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Extraction of chitin and chitosan from housefly, Musca domestica , pupa

shells: Production of chitin from housefly

Article  in  Entomological Research · September 2016

DOI: 10.1111/1748-5967.12175

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Entomological Research 46 (2016) 324–328

S H O R T CO M M U N I C A T I O N

Extraction of chitin and chitosan from housefly, Musca

domestica, pupa shells
Min-Woo KIM1, Yeon Soo HAN2, Yong Hun JO2, Myung Hyo CHOI3, Seung Ho KANG3, Sun-Am KIM4
and Woo-Jin JUNG1
1
Department of Agriculture 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

Correspondence
Woo-Jin Jung, Department of Agriculture
Chemistry, Institute of Environmentally
Friendly Agriculture (IEFA), College of
Agricultural and Life Science, Chonnam
National University, Gwangju 61186, South
Korea.
Email: woojung@jnu.ac.kr
Received 21 January 2016;
accepted 8 April 2016.
doi: 10.1111/1748-5967.12175
Abstract
Chitins and chitosans are some of the most abundant natural polysaccharide
materials, and are used to increase innate immune response and disease resistance
in humans and animals. In this work, chitin and chitosan from housefly, Musca
domestica, pupa shells were obtained by treatment with HCl and NaOH. For chitin
extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve decalcification
and deproteinization, respectively. For chitosan extraction, 50% NaOH solution was
used to achieve deacetylation. The yields of chitin and chitosan from pupa shells of
M. domestica were 8.02% and 5.87%, respectively. The deacetylations of chitosan
(from chitin C1 and C2) were 89.76% and 92.39%, respectively, after the first alkali
treatment with 50% NaOH (w/w) solution at 105 °C for 3 h and 5 h, respectively. The
viscosities of the chitosans (from chitin C1 and C2) were 33.6 and 19.2 cP,
respectively.

Key words: chitin, chitosan, housefly, Musca domestica, pupa shells.

Introduction Housefly, M. domestica, is widely distributed as a

Chitin is the second most abundant naturally occurring
polysaccharide after cellulose. It is obtained from the cell
walls of some fungi, exoskeletons of crustaceans, internal
structure of invertebrates and exuvium of insects. Chitin and
chitosan produced from Musca domestica larval shells have
been used as sorbents for heavy metal ions (Gyliene et al.
2002). It has been reported that chitosan exhibits biological
activities against microorganisms. Chitosan is produced from
housefly M. domestica larval cuticles (Jing et al. 2007) and
shrimp shells (Teli & Sheikh 2012; Younes et al. 2014). It
was reported that Cirrhinus mrigala, a freshwater fish, fed
with 1% chitin or chitosan obtained from the exoskeleton of
the giant freshwater prawn, Macrobrachium rosenbergii,
shows increased immunity and disease resistance against
Aphanomyces invadans (Mari et al. 2014).
livestock pest in Korea and around the world. The lifecycle
of a housefly is egg (1 day), larva (5 – 7 days), pupa (5 days)
and adult as a heteromorphism. A housefly lays around 600
eggs on livestock feces. In order to control M. domestica,
its natural enemy Muscidlifurax raptor has been used
against it. To this end, Korea Beneficial Insects Lab. Co.,
Ltd has undertaken mass rearing of M. raptor.
Muscidlifurax raptor inserts its eggs into the pupae of the
housefly. Musca domestica pupa are used as a food source
for M. raptor larvae hatched from the eggs. Musca
domestica pupa shells can be obtained in large quantities
from Korea Beneficial Insects Lab. Co., Ltd. Therefore
the M. domestica pupa shells are a byproduct of breeding
M. raptor larvae. These chitin-containing M. domestica pupa
shells could be a beneficial resource for use in the insect-
based animal feed industry.

