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Extraction and Characterization of Chitin from the Beetle Holotrichia parallela Motschulsky

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Insect chitin was isolated from adult Holotrichia parallela by treatment with 1 M HCl and 1 M NaOH, following by 1% potassium permanganate solution for decolorization. The yield of chitin from this species is 15%. This insect chitin was compared with the commercial a-chitin from shrimp, by infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and elemental analysis. Both chitins exhibited similar chemical structures and physicochemical properties. Adult H. parallela is thus a promising alternative source of chitin.
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Molecules 2012, 17, 4604-4611; doi:10.3390/molecules17044604
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Extraction and Characterization of Chitin from the Beetle
Holotrichia parallela Motschulsky
Shaofang Liu, Jie Sun, Lina Yu, Chushu Zhang, Jie Bi, Feng Zhu, Mingjing Qu, Chen Jiang
and Qingli Yang *
Shandong Peanut Research Institute, No. 126 Fushan Road, Qingdao 266100, China
* Author to whom correspondence should be addressed; E-Mail: rice407@163.com;
Tel.: +86-532-8761-5601; Fax: +86-532-8761-1087.
Received: 6 March 2012; in revised form: 31 March 2012 / Accepted: 11 April 2012 /
Published: 17 April 2012
Abstract: Insect chitin was isolated from adult Holotrichia parallela by treatment with 1 M
HCl and 1 M NaOH, following by 1% potassium permanganate solution for decolorization.
The yield of chitin from this species is 15%. This insect chitin was compared with the
commercial -chitin from shrimp, by infrared spectroscopy, X-ray diffraction, scanning
electron microscopy, and elemental analysis. Both chitins exhibited similar chemical
structures and physicochemical properties. Adult H. parallela is thus a promising
alternative source of chitin.
Keywords: insect; chitin; Holotrichia parallela; characterization; extraction
1. Introduction
Chitin, found in arthropods (insects, crustaceans, arachnids and myriapods), is the second most
abundant biopolymer after cellulose [1]. Structurally, it is a derivative of cellulose with only an
acetamido group replacement at position C-2, i.e., β-(1,4)-2-acetamido-2-deoxy-D-glucose. Chitin is
classified into three types according to the different orientations of its microfibrils: -chitin (anti-parallel
chains), β-chitin (parallel chains), and γ-chitin (the combination of parallel and anti-parallel chains) [1,2].
Chitin is insoluble in most solvents because of its compact structure. Therefore, chemical modifications
of chitin are performed to obtain more soluble analogs, among which, chitosan, derived by partial
N-deacetylation of chitin, is the most common such derivative.
OPEN ACCESS
Molecules 2012, 17 4605
Chitin and chitosan are attracting great interest because of their beneficial biological properties,
such as biodegradability, biocompatibility, non-antigenicity and non-toxicity [3,4]. Since they are
versatile biopolymers, their potential applications in various industrial fields are being actively
investigated. For example, chitin and chitosan have been documented to be useful as antimicrobial,
emulsifying, thickening and stabilizing agents in the food industry [5]. They have also shown notable
bioactivity in biomedical fields, including wound healing promotion, immune system enhancement,
and hemostatic, hypolipidemic and antimicrobial activity [6,7].
Traditionally, chitin is prepared mainly from crab and shrimp shells obtained as byproducts in the
seafood industry. Chitin is also a primary component in insect cuticles. Therefore, insects are an
alternative source of chitin and, consequently, of chitosan. Recently, the production of chitin and
chitosan from insect sources has drawn increased attention. First, insects possess enormous
biodiversity and represent 95% of the animal kingdom [8]. Therefore, they offer a tremendous
potential as a natural resource for chitin and chitosan production. Furthermore, insect cuticles have
lower levels of inorganic material compared to crustacean shells, which makes their demineralization
treatment more convenient [9]. Until now, however, only limited numbers of insect species have been
documented to be sources of chitin [9–13].
