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Production of chitin and chitosan from the exoskeleton of adult two‐spotted

field crickets (Gryllus bimaculatus)

Article  in  Entomological Research · September 2017

DOI: 10.1111/1748-5967.12239

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Entomological Research 47 (2017) 279–285

SHO RT C OM MU NI CA TIO N

Production of chitin and chitosan from the exoskeleton of

adult two-spotted field crickets (Gryllus bimaculatus)
Min-Woo KIM1, Yong-Su SONG1, Yeon Soo HAN2, Yong Hun JO2, Myung Hyo CHOI3, Young-Kyu PARK3,
Seung Ho KANG3, Sun-Am KIM4, Changyong CHOI5 and Woo-Jin JUNG1
1
Department of Agricultural Chemistry, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Science, Chonnam
National University, Gwangju, South Korea
2
Division of Plant Biotechnology, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Science, Chonnam National
University, Gwangju, South Korea
3
Korea Beneficial Insects Lab. Co. Ltd., Gokseong-gun Jeollanam-do, South Korea
4
Jeonnam Bioindustry Foundation Bio Control Research Center, Gokseong, South Korea
5
Department of Polymer Science and Engineering, Sunchon National University, Jeonnam, South Korea

Correspondence Abstract
Woo-Jin Jung, Department of Agricultural
Chemistry, Institute of Environmentally-Friendly Chitin and chitosan were extracted from all specimens of Type I and II two-spotted
Agriculture (IEFA), College of Agricultural and field crickets (Gryllus bimaculatus) following chemical treatment with an acid and
Life Science, Chonnam National University, alkali. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to
Gwangju 61186, South Korea. achieve demineralization and deproteinization, respectively. For chitosan extraction,
Email: woojung@jnu.ac.kr 50 % NaOH (w/v) and 50 % NaOH (w/w) solutions were used to achieve
deacetylation. Chitosan yielded from adult exoskeletons of G. bimaculatus in Test
Received 28 May 2016;
accepted 18 April 2017.
A of Type I was 1.76 and 8.40 % on a fresh weight (FW) and dry weight (DW) basis,
respectively, after treatment with 50 % NaOH (w/v) at 95°C for 3 h. Furthermore, the
doi: 10.1111/1748-5967.12239 chitosan yielded in Test D of Type II was 1.79 and 7.06 % on FW and DW basis,
respectively, after treatment with 50 % NaOH (w/w) at 105°C for 3 h. The average
yield of chitin and chitosan was 2.42 and 1.65 % on a FW basis, and 10.91 and
7.50 % on DW basis, respectively. The deacetylation (%) of chitosan extracted from
adult exoskeletons in Tests A, B, C1, C2, D1, and D2 were 81.2 %, 14.5 %, 19.6 %,
90.7 %, 17.1 %, and 95.5 %, respectively. The viscosities of the chitosans extracted
from adult exoskeletons in Tests A, C2, and D2 were 32.0, 21.6, and 62.4 cP (centi
Poise), respectively. The molecular weight of chitosan from adult exoskeletons of
G. bimaculatus was 308.3 kDa. Our results indicate that adult exoskeletons of G.
bimaculatus could be used as a source of chitin and chitosan for use as functional
additives in industrial animal feeds.

Key words: adult exoskeleton, chitin, chitosan, Cricket (Gryllus bimaculatus).

Introduction chitosan-based feed additive improved the growth and quality

Chitin is most commonly obtained from the exoskeletons of
crustaceans, such as crabs and shrimp, and may also be
partially obtained from the exoskeletons and exuviae of
insects. The insect exoskeleton also provides support as a solid
shell for the attachment of muscles. A chitosan coating
material obtained from crab shells was used for the
environmentally friendly improvement of moist pellets used
to feed cultured flounder fish (Cha et al. 2006). The use of a
of cultured Japanese flounder (Kim et al. 2005). In addition,
chitin and chitosan extracted from a prawn was fed to fish to
reinforce their immune system and promote disease resistance
(Mari et al. 2014), and chitosan extracted from the
exoskeleton of shrimp, crabs, and insects has been used as
an additive in poultry and pig feedstocks (Swiatkiewicz
et al. 2015). These compounds have beneficial biological
effects, including immunomodulatory, anti-oxidative,
antimicrobial, and hypocholesterolemic properties.