© 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd

However, extraction of chitin and chitosan from M.
domestica pupa shells has not been extensively studied. Thus,
the objective of this study was to obtain the chitin and chitosan
from M. domestica pupa shells for the development of a
functionally improved insect-based feed for livestock. In this
study, chitin and chitosan were isolated from M. domestica
pupa shells using a chemical manufacturing process.
Materials and methods
Preparation of chitin and chitosan
The pupa shells of M. domestica after use as feed for M. raptor
larvae were supplied from Korea Beneficial Insects Lab. Co.,
Ltd. (Goksung, Junnam, Korea). Chitin was prepared from the
pupa shells of M. domestica by the modified method of
Hackman (1954). The pupa shells were prepared with
(G form) and without (NG form) grinding. The NG form
(50 g) and G form (10, 50 and 100 g) of pupa shells were then
decalcified for 3 h in 500 mL of 2 N HCl solution at room
temperature. After washing, the samples were incubated in
500 mL of 1.25 N NaOH at 95 °C for 3 h to remove the
proteins, then washed with water to a neutral pH and then
dried for 24 h at 70 °C in an oven.
To obtain chitosan, chitin particles separated from the M.
domestica pupa shells were boiled in 500 mL of 50% NaOH
(w/v) and 50% NaOH (w/w) solution at 95 °C and 105 °C
for 3 h and 5 h (Song et al. 2014). The chitosan produced from
chitin was washed to a neutral pH with tap water and then
dried for 24 h at 70 °C in an oven.
Determination of viscosity
Molecular weight of chitosan samples (0.5 g) was monitored
by viscosity measurements in 1% acetic acid solution. The
measurements were made at room temperature using a
viscometer (Brookfield Model DV-II+ Pro, Brookfield, MA,
USA) with spindle #63 (100 rpm). The unit of viscosity is cP
(centipoise = mPa · s).
Determination of degree of deacetylation
Determination of deacetylation (DAc) of chitosan used the
colloidal titration method of Terayama (1952) for determining
the free amino group content in chitosan. Chitosan (1 g) was
added to 100 mL water, and was then mixed with 100 mL
acetate buffer (82 mL of 0.4 M acetic acid + 18 mL of 0.4 M
sodium acetate). Dissolved chitosan solution 1 g was added
to 30 mL water with 2 – 3 drops of indicator (3.82 g toluidine
blue per 100 mL DW + 0.57 g methylene blue per 100 mL
ethanol). The mixture was titrated with colloidal solution of
Entomological Research 46 (2016) 324–328
© 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
Production of chitin from housefly
N/400 polyvinyl sulfate potassium salt (PVSK). DAc was
calculated using equation (1):
DAc ð%Þ ¼ 1=f½ð50=4:03  V Þ  1  0:793 þ 1g  100
ðV ¼ Titration value of N =400 PVSK Þ
(1)
Results
The process of extracting chitin and chitosan from the pupa
shells (G form, fresh weight 100 g) of M. domestica is shown
in Fig. 1. The yields of chitin and chitosan from the pupa shells
were 7.71% and 6.77%, respectively.
Both the NG and G forms of the pupa shells were used for
the manufacture of chitin and chitosan (Table 1). The moisture
content of the raw pupa shells was 4.5%. The pupa shells were
treated with 2 N HCl solution at room temperature for 3 h.
Afterwards, the dry weights of the pupa shells were 47.20 g
for the original form (from 50 g) and 3.74, 30.82 and 60.42 g
for the G forms (from 10, 50 and 100 g, respectively).
Demineralization of the NG form (50 g) was 5.6%, while
those of the G forms (10, 50 and 100 g) were 62.60%,
38.36% and 39.58%, respectively. From this result, only the
G form of the pupa shells obtained from acid treatment in
the next step of chitin manufacture was used for
deproteinization under alkali treatment. After the first alkali
treatment, the dry weight contents of the pupa shells were
0.85, 3.93 and 7.71 g from 10, 50 and 100 g, respectively.
Deproteinization (%) of the three G forms of the pupa shells
was measured as 77.28, 87.25 and 87.24%, respectively, while
chitin yields were 8.50, 7.86 and 7.71%, respectively.
Chitin obtained from G forms of the pupa shells were used
for the manufacture of chitosan (Table 2). For deacetylation,
the chitin obtained from the pupa shells was boiled in 50%
NaOH (w/v) and 50% NaOH (w/w) solution at 95 °C and
105 °C for 3 h and 5 h, respectively. For chitin A, after the
alkali treatment the deacetylations of chitosan were 7.04%,
11.60% and 33.98%, from the first, second and third
treatments, respectively. For chitin B1, after the alkali
treatment the deacetylations of chitosan were 7.60% and
33.43% from the first and second treatments, respectively.
For chitin B2, after the alkali treatment the deacetylations of
chitosan were 7.60% and 96.65%, from the first and second
treatments, respectively. For chitin C1 and C2, the
deacetylation of chitosan was 89.76% and 92.39%,
respectively, after the first alkali treatment. From these results,
the viscosities of chitosan in chitin B2, C1 and C2 were 36.0,
33.6 and 19.2 cP, respectively.
From the process of chitin and chitosan extraction from
pupa shells, using 10, 50 and 100 g of the G form, the relative
yields of chitin and chitosan were 8.02% and 5.87%,
respectively (Fig. 2A). For the production of chitin from the
ground pupa shells (both 50 g and 100 g samples), the average
weight removed from the pupa shells after demineralization
325
M–W. Kim et al.

