Characterization of chitosan and chitin produced

from silkworm crysalides

Alexandre T. Paulino, Julliana I. Simionato, Juliana C. Garcia, Jorge Nozaki *

Chemistry Department, Maringa State University. Av. Colombo, 5790 CEP, 87020-900 Maringa PR, Brazil

Received 29 October 2004; received in revised form 13 September 2005; accepted 28 October 2005

Available online 5 December 2005

Abstract

Chitin, extracted from silkworm chrysalides, was employed for the production of a high purity and porous chitosan, as observed by scanning

electron microscopy (SEM). Chitin and chitosan produced were characterized by infrared (FTIR), nuclear magnetic resonance (13C-NMR)

spectroscopy, thermal analysis (TGA), differential scanning calorimetry (DSC), and SEM. Two methods of chitin and chitosan extractions were

investigated and compared, and although these were of high purity, the yield of chitin and chitosan were low if compared with the chitin and

chitosan produced from crustacean shells. The yield of chitosan production by chitin deacetylation or degree of deacetylation (DD) was an average

83%. The molecular weight (MW) was determined by viscosimetric methods.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Silkworm; Chitin; Chitosan; Chrysalides; Spectroscopy

1. Introduction

Chitin is obtained in industrial scale from shrimps and

crustaceans in general (Yanga et al., 2000). However, the

chrysalides of the silkworm are an alternative source of chitin

and, consequently, of chitosan (Zhang et al., 2000). These

chrysalides are the adult form of the larvae responsible for the

production of the silk thread, and the chrysalides itself

constitute a by-product from the silk industry, which is of

low price and easily available. China and Brazil are the

principal exporting countries of cocoons and raw silk (Table 1),

and the Chinese silkworm (Bombyx mori) has been used in

commercial silk production for centuries (Dingle, 2000).

The complete metamorphosis of butterflies, moths, and

some other insects involves four stages: egg, larva (caterpillar),

pupa (chrysalides or cocoon), and adult. In commercial use, it

refers almost entirely to filaments from cocoons produced by

the caterpillar of several moth species of the genus Bombyx,

commonly called silkworms. Silk is a continuous protein

filament around each cocoon, and in the silk production

industry, it is freed by softening the cocoon in water. Table 1

shows the world silk production by country, and China is the

0144-8617/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.carbpol.2005.10.032

* Corresponding author. Tel.: C55 442235392; fax: C55 442635784.

E-mail address: jnozaki@cybertelecom.com.br (J. Nozaki).

main country of silk producers. The Northwest of Parana state

is the main silkworm producers in Brazil, and Parana State is

responsible for 89% of the total Brazilian production.

Chitosan (ß-(1,4)-N-acetyl-D-glucosamine) is a derivative of

chitin after deacetylation, as shown in Fig. 1. Chitosan is a

biodegradable cationic biopolymer and could assist in the

reduction of pollutants in residual waters by adsorption and

chelating with heavy metallic ions, and can also act in the

coagulation of colloidal particles.

In this work, chitin was isolated from chrysalides of

silkworm (B. mori), using a modified process employed for

the extraction of chitin from crustaceans (Acosta et al., 1993).

The products were characterized by infrared spectroscopy

(FTIR), nuclear magnetic resonance spectroscopy

(MAS/13CMNR), thermal analysis (TGA), differential scan-

ning calorimetry (DSC), and scanning electron microscopy

(SEM).

2. Materials and methods

2.1. Reagents

All of the reagents used were of a highly pure grade,

without further purification, and the deionized water was

used for all reagent solution. The standard samples of

chitosan, 86% degree of deacetylation (DD), were purchased

from Aldrich-USA.