© 2017 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
M.-W. Kim et al.
In this study, chitin and chitosan were extracted from the
adult exoskeleton of the two-spotted field cricket (Gryllus
bimaculatus). G. bimaculatus (Orthoptera: Gryllidae) is
widely distributed in Korea and around the world. Recently,
G. bimaculatus was temporarily approved as an edible insect
in Korea. In addition, new cooking techniques are being
developed using insects such as G. bimaculatus. The lifecycle
of the two-spotted field cricket is composed of an egg
(13 days), nymph (40–50 days), and adult (20 days) stage.
Using G. bimaculatus as a feed additive for poultry resulted
in an increase of unsaturated fatty acids (12–23 %) in chicken
meat (Ahn et al. 2000). Methanol extracts from G.
bimaculatus display liver protection properties in mice (Ahn
et al. 2002). The quality characteristics of G. bimaculatus
reported on the aspect of nutrient content including
polyunsaturated fatty acids and amino acid (Kim et al. 2015).
Exoskeletons of adult G. bimaculatus can be obtained in
large quantities from Korea Beneficial Insects Lab. Co., and
serve as a beneficial resource for insect-based animal feed
and in the food industry. However, the extraction of chitin
and chitosan from G. bimaculatus adult exoskeletons has not
been extensively studied. In this study, chitin and chitosan
were isolated from the exoskeletons of G. bimaculatus adults
using a chemical manufacturing process. The objective of this
study was to obtain chitin and chitosan from these
exoskeletons to develop a functionally improved insect-based
feed for livestock, cultured fish, and the food industry.
Materials and methods
Preparation of chitin and chitosan
Entire adult (Type I and II) two-spotted field crickets (G.
bimaculatus) were supplied from Korea Beneficial Insects
Lab. Co. (Goksung, Junnam, Korea). Type I G. bimaculatus
(fresh weight: 1.15 g/three insects) were smaller in size and
weight than Type II (fresh weight: 2.23 g/three insects)
specimens. Adult G. bimaculatus were obtained in large
quantities after breeding (Park et al. 2013). Chitin was
extracted from the exoskeletons of entire adult G. bimaculatus
using the method of Hackman (1954) with modifications.
Entire adults were chopped into pieces [fresh weight of Test
A (50 g, Type I), Test B (50 g Type II), Test C (100 g, Type
II), and Test D (200 g, Type II)] and washed with tap water.
The exoskeletons were then decalcified for 3 h in 500 mL
2 N HCl at 21°C. After decalcification, the samples were
incubated in 500 mL 1.25 N NaOH at 95°C for 3 h to remove
proteins, and then washed with water until around pH 7 and
dried in an oven at 70°C for 24 h.
To obtain chitosan, chitin particles from G. bimaculatus
were boiled in 500 mL 50 % NaOH (w/v) and 50 % NaOH
(w/w) solutions at 95 and 105°C for 3 h (Song et al. 2014),
280
respectively. The resulting chitosan was washed with tap
water until around pH 7 and then dried for 24 h at 70°C in
an oven.
Determination of the viscosity and molecular weight
of chitosan
The viscosity of chitosan samples obtained from G.
bimaculatus was determined via viscosity measurements
taken in acetic acid solution. The measurements were made
at 21°C using a Brookfield DV-II+ Pro Viscometer
(Brookfield AMETEK, Inc., Middleboro, MA, USA)
equipped with an LV-3 spindle (model no. 63; rpm = 100).
Viscosity was measured in cP units (centipoise = mPa·s).
The molecular weight of chitosan samples obtained from G.
bimaculatus was determined by gel permeation
chromatography equipped with multi-angle laser light
scattering detector (GPC-MALLS, 18 angle detector, Wyatt,
MO, USA) (Park et al. 2014). Eluent in mobile phase was
0.5 M ammonium acetate (pH 5.5) on Shodex Ohpak columns
(SB802HQ and SB803HQ).
Determination of the degree of deacetylation
To determine the free amino group content of chitosan, the
degree of deacetylation (DAc) was measured using the
method of Terayama (1952) with modifications. One gram
of chitosan was suspended in 100 mL water and then mixed
with 100 mL 0.4 M acetate buffer. One gram of dissolved
chitosan solution was added to 30 mL water containing 2 to
3 drops of indicator. The mixture was titrated with a colloidal
solution composed of N/400 PVSK [Poly(vinyl sulfate)
potassium salt] using a digital burette (Model HWA-162007;
VITLAB, Grossostheim, Germany). The DAc was
determined using the following equation:
DAc ð%Þ ¼ 1=f½ð50=4:03V Þ  10:793 þ 1g100
ðV ¼ Titration value of N =400 PVSK Þ
Results
Type I and II G. bimaculatus were compared by body weight.
The fresh weight (FW) of entire Type I and II G. bimaculatus
measured 0.383 g and 0.743 g per insect, respectively. The
process of extracting chitin and chitosan from entire
specimens (FW = 100 g, Type II) is shown in Figure 1. The
relative yield of chitin and chitosan from adult exoskeletons
was 2.35 % and 1.79 % FW, respectively.
Exoskeletons of adult G. bimaculatus were used to compare
the degree of demineralization (DM) and deproteinization
Entomological Research 47 (2017) 279–285
© 2017 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
 Chitin and chitosan from G. bimaculatus