Figure 1 Process for producing chitin and

chitosan from housefly, Musca domestica,
pupa shells.

Table 1 Comparison of chitin yields after treatment of both the NG and G forms of M. domestica pupa shells with acid and alkali treatments

Items Pupa shells† fresh weight (g) Treatment A, dry weight (g) Yield (%) Treatment B, dry weight (g) Chitin yield (%)

Type A 50 47.20 94.40 – –

Type B 10 37.74 37.40 0.85 8.50
Type B 50 30.82 61.64 3.93 7.86
Type B 100 60.42 60.42 7.71 7.71

†Moisture content of pupa shells = 4.5%.

Type A, NG form of pupa shells; Type B, G form of pupa shells; Treatment A, acid treatment with 2 N HCl for 3 h for RT; Yield, dry weight of pupa shells
after acid treatment; Treatment B, first alkali treatment with 5% NaOH (w/v) for 3 h for 95 °C; –, no data.

was 38.97 g/100 g dry weight (Fig. 2B), and after
deproteinization it was 52.95 g/100 g dry weight. The average
chitin content was 7.79 g/100 g dry weight.
The NG and G forms of the pupa shells are shown in Fig. 3A
and 3B, respectively, while the final products extracted from
pupa shells, chitin and chitosan, are shown in Fig. 3C and
3D, respectively.
Discussion
To produce chitin, two different forms (NG and G forms) of
M. domestica pupa shells were used in the procedure
(Table 1). The demineralization of the unground pupa shells
(NG forms) was very inefficient. Thus, the ground form was
used for the production of chitin and chitosan.
Research into commercial chitin production in crustaceans,
such as crab and shrimp, has mainly been conducted for use in
the food industry. The chitin contents found were about 13%
in brown crab, Cancer pagurus (Harkin et al. 2015), about
14% in Atlantic blue crab, Callinectes sapidus (Muralidhara
& Maggin 1985), about 21% in blue crab, Portunus
trituberculatus (Lee et al. 1984), about 27% in red crab,
Chionoecetes opilio (No & Lee 1995), about 27% in shore
crab, Carcinus mediterraneus (Hajji et al. 2015) and about
35% in prawn, Litopenaeus vannamei (Mohammed et al.
2013). Compared with those results, the chitin content
obtained from pupa shells, averaging 8.02%, was much lower.
For deacetylation, the chitin obtained from M. domestica
pupa shells was used with different alkali concentrations
and temperatures (Table 2). Deacetylation in chitin A was
33.98% after three treatments with 50% NaOH (w/v) at
95 °C for 3 h. Deacetylation in chitin B1 was 33.43% after
two treatments with 50% NaOH (w/v) at 95 °C for 5 h and
50% NaOH (w/v) at 105 °C for 3 h. Deacetylation
percentage in chitin B2, chitin C1, and chitin C2 was

326 Entomological Research 46 (2016) 324–328

© 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
 Production of chitin from housefly

Table 2 Comparison of deacetylation and viscosity† after treatment of chitin obtained from M. domestica pupa shells with different alkali
concentration and temperature treatments.