In this study, the isolation and characterization of chitin from adult Holotrichia parallela
Motschulsky is described. H. parallela is a beetle species belonging to the Melolonthidae subfamily of
the Scarabaeoidea family. These insects are field crop pests in China. In addition, they have also been
used as food and traditional medicinal herbs in China and East Asia [14–16]. Every year, large
amounts of H. parallela adults are captured for the control of this pest in fields. Our institute is
investigating the possibility of rearing H. parallela [17]. This will provide a source for studies on the
identification of active components from this beetle, including chitin and its derivative chitosan. Here,
chitin was purified from H. parallela adults using acid and alkaline treatments, followed by decolorization
with potassium permanganate. The physiochemical properties of chitin were characterized by infrared
spectroscopy (IR), X-ray diffraction (XRD), elemental analysis and scanning electron microscopy
(SEM) methods. These physicochemical properties were also compared with those of shrimp chitin.
2. Results and Discussion
2.1. Chitin Extraction
Chitin is a major component of the insect cuticle, which is always covalently bound to catechol
compounds and sclerotin-like proteins [10]. The most common method for chitin extraction from
insects involves two steps, an acidic step to remove catechols and a basic step to remove the cuticle
proteins, as had been mentioned elsewhere for insect chitin isolation [9,10]. Generally, the acidic
treatment conditions used for extraction from insects are moderate in comparison to crustacean
exoskeletons. The reason for this is that insects have low levels of inorganic material (less than 10%)
as compared to crustacean shells (20–40%) [18,19].
The ash content of chitin is indicative of the effectiveness of the method used for removal of
inorganic materials. From Table 1, the adult H. parallela contained 5.5% ash on a dried basis, while
the ash content of chitin extracted from this species dropped to 2.2%. This result suggested that the
Molecules 2012, 17 4606
demineralization with 1 M HCl for 30 min was an effective method for decreasing the ash content in
adult H. parallela. The nitrogen content of chitin from adult H. parallela decreased to 6.3%, which is
lower than the theoretical value of 6.9% for pure chitin. This also indicated an effective deproteinization
process in chitin extraction. In comparison, the commercial chitin used as standard contained 1.6% ash
and 6.2% nitrogen.
Table 1. Chemical composition of dried raw materials and chitin from adult H. parallela
and shrimp chitin.
Moisture (%) Ash (%) Nitrogen (%) a
Dried raw materials 3.63 ± 0.04 5.53 ± 0.23 11.29 ± 0.03
Beetle chitin 7.12 ± 0.16 2.20 ± 0.13 6.33 ± 0.06
Shrimp chitin 7.64 ± 0.06 1.59 ± 0.12 6.22 ± 0.06
a measured by the Kjeldahl method.
The yield of chitin from adult H. parallela is around 15%. The yields of chitin from other insects
varied with species and their development stages. Bombyx mori larva cuticle and silkworm pupa
exuviae were reported to yield 15–20% of chitin [9]. A higher chitin yield of 36% was reported for
cicada sloughs [12]. In comparison, the yields of -chitin from crustacean shells are about 7–40%,
depending on the species [18]. Adult H. parallela is thus a promising alternative source of chitin.
2.2. Characterization of Chitin
2.2.1. IR Analysis
The IR spectra of chitin from shrimp and H. parallela are shown in Figure 1. They were quite
similar, and comparable to those of -chitin from other sources in the literature [10,20]. The spectra
were characterized by three significant amide bands at 1,654, 1,560 and 1,310 cm1, which correspond
to the amide Ι stretching of C=O, the amide ΙΙ of N-H and amide ΙΙΙ of C-N, respectively. It is
important to note that the amide Ι band of -chitin splits at 1,654 and 1,627 cm1, which is attributed to
the two types of H-bonds formed by amide groups in the antiparallel alignment present in crystalline
regions of -chitin [21,22]. Table 2 showed the assignment of the relevant bands of chitins from
shrimp and H. parallela. From this result, it was demonstrated that both chitins are in form. The
degree of acetylation (DA) values of chitin from shrimp and H. parallela calculated using the formula
(1) were 94.3% and 93.1%, respectively.
Table 2. Assignments of the relevant bands of IR spectra of chitin from shrimp and adult H. parallela.
Assignments Wave Number (cm1)
Beetle Chitin Shrimp Chitin
ν (O-H) 3,424 3,440
ν (N-H) 3,262 3,268
ν (COCH3) 2,934 2,932
ν (C-H) 2,884 2,874
ν (C-O) 1,655 1,654
ν (C=O of N-acetyl group) 1,624 1,627
δ (N-H of N-acetyl group) 1,560 1,560
Molecules 2012, 17 4607
Figure 1. IR spectra of -chitin from shrimp (A) and from adult H. parallela (B).
2.2.2. Elemental Analysis
Elemental analysis of chitins from shrimp and H. parallela, including the carbon, nitrogen and
hydrogen contents and C/N ratio are shown in Table 3. From chitin, the nitrogen contents of the
sample come mainly from protein and chitin. The total nitrogen content of the beetle before chitin
extraction was ~11% (by Kjeldahl method). After extraction, the resultant nitrogen content dropped to
6.45%. This value was lower than the theoretical value (6.9%) calculated for a completely acetylated
chitin, indicating the minimum amount of protein left.
Table 3. The elemental analysis of chitin from shrimp and H. parallela and the
corresponding degree of acetylation (DA).
Samples Content (%) C/N
N C H
Shrimp chitin 6.24 43.75 6.40 7.07
Insect chitin 6.45 44.36 5.92 6.88
2.2.3. XRD Analysis
Figure 2 represents the XRD pattern for -chitin from shrimp and H. parallela. From the results,
both chitin samples showed similar XRD patterns, with strong reflections at 9.2 and 19.1° and minor
reflections at 12.6, 22.9 and 26.2° (Figure 2A,B). Chitin from other beetle species like cicada sloughs was
reported to show a similar result, with reflections at 2θ 9.20, 12.60, 19.18, 20.68, 23.30 and 26.48° [12].
The crystalline index (CrI) of chitin was calculated from the XRD data. The results showed that both
chitins have nearly the same crystallinity, 89.17 and 89.05% for chitin from shrimp and H. parallela,
respectively. Chitin from cicada sloughs had a similar crystallinity of 89.7% (CrI110). However, a
much lower crystallinity, only 54 and 58%, was found in chitins from larva cuticles and silkworm
pupa exuviae (Bombyx mori), respectively [9]. The authors inferred that the low molecular weight
compound catechol that remained in the insect chitin was probably the reason for the low crystallinity.
Molecules 2012, 17 4608
Figure 2. The XRD patterns of chitin from shrimp (A) and H. parallela (B).
2.2.4. Scanning Electron Microscopy (SEM)
Figure 3 shows the SEM photographs of commercial chitin from shrimp and chitin from H. parallela.
Both samples exhibited rough and thick surface morphology under electron microscopic examination
at 50× magnification.
Figure 3. SEM micrographs for -chitin from shrimp at 50× magnification (A) and at
3,000× magnification (B) and from adult H. parallela at 50× magnification (C) and at
3,000× magnification (D).
Molecules 2012, 17 4609
Similar results can be seen in the SEM photographs of the beetle chitin from cicada sloughs [12]. At
higher magnification (3,000×), chitin from shrimp (Figure 3B) was found to be distinctly arranged in a
microfibrillar crystalline structure, more noticeable than chitin from adult H. parallela (Figure 3D).
3. Experimental
3.1. Isolation of Chitin from Adult H. parallela
Adult H. parallela were captured in July in peanut fields in the suburbs of Qingdao, Shandong
Province, China, as part of the usual control of this pest. The beetles were starved for 48 h to eliminate
gut contents, washed with water and killed by freezing. They were allowed to thaw at room
temperature and then air-dried at 50 °C for 2 days. The dried beetles were milled to a powder to pass
through a 20-mesh screen and stored at 4 °C in airtight containers. The powder (5 g) was treated
with 1 M HCl solution (250 mL) at 100 °C for 30 min to remove minerals and catechols. The
demineralization step was followed by rinsing with distilled water until neutrality was reached.
Deproteinization was performed using alkaline treatment with 1 M NaOH (250 mL) solution at 80 °C for
24 h, and the product was washed with distilled water until the pH became neutral. For the purpose of
decolorization, the precipitate was treated further with 1% potassium permanganate solution (100 mL)
for 1 h. Finally, lightly brown chitin was washed with distilled water and dried at 50 °C in a dry
heat sterilizer.
3.2. Infrared Spectra (IR) Analysis
Chitin samples were characterized from 4,000 to 400 cm1 by infrared spectrophotometry
(WGH-30/30A, Gangdong Technology Ltd., Tianjin, China) in KBr pellets. Commercial chitin from
shrimp (Sigma) was used as standard. The DA of both chitin samples was determined by comparing
the absorbance of the measured peak to that of the reference peak. The DA was calculated from the
absorbance (A) ratios according to the following equation [10]:
DA = (A1655/A3450) × 100 (1)
3.3. Elemental Analysis
Elemental analysis was performed using a Vario EL III analyzer (Elementar, Hanau, Germany) at
the Institute of Chemistry, Qingdao University of Science and Technology according to the standard
operation procedures provided by the manufacturer.
3.4. X-ray Chitin Powder Diffraction
XRD analysis was used to detect the crystallinity of chitins prepared, and their patterns were
recorded using a D/Max-rA diffractometer (Rigaku, Tokyo, Japan) with Cu radiation at the Institute of
Chemistry, Qingdao University of Science and Technology. Data were collected at a scan rate of
1°/min with the scan angle from 5° to 40°. The crystalline index (CrI) was determined by the following
equation:
Molecules 2012, 17 4610
CrI110 = [(I110 Iam)/I110] × 100 (2)
where I110 is the maximum intensity at 2θ 20° and Iam is the intensity of amorphous diffraction at
2θ 16°.
3.5. Scanning Electron Microscopy (SEM)
The surface morphology of chitin was examined with JSM-6700F scanning electron microscope
(JEOL, Tokyo, Japan) at the Institute of Chemistry, Qingdao University of Science and Technology.
The dried samples were ground, fixed on an adhesive tape and then coated with a thin gold layer by a
sputter coater. The SEM was conducted at 5.0 kV.
3.6. Composition Analysis
Moisture and ash were assayed according to the Association of Official Analytical Chemists
methods (AOAC, 2006) (AOAC methods 934.01 and 942.05). Nitrogen content was measured by the
Kjeldahl method (AOAC method 984.13).
4. Conclusions
Chitin was isolated from adult H. parallela using standard methods. The low levels of ash and
nitrogen contents are indicative of the effectiveness of the chitin extraction method. The characteristics
of chitin from adult H. parallela were similar to those of commercial chitin from shrimp by IR, XRD,
SEM and elemental analysis. The chitin extracted from adult H. parallela is thus suitable for chitosan
production. Adult H. parallela is an alternative source of chitin. The large numbers of H. parallela
adults captured for the control of this pest in fields every year provide an abundant source for the
production of chitin. In addition, attempts to domesticate the beetle will help relieve the impact to
ecological systems in the future.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31100205;
31000728), the China Agriculture Research System (CARS-14), the National Key Technology R&D
Program (2012BAK17B13), the Promotive Research Fund for Young and Middle-aged Scientists of
Shandong Province (BS2010NY023), the Natural Science Fund of Shangdong Province (ZR2009DQ004;
ZR2011CQ036), Qingdao Municipal Science and Technology Plan Project (11-2-3-26-nsh;
11-2-4-9-(3)-jch).
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© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
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... The chitin content increased gradually during the transition from larvae to adult black soldier flies, as predicted, with the pupal stage having the highest chitin content, contributing to the hardening process of the insect cuticle. In comparison, chitin extracted from other sources showed that the percentage of chitin was about 23% to 32% in Apis mellifera (western honey bees) (Nemtsev et al. 2004), 15% in Holotrichia parallela (beetle) (Liu et al. 2012), and an average of 20% in silkworm chrysalides (Paulino et al. 2006). Kaya et al. (2016) found that the chitin contents in larvae, pupa, and adults of Vespa crabro (wasp) were 2.2%, 6.2%, and 10.3%, respectively. ...
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In this paper, from many new examples, our approach on the preparation of chitins and chitosans with controlled physico-chemical characteristics was presented. The chitosan samples were prepared from α-chitin from crustacean shells and β-chitin from squid pens. The chitin deacetylation was carried out according to two methods using, respectively, as alkaline agent, the aqueous sodium hydroxide solution and anhydrous potassium hydroxide. The role of the source and of the process on the N-deacetylation reactions is confirmed. The effect of the addition of sodium borohydride or thiophenol within the reaction medium was studied. One of the parameters conditioning the physico-chemical characteristics of chitosan being in relation with the nature and the quality of original chitin, the role of this parameter was examined and the isolation process was discussed to put in evidence its advantages compared to other processes quoted in the literature by relying on new results.