Figure 1 Process for producing chitin and

chitosan from entire specimens of Type II
two-spotted field crickets (Gryllus
bimaculatus).

(DP) after acid and alkali treatments (Table 1). After washing
chopped pieces of adult specimens, the DWs of Tests A, B, C,
and D adult exoskeleton were 10.47, 10.84, 21.35, and
50.70 g, respectively. The average yield of the adult
exoskeleton on a DW basis was 21.82 %. After acid treatment,
the DWs of Tests A, B, C, and D adult exoskeleton were 9.05,
9.88, 19.27, and 40.07 g, respectively. The average yield of
the adult exoskeleton on a DW basis was 86.72 %. The DM
(%) of Tests A, B, C, and D adult exoskeleton was 13.56,
8.86, 9.74, and 20.97 %, respectively. The DP (%) of Tests
A, B, C, and D adult exoskeleton was 86.30, 87.75, 87.81,
and 87.82 %, respectively. The average DM (%) and DP (%)
of adult exoskeletons on a DW basis was 13.28 and
87.42 %, respectively.
Chitin obtained from the adult exoskeleton was then used
for the manufacture of chitosan (Table 2). For chitin
manufacture, the adult exoskeleton was boiled in 1.25 N
NaOH at 95°C for 3 h. After alkali treatment in Test A, the
chitin yielded from adult exoskeletons on a FW and DW basis
was 2.48 and 11.84 %, respectively. In Test B, the chitin
yielded from adult exoskeletons on a FW and DW basis was
2.42 and 11.16 %, respectively. In Test C1, the chitin yielded
from adult exoskeletons on a FW and DW basis was 2.35 and
11.01 %, respectively. In Test D0, the chitin yielded from

Table 1 Demineralization (DM), deproteinization (DP), and yield after treating Gryllus bimaculatus with HCl and NaOH

G. bimaculatus G. bimaculatus Yieldc Acid treatmentd Yielde DMf DPg

Items Whole FWa (g) DWb (g) (%) DW (g) (%) (%) (%)

Test Ah 50 10.47 20.91 9.05 86.44 13.56 86.30

Test Bi 50 10.84 21.68 9.88 91.14 8.86 87.75
Test C 100 21.35 21.35 19.27 90.26 9.74 87.81
Test D 200 50.70 23.35 40.07 79.03 20.97 87.82

a
FW: fresh weight of entire G. bimaculatus specimens.
b
DW: dry weight of the G. bimaculatus exoskeleton.
c
Yield (%) = (DW of G. bimaculatus / Whole FW of G. bimaculatus) × 100.
d
Acid treatment with 2 N HCl at room temperature for 3 h.
e
Yield (%) = (DW after acid treatment / DW of G. bimaculatus) × 100.
f
DM (%) = 1 – (DW after acid treatment / DW of G. bimaculatus) × 100.
g
DP (%) = 1 – (DW after alkali treatment / DW after acid treatment of G. bimaculatus) × 100.
h
Test A: Type I G. bimaculatus.
i
Test B, C, and D: Type II G. bimaculatus.