Carbohydrate Polymers 64 (2006) 98–103

www.elsevier.com/locate/carbpol

Table 1

World silk production by country (tones), and the main exporting countries

(China, India, and Brazil)

Year China India Japan Brazil Others Total

1978 19,000 3475 15,960 1250 5440 45,125

1985 32,000 7029 9582 1458 6738 56,807

1993 71,845 14,000 4254 2326 7750 100,175

1996 59,000 13,000 2250 2360 5100 81,710

Table 2

Chitin extraction from silkworm chrysalides (dry weight basis)

Sample Time (h) T (8C) Yield (%) GSD

(Basic

reaction)

Chit-A1 24 80 2.59 0.22

Chit-A2 18 80 2.78 0.14

Chit-A3 12 80 2,89 0,27

Chit-A4 6 100 3.16 0.26

Chit-A5 3 80 4,18 0.18

Chit-A6 1.5 80 4.16 0.38

Chit-B1 3 65 3.23 0.16

Chit-B3 2 65 3.99 0.12

Chit-B5 1 65 4.23 0.56

Chit-A1, Chit-A2, Chit-A3, Chit-A4, Chit-A5, and Chit-A6Zclosed reactor

and heating using an oven. Time of acid reactionZ20 min, and TZ100 8C.

Chit-B1, Chit-B2, and Chit-B3Zopen reactor using heating plate with stirring.

Time of acid reactionZ120 min, and TZ25 8C.

Table 3

Chitosan preparation: Chitos0, Chitos1, Chitos2, Chitos3, Chitos4, and

Chitos5Zclosed reactor and oven, reaction temperatureZ100 8C

Samples Time (min) Average yielda(%)

Chitos0 360 96.75

Chitos1 300 73.00

Chitos2 240 82.74

Chitos3 180 79.15

Chitos4 120 78.33

Chitos5 60 88.40

a Zor degree of deacetylation (DD).

A.T. Paulino et al. / Carbohydrate Polymers 64 (2006) 98–103 99

2.2. Chitin extraction

The chrysalides of the silkworm were kindly given by the

silk production department of COCAMAR (Cooperative of

Agriculturist and Coffee producer of Maringa-PR, Brazil). The

chrysalides were dried by lyophilization (Martin Christ, Freeze

Dryer, Alpha 1–2/LD) for 12 h. Two methods of extraction

were used to compare the yield and the purity of final products.

The extraction of chitin from chrysalides were performed using

a closed reactor made of Teflon, with 35 mm of inside

diameter, and 50!140 mm of external diameter and height,

respectively. This reactor was put inside the stainless steel

reactor, before heating in the oven. For the second method the

extraction was performed with an open system (beaker), using

a heating plate and stirring the solution.

Firstly, the dried chrysalides were treated with HCl

1.0 mol LK1 for 20 min at 100 8C in a closed reactor for the

elimination of catechols and also Ca, Mg and K. The weighed

samples were reacted with a proportion of 10 mL (HCl)/g of

dried chrysalides. Using the vacuum pump, it was filtrated and

the residue washed repeatedly with deionized water in order to

neutralize the excess of acid. In the sequence, the treatment

with NaOH 1.0 mol LK1, at the same proportion used for the

acid was performed during 24 h at 80 8C for proteins

elimination. The hot solution was filtrated in Buchner funnel,

washing several times with deionized water to remove the

excess of NaOH. The crystals of chitin were washed with

Na2CO3 0.4% several times, dried in an oven at 80 8C and,

afterwards, prepared for spectroscopic characterization. The

conditions such acid and basic concentrations, time, and

temperature of reactions of the two methods are presented in

Table 2.

2.3. Chitosan production

The deacetylation reaction of chitin was made using NaOH

(40 wt%) solution, with NaBH4 (0.83 g LK1) as reducing and

NH

C O

CH3

O

OH O

CH2OH

12

3

45

n

NaOH 40%

NaBH4 0.83gL–1

54

3

2 1

CH2OH

OOH

O

NH2n

Fig. 1. General mechanism of chitosan production from chitin.

protecting reagents, using a closed reactor and oven for

heating. The conditions of reaction times, temperature, and the

average yields are shown in Table 3.

2.4. Chitin and chitosan characterization

The samples of chitin and chitosan produced were

characterized, in KBr pellets, by infrared spectrophotometry

(FTIR, FT–GOmax Bomem Easy MB-100, Nichelson). The

nuclear magnetic resonance spectroscopy was investigated

with diluted samples in CD3COOD solution, and solid samples

(RMN Varian, Mercury plus 300 MHz BB spectrometer), the

thermogravimetric analysis (TGA-Shimadzu), differential

scanning calorimetry (DSC-Shimadzu), and the scanning

electronic microscopy with Shimadzu SS-550 Superscan.