Items 1st Treatment DAc Viscosity 2nd Treatment DAc Viscosity 3rd Treatment DAc Viscosity
condition (%) (cP) condition (%) (cP) condition (%) (cP)

Chitin A 95 °C/3 h, 50% NaOH 7.04 – 95 °C/3 h, 50% NaOH 11.60 – 95 °C/3 h, 50% NaOH 33.98 –
(w/v) (w/v) (w/v)
2
Chitin B1 95 °C/5 h, 50% NaOH 7.60 – 105 °C/3 h, 50% NaOH 33.43 – n.a. n.a. n.a.
(w/v) (w/v)
Chitin B2 95 °C/5 h, 50% NaOH 7.60 – 105 °C/3 h, 50% NaOH 96.65 36.0 n.a. n.a. n.a.
(w/v) (w/w)
Chitin C1 105 °C/3 h, 50% NaOH 89.76 33.6 n.a. n.a. n.a. n.a. n.a. n.a.
(w/w)
Chitin C2 105 °C/5 h, 50% NaOH 92.39 19.2 n.a. n.a. n.a. n.a. n.a. n.a.
(w/w)

Chitin A, chitin obtained from NG form pupa shells (50 g); Chitin B1, B2, C1 and C2, Chitin obtained from G form pupa shells (100 g); DAc,
deacetylation; –, no data; n.a., not applicable.
†Viscosity was measured at room temperature using a Brookfield viscometer (Model DV-II+ Pro) with spindle number #63 (100 r.p.m.); viscosity unit is
cP (centipoise = mPa · s).

Figure 3 Photographs of M. domestica pupa shells in their (A) NG form

and (B) G form; (C) chitin and (D) chitosan extracted from the pupa
shells.

Figure 2 (A) Relative yield of chitin and chitosan extracted from ground
pupa shells (from 10, 50 and 100 g) of M. domestica. (B)
Demineralization and deproteinization, and chitin content per 100 g of
dry pupa shells.
greater than 70% deacetylation with permission range as
chitosan. From these results, the concentration of alkali
plays an important role in converting chitin to chitosan.
The yields for chitin and chitosan from the pupa shells were
8.02% and 5.87%, respectively (using 10, 50 and 100 g of the
ground form; Fig. 2A). Larval and pupal exuviae of the
hornworm, Manduca sexta, contained 11% and 34% chitin,
respectively (Kramer et al. 1995), larval and pupal exuviae
of beetle (Tenebrio molitor) contained 11% and 25% chitin,
respectively (Kramer et al. 1995, and honeybees were shown
to contain 11.4% chitin after treatment with 4% NaOH at
99 °C for 2 h (Nemtsev et al. 2004). In this study, the chitin
content of M. domestica pupa shells was found to be 8.04%

Entomological Research 46 (2016) 324–328 327

© 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd
 M–W. Kim et al.
after treatment 1.25 N NaOH at 95–100 °C for 3 h. The yield
of chitin from M. domestica larva shells was previously
identified as 40–45% of the original weight of the raw material
(Glyiene et al. 2002). Chitosan produced from M. domestica
larva shells has been investigated as a sorbent for heavy metal
ions such as Cu and Ni (Glyiene et al. 2002).
Current research suggests that insect-based materials could
be used as food for human and feed for fish and livestock
(FAO, 2013; Moon and Lee 2015; Verbeke et al. 2015).
However, problems preventing their use include the high cost
of the insect materials and regulations for applying the
materials as feed. To improve the value of insect-based
materials, development of highly functional insect-based
materials is necessary. Here, we investigated the production
efficiency of chitin and chitosan from housefly pupa shells,
with our results demonstrating 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 (Agri-
Bioindustry Technology Development Program), funded by
Ministry of Agriculture, Food and Rural Affairs (MAFRA)
(no. 315034031SB050).
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© 2016 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd