z. morio were relatively lower than other insect species such as cicada sloughs
(82.7%-89.7%), beetle Motschulsky (89.05%), and Colorado potato beetle (72%-76%) [29,48,49].
Meanwhile, the 푋푐푟 of butterfly chitin (64%-66%) was closer to the Z. morio chitin [28].
14

3.7 Antioxidant analysis
The scavenging ability on DPPH and metal-chelating ability of chitosan were shown in Table
3. Chitosan 1, 2 and 3 were partially dissolved whereas commercial chitosan was fully dissolved in 1%
of acetic acid solution. This indicated the chitosan isolates had relatively high molecular weight due to
its high intermolecular attraction force between the chitosan fibres. Hence, the solubility of the Z. morio
chitosan in diluted weak acid was low [50,51]. Notably, the higher DD value of partial deacetylated
chitosan, the lower IC50 and CEC50 concentration. Meanwhile, the commercial chitosan with DD 75-
85% and molecular weight (Mw = 190-310 kDa) [52] showed the lowest IC50 (1.68 mg/mL) and CEC50
(12.58 µg/mL). This might due to its high DD value, medium molecular weight and consequently, the
strong antioxidant activity of commercial chitosan [53]. The adult Colorado potato beetle (L.
decemlineata) (DD: 82%) reported lower IC50 than Z. morio chitosan (65.87 mg/mL – 140.73 mg/mL),
which were 4.15 mg/mL. The IC50 of chitosan from grasshoppers (C. babarus and O. decorus) was
slightly lower than Chitosan 1, which were only 10.68 and 10.91 mg/ml, respectively [31].
From the ferric chelating assay, the nitrogen in amine group of D-glucosamine acted as the lone
pair of electron donor to form coordinate bond with positive charge iron ions which had vacant d orbitals
[51]. The higher DD of chitosan indicated higher molar fraction of D-glucosamine unit [10], and hence
greater chelating activity due to the presence of higher amount of free electron donor amine group. The
chelating effect of the chitosan samples had minimal range of difference in CEC50 (17.75 µg/mL to
21.70 µg/mL) and were close to the commercial chitosan (12.58 µg/mL). Hence, the chitosan samples
might have the potential to be stable toxic heavy metal chelating agent.
4. Conclusion
15

The physicochemical properties of the chitin and chitosan samples extracted from Z. morio
superworm was determined after the treatment of different NaOH concentration (0.5 M to 2.0M) during
deproteinization. The yield of superworm chitin increased when treated with lower NaOH concentration.
Nonetheless, the chitin samples with higher NaOH concentration treatment had higher DA value. The
higher DA value of Z. morio chitin yielded higher amount of chitosan after partial deacetylation. The
DD values of Z. morio chitosan were not affected by the DA of the chitin. The structure of chitin was
confirmed as α-chitin based on FT-IR and TGA. The partial deacetylated chitosan samples from
superworm showed double thermal degradation stages in the range of 300°C to 400°C, which denoted
the degradation of D-glucosamine and N-acetylglucosamine. 0.5 M of NaOH was not sufficient to
remove the protein impurities entirely from Z. morio larvae, proven with wider endothermic heating
peak in DSC analysis. The extracted chitin from superworm were highly amorphous, which had the
degree of crystallinity from 57.62% to 67.82%. The higher DA value of the chitin samples showed
lower crystallinity structure. The free radical scavenging and ferric chelating ability of partial
deacetylated chitosan samples increased when the DD values increased. Overall, 2.0 M of NaOH was
sufficient enough to produce the chitin from Z. morio with relatively high purity compared to the other
sources that used up to 4.0M of NaOH. Hence, it has the potential as the alternate source of chitin and
chitosan production for biotechnological, biomedical, packaging and industrial catalytic applications.
Authors’ contribution
Chu Yong Soon
*Yee Bond Tee
Choon Hui Tan
A. Talib Rosnita
Abdan Khalina
: Main author and conduct the research
: Manuscript editing and experimental consultation
: Consultation on the manuscript content
: Laboratory support for the experiment
: Laboratory support for the experiment
Acknowledgement
16

The present research was financially supported by UCSI University Pioneer Scientist Initiative Fund
(PROJ-in-FAS-030) and University Putra Malaysia.
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Captions to illustrations
Fig. 1: The attenuated FT-IR spectrum of (a) chitin and (b) chitosan with wavelength ranging from 4000
cm-1 to 400 cm-1 with 4 cm-1 spectral resolution.
Fig. 2: SEM image of (a) grinded Z. morio (before chitin extraction), (b) Chitin 1, (c) Chitin 2, and (d)
Chitin 3 at 1, 000x magnification.
Fig. 3: SEM image of (a) commercial chitosan from shrimp, and (b) chitosan 3 from Z. morio at 3, 000x
magnification.
Fig. 4: The (a) TGA and DTG of chitin samples, and (b) TGA and DTG of chitosan samples with the
range of 30°C to 600°C at constant heating rate of 10°C min-1 under nitrogen atmosphere.
22

Fig. 5: The XRD spectrum of (a) Chitin 1, (b) Chitin 2, and (c) Chitin 3 with scanning angle from 5° to
45° at 30 kV, 30 mA.
Illustrations
Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, A. Talib Rosnitab, Abdan Khalinac
Fig. 1 (a)
23

Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, A. Talib Rosnitab, Abdan Khalinac
Fig. 1 (b)
24

Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, A. Talib Rosnitab, Abdan Khalinac
Fig. 2
25

Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, A. Talib Rosnitab, Abdan Khalinac
Fig. 3
26

a
Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, A. Talib Rosnitab, Abdan Khalinac
Fig. 4 (a)
27

b
Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, A. Talib Rosnitab, Abdan Khalinac
Fig. 4 (b)
28

a
Extraction and physicochemical characterization of chitin and chitosan from Zophobas morio larvae in
varying sodium hydroxide concentration
Chu Yong Soona, Yee Bond Teea,*, Choon Hui Tana, A. Talib Rosnitab, Abdan Khalinac
Fig. 5 (a)
29