3. Results and discussion

3.1. Chitin extraction and chitosan preparation

The silkworm presents an average of 20% of chitin in its

structure, besides proteins, minerals, and fat (Zhang et al.,

2000). In this way, the acidic stage of the treatment removed

both the catechols and the minerals present in the structure of

the chrysalides. The goal of the basic step was the removal of

the cuticle protein, and the reduction of the fat content. Due to

the fat contents, the hot filtration of the acidic stage was made

to decrease the saponification. When the samples were filtrated

4000 3000 2000 1000

10

20

30

40

50

60

70

80

90

Chitosan

Chitin

% T

rans

mita

nce

Wave number (cm–1)

Fig. 3. (A) FTIR of chitin and chitosan produced from silkworm chrysalides;

(B) range of 1400–1700 cmK1.

A.T. Paulino et al. / Carbohydrate Polymers 64 (2006) 98–103100

at room temperature, the samples acquired an aspect similar to

soap, making the filtration practically impossible. Related to

the reaction yield, the calculations were based on crude and

dried mass of the chrysalides, and the reaction yield of chitin

increased with the decrease of the basic reaction time. On the

other hand, no significative differences were observed

regarding the yields of chitosan production with basic reaction

for different times: 24, 18, 12, 15, 6, and 3 h, by the Tukey test

(pZ0.05), as shown in Table 3.

A higher yield of chitin production was obtained with the

extraction using an open reactor and 1 h of the acidic reaction

(Table 2). Although the yield was higher, the FTIR spectrum

showed the presence of impurity, while the product obtained

from the extraction in closed reactor with 24 h of the basic

reaction, practically free from impurities, although the chitin

yield was lower. The best conditions of chitosan preparation

were obtained using a solution of NaOH (40 wt%) and NaBH4

as protective agent against oxidation and the degradation of the

polymeric chain, reducing the polysaccharide aldehyde to an

alditol group (Muzzarelli & Petrarulo, 1994; Tolaimate et al.,

2003).

Regarding the yield for chitosan production in different

deacetylation times, homogeneity in the results was not

observed, and this fact can be attributed to the losses during

the filtration steps, with significative differences (pZ0.05)

among the experiments with different times of reaction

(Table 3).

3.2. Chitin FTIR

Studies indicate that chitin, in the crystalline state, shows

only one intense peak at 1626 cmK1. However, the spectra of

the samples indicated the presence of two bands, one at

1626 cmK1 and another at 1656 cmK1, probably, indicating an

amorphous state. These bands are attributed to the vibrations of

the amide I band, and the band at 1656 cmK1 corresponds to

4000 3500 3000 2500 2000 1500 1000

20

30

40

50

60

70

80

90

1.5h

12h

18h 6h

3h24h

% tr

ansm

itanc

e

Wave number (cm–1)

Chit-A1Chit-A5Chit-A4Chit-A2Chit-A3Chit-A6

Fig. 2. FTIR of chitin extracted using closed reactor and oven, with different

times of basic reaction: (Chit-A1:24h, Chit-A2:24h, Chit-A3:12h, Chit-A4:6h,

Chit-A5:3h, Chit-A6:1.5h).

the amide I stretching of CaO. The band at 1626 cmK1 could

be attributed to the stretching of C–N vibration of the

superimposed CaO group, linked to OH group by H bonding.

These bands can be clearly observed in all samples.

The bands observed at 3474 and 3434 cmK1 correspond to

the vibrational stretching of the hydroxyl groups. When these

two peaks appeared with certain intensity, we observed two

bands at 1626 and 1656 cmK1. The wide peak at 3500 and

1650 cmK1 indicated that the hydrogen interactions are

less accentuated, or the presence of free hydroxyl groups

(Duarte et al., 2002).

The band at 1345 cmK1 corresponds to a CO–NH

deformation and to the CH2 group (amide III), due to the

formation of CO–NH group. The sharp band at 1377 cmK1

corresponds to a symmetrical deformation of the CH3 group,

and at 1557 cmK1 corresponds to the stretching or N–H

deformation of amine II (Duarte et al., 2001; Ravindra et al.,

1998). The results of FTIR spectra of chitin are shown in Figs.

2 and 3.