Entomological Research 47 (2017) 279–285 281

© 2017 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
M.-W. Kim et al.

Table 2 Processing chitin and chitosan after treating Gryllus bimaculatus with HCl and NaOH

Test A: Type I G. bimaculatus.

Test B, C, and D: Type II G. bimaculatus.
Chitosan of C2 is obtained from chitin C1, and Chitosans of D1 and D2 were obtained from chitin D0.

adult exoskeletons on a FW and DW basis was 2.44 and
9.63 %, respectively.
For deacetylation, the chitin extracted from the adult
exoskeleton was boiled at two different alkali concentrations
composed of both 50 % NaOH (w/v) and 50 % NaOH (w/w)
solutions at 95°C or 105°C for 3 h. In Test A, after 50 %
NaOH (w/v) treatment, the yield of chitosan on a FW and
DW basis was 1.76 and 8.40 %, respectively. In Test B after
50 % NaOH (w/v) treatment, the yield of chitosan on a FW
and DW basis was 1.92 and 8.86 %, respectively. In Test C1
after 50 % NaOH (w/v) treatment, the yield of chitosan on a
FW and DW basis was 1.87 and 8.81 %, respectively. In Test
C1 after 50 % NaOH (w/w) treatment, the yield of chitosan on
a FW and DW basis was 1.14 and 5.32 %, respectively. In Test
D1 after 50 % NaOH (w/v) treatment, the yield of chitosan on
a FW and DW basis was 1.89 and 7.46 %, respectively. In Test
D2 after 50 % NaOH (w/w) treatment, the yield of chitosan on
a FW and DW basis was 1.79 and 7.06 %, respectively.
During the extraction of chitin and chitosan from Type I and
II adult exoskeletons, the relative yields of exoskeletons

282 Entomological Research 47 (2017) 279–285

© 2017 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
 Chitin and chitosan from G. bimaculatus

washed with tap water (WA), DM, chitin, and chitosan were
21.82, 19.36, 2.42, and 1.65 % on a FW basis, respectively
(Fig. 2). The relative yield of chitin and chitosan was 10.91
and 7.50 % on a DW basis, respectively (inset of Fig. 2).
After alkali treatment, the deacetylation percentage and
viscosity of chitosan were measured to determine the DAc
and molecular weight (Fig. 3). The deacetylation percentage
of chitosan extracted from adult exoskeletons of Test A, B,
C1, C2, D1, and D2 was 81.2, 14.5, 19.6, 90.7, 17.1, and
95.5 %, respectively (Fig. 3A). The viscosity of chitosan
extracted from adult exoskeletons of Test A, C2, and D2
was 32.0, 21.6, and 62.4 cP, respectively (Fig. 3B). The
molecular weight (Mw) and polydispersity index (PDI) of
chitosan obtained Test D2 of Table 3 was 308.3 kDa and
1.172, respectively (Table 3).

Discussion
For the production of chitin, adult exoskeletons of Type I and
II G. bimaculatus were used in a manufacturing process
(Table 1). DP (%) of G. bimaculatus chitin was higher than
DM based on this procedure. Chitin obtained by this process
was used to produce chitosan. Research into insect-based
chitin and chitosan production has been conducted to
Figure 3 (A) Degree of deacetylation (DAc) and (B) viscosity (cP) of
determine their biological activities against microorganisms
chitosan extracted from the exoskeleton of Type I (Treatment A) and
(Hou et al. 2007; Jing et al. 2007). Type II (Treatment B, C1, C2, D1, and D2) adult two-spotted field crickets
For the production of chitosan, the deacetylation process of (Gryllus bimaculatus).
the chitin obtained from G. bimaculatus was carried out with a
strong alkali (Table 2). The two types (Type I and II) of adult
exoskeletons were boiled in two different alkali
Table 3 Molecular weight of chitosan separated from whole body
shells of adult two-spotted field crickets (Gryllus bimaculatus)

Sample† Mn (Da) MW (Da) PDI (Mw/Mn)‡

G. bimaculatus 262,900 308,300 1.172

†Chitosan D2 treated with 50 %(w/w) NaOH at 105°C, for 3 h from

Table 2.
‡Polydispersity index by GPC (0.5 M ammonium acetate, pH 5.5, 25°C).

concentrations: 50 % NaOH (w/v) and 50 % NaOH (w/w)

Figure 2 Relative yield (%) of washing with tap water (WA),
demineralization (DM), chitin, and chitosan from Type I and II two-
spotted field crickets (Gryllus bimaculatus) on a fresh weight basis.
Relative yield (%) of chitin and chitosan extracted from G. bimaculatus
adult exoskeletons on dry weight basis (inner graph).
solution at 95°C (in Type I) or 105°C (in Type II) for 3 h.
The N-deacetylation of chitin obtained from Type I specimens
was easier than that of Type II. The DW content and
deacetylation (%) of chitosan in Test A (Type I) was 0.88 g
and 81.2 %, respectively, after treatment with 50 % NaOH
(w/v) at 95°C for 3 h. In a previous study, the N-acetyl groups
on the cuticle of beetle larvae were removed by up to 94 %
after one treatment with 40 % NaOH for 4 h at 110°C (Zhang
et al. 2000). The DW content and deacetylation (%) of
chitosan in Test C2 (Type II) was 1.37 g and 90.7 %,
respectively, after treatment with 50 % NaOH (w/w) at

Entomological Research 47 (2017) 279–285 283

© 2017 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
M.-W. Kim et al.
105°C for 3 h. The DW content and deacetylation (%) of
chitosan in Test D2 (Type II) was 1.17 g and 95.5 %,
respectively, after treatment with 50 % NaOH (w/w) at
105°C for 3 h. The adult exoskeleton of Type I G. bimaculatus
formed chitosan when treated with 50 % NaOH (w/v);
however, chitosan was not formed in Type II exoskeletons at
the same alkali concentration. Based on these results, the adult
Type of G. bimaculatus and concentration of alkali plays an
important role in the conversion of chitin to chitosan.
The yield of chitin and chitosan obtained from G.
bimaculatus was 10.91 and 7.50 % on a DW basis,
respectively (Fig. 2). In a similar study, the chitin content of
G. bimaculatus powder was 9.77, 12.25, 11.27, and 6.36 %
following lyophilization, hot-air, roasting, and frying
processes, respectively (Kim et al. 2015). In the manufacture
of chitin using sodium hydroxide and oxalic acid, the yield
of chitin and chitosan obtained from G. bimaculatus powder
was 5.10 and 2.13 % on a DW basis, respectively (Chae
2015). The DW of the chitin content of insects has been
reported for various species. The dry weight of chitin was
reported to be 18 % in the wings of the cockroach Periplaneta
americana (Kaya & Baran 2015), 15–20 % in the pupal
exuvia of the silkworm Bombyx mori (Zhang et al. 2000),
15 % in the beetle Holotrichia parallela (Liu et al. 2012),
10.8 % in the green shield bug Palomena prasina (Kaya et al.
2015b), 8–8.5 and 6.5–7 % in the spiders Geolycosa vultuosa
and Hogna radiata (Kaya et al. 2014), and 5.3–8.9 % in a
grasshopper species (Kaya et al. 2015a). In addition, the chitin
content of cicada sloughs was 36.6 % (Sajomsang & Gonil
2010).
In this study, insect-based materials could be used as a food
source and feed source for humans and fish and livestock,
respectively (FAO 2013; Verbeke et al. 2015). However,
problems for their application include the high marketing cost
of the insect materials and suitable regulations for applying the
materials as feed (Moon & Lee 2015; Jo & Lee 2016). To
improve the value of insect-based materials, the research for
functional value of insect-based materials is necessary. In this
study, the production efficiency of chitin and chitosan
obtained from the exoskeleton of adult two-spotted field
crickets (G. bimaculatus) was investigated, and our results
demonstrate that these compounds are a potential source of
feed for stock farming.
Acknowledgments
This work was supported by a grant from the Korea Institute
of Planning & Evaluation for Technology in Food,
Agriculture, Forestry, and Fisheries (iPET) through the Agri-
Bioindustry Technology Development Program, funded by
the Ministry of Agriculture, Food, and Rural Affairs
(MAFRA) (no. 315034031SB050).
284
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