The best spectra were obtained with the samples submitted

to the extraction in closed reactor into the oven, and times for

the basic reaction above 18 h (Fig. 2). The samples obtained by

the extraction in open reactor did not demonstrate significant

changes with the time variation of the basic reaction (Fig. 3).

4000 3500 3000 2500 2000 1500 1000

10

20

30

40

50

60

70

80

5h

3h6h4h

1h

2h

% T

rans

mita

nce

Wave number (cm–1)

Fig. 4. (a) Chitosan FTIR of samples with different times of deacetylation. (1, 2,

3, 4, 5, and 6 h).

250 200 150 100 50 0ppm

CPMAS/13CNMR-chitin

Fig. 5. 13C-NMR of chitin extracted from chrysalides silkworm.

0 200 400 600 800 1000

40

60

80

100

DTG

TG

Temperature (ºC)

resi

dual

mas

s (%

)

–0.8

–0.6

–0.4

–0.2

0.0

DT

G (%

/ºC)

Fig. 7. TGA of chitin extracted from silkworm chrysalides.

A.T. Paulino et al. / Carbohydrate Polymers 64 (2006) 98–103 101

Chitosan FTIR: The spectra of Fig. 3 (A) and (B) correspond

to a chitin, and the deacetylated sample with NaOH (40 wt%)

in the presence of NaBH4 for 5 h. Note that for chitosan, the

band at 1590 cmK1 has a larger intensity than at 1655 cmK1,

which suggests effective deacetylation. When chitin deacetyla-

tion occurs, the band observed at 1655 cmK1 decreases, while a

growth at 1590 cmK1 occurs, indicating the prevalence of NH2

groups (Bordi et al., 1991).

When the same spectrum is observed (Fig. 3(B)), in which

the band from 1500 to 1700 cmK1 is stressed, indicated that

there was an intensification of the peak at 1590 and a decrease

at 1655 cmK1, that suggests the occurrence of deacetylation.

Fig. 4 shows the spectrum of chitosan obtained with different

times of deacetylation, and was observed that even after 4 h of

reaction, the deacetylation was very small. After 5 h of

reaction, an intensification of the peak at 1590 cmK1 occurs,

indicating the efficiency of deacetylation.

NMR13C/MAS of chitin and chitosan: The structural

analyses of NMR13C in the solid state is very useful for

compounds such as chitin, because does not destroy its

conformation. The spectrum of Fig. 5, correspond to the

structure of chitin and have defined C1 peaks (d104.5), C2

(d55.6), C3 (d73.8), C4 (d83.5), C5 (d76.1), C6 (d61.4), C7

(d23.2), 13C signals of catechols compounds at (d173.5), and the

(CaO) at d 173.8 (Zhang et al., 2000; Lavertu, et al., 2003). The

peaks of C3 and C5 appear as a doublet centered at 75 ppm.

Note in the spectrum that the removal of proteins and the

catechols were efficient during the extraction, once the peaks at

d174, 146, and 117 ppm are practically imperceptible,

200 150 100 50 0ppm

CPMAS/13CNMR-chitosan

Fig. 6. 13C-NMR of chitosan produced by deacetylation of chitin.

suggesting a great purity of the product. Fig. 6 shows the

chitosan spectrum, in which the deacetylation of chitin is

evident, since there are no peaks at d23 and 174 ppm, that

correspond to the CH3 and CaO groups, respectively. The

other peaks correspond to C1 (d105.3), C2 (d57.9), C3 (d75.8),

C4 (d82.3), C5 (d75.8) and C6 (d61.1). C3 and C5 peaks appear

as an only signal at d75.6 ppm. The presence of a peak at

d33.5 ppm (not expected), could be due to the presence of a

possible by–product or impurity in the sample (Fig. 6).

Thermogravimetric analysis: In the thermogram of chitin

(Fig. 7) two decomposition steps could be observed, the first

occurs in the range of 50–110 8C, and is attributed to water

evaporation. The second occurs in the range of 300–400 8C and

could be attributed to the degradation of the saccharide

structure of the molecule, including the dehydration of

saccharide rings and the polymerization and decomposition

of the acetylated and deacetylated units of chitin. The

percentage of residual mass after heating at 1000 8C was

36%, and could suggest the presence of minerals that were not

extracted in the acidic stage.

In the chitosan thermogram (Fig. 8), three decomposition

steps were observed, and the first and the third peaks were

similar to found in chitin, occurring in the range of 50–110 8C

and 300–400 8C, respectively. The second decomposition step

0 200 400 600 800 1000

0

20

40

60

80

100

DTG

TG

DT

G (%

/ºC)

Temperature (ºC)

resi

dual

mas

s (%

)

–0.8

–0.6

–0.4

–0.2

0.0

Fig. 8. TGA of chitosan produced from silkworm chitin.

Fig. 9. Scanning electron microscopy (SEM) of pure (a) chitin and (b) chitosan.

A.T. Paulino et al. / Carbohydrate Polymers 64 (2006) 98–103102

occurred at a lower temperature than observed for the chitin

decomposition. It suggests that chitosan has poor thermal

stability. The peak that appears around 300 8C could be due to

the degradation of part of the molecule that was deacetylated.

Determination of the molar mass by viscosimetry: chitosan

sample was dissolved in solutions of acetic acid 0.02 M and

NaCl 0.1 M with the ionic strength of 0.12 M. The viscosity

was measured in a viscosimeter (Ubbelohd Cannon, State

College Pa 16804 0016 USES 150.D786) coupled with a

thermostatic bath. Firstly, the time of pure solvent flow was

measured and soon after, the time of chitosan solution flow was

measured. Several dilutions were performed until the flow time

of chitosan approaches the flow time of the solvent (Kaplan,

1998).

½hred�ZTKT0T0½Q�

(1)

Eq. (1) was used for the reduced viscosity determination, in

which [hred] is the reduced viscosity, T is the chitosan solution

time of flow, T0 is the solvent time of flow and [Q] is the

concentration of chitosan solution in time T0. Plotting

the calculated relative viscosity from (1) versus concentration,

the intrinsic viscosity was determined. With the determination

of intrinsic viscosity, the constants k and a were obtained from

the literature for the conditions used in the experiment. The

chitin and chitosan molar mass were determined using the

Eq. (2) from the values of intrinsic viscosity [hint], k and a are

Table 4

Molar mass determination by viscosimetric methods

Sample R2 a K (!10K3) hiintrinsic(mL gK1)

Molar

mass

QA standard 0.9998 0.93 1.81 447 6.29!105

A1 0.8667 0.93 1.81 3291 5.9!106

A3 0.9139 0.93 1.81 2329 3.76!106

QB standard 0.9917 0.93 1.81 263 3.56!105

B1 0.9485 0.93 1.81 3354 5.48!106

B3 0.9978 0.93 1.81 2073 3.29!106

QA,QB, commercial sample of chitosan (Aldrich-USA), 86% DD.QA standard,

commercial chitosan sample, 86%DD, (Aldrich-USA); ionic strengthZ0.12 M

(acetic acid 0.02 mol LK1CNaCl 0.1 mol LK1); A—molar mass of chitosan by

plotting ln(hr)/C vs. C; B—molar mass of chitosan by plotting hred vs. C; QB

standard, commercial chitosan sample, 86% DD, (Aldrich-USA).

constants and M is the molar mass (Table 4).

½hint�Z kMa (2)

3.3. Scanning electronic microscopy

Fig. 9 (A) and (B) show the SEM of chitin (A) and chitosan

(B) prepared from chrysalides of silkworm. The chitin structure

appears as several fine loosely united leaves, and similar results

were obtained previously with chitin isolated from crustaceans.

Chitosan prepared from silkworm chitin shows a highly porous

structure.

4. Conclusion

The efficiency of chitin extraction from silkworm chrysa-

lides and chitosan production by deacetylation of chitin were

investigated. The yield of chitin extraction was low in both

methods investigated. However, although the low yields, the

final products using a closed reactor were of high purity chitin

and high purity and porous chitosan. The average yield of

chitosan production by chitin deacetylation (% DD) was 83%.

Acknowledgements

We thank CNPq (Brazil), CAPES (Brazil), and Fundacao

Araucaria-PR (Brazil) for financial support. We are also

grateful to COCAMAR, Maringa, PR (Brazil) for the samples

of silkworm chrysalides.

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