International Journal of Science Research Volume 01, Issue 03, December 2012, pp.
116-120
Preparation of sericin nano particles from waste of
silk industry
Pushpa Agrawal*, Sushant Bhushan
1
R. V. College of Engineering, Bangalore-560059, Karnataka, India
2
Tumkur University, Tumkur-572103, Karnataka, India
Author for Correspondence: pushpa_agr@yahoo.co.in;
sushantbhushan@gmail.com
Received on: 15 June 2012; Accepted: 08 September 2012
Abstract Silk is a natural protein fiber produced by the
domesticated silk moth. Silk secreted by the silkworm,
Bombyx mori, consists of two main group of proteins, sericin
and fibroin. Sericin is the minor component of the cocoon.
The silk industry obtains silk fibers after degumming of
cocoons. In this process of degumming, sericin is removed in
the waste water which is released into field without treatment
leading to environmental problems. Sericin can be used as
biomaterial for various applications therefore; a new protocol
has been developed for the efficient recovery of sericin and to
produce nano sericin material. In the present research work we
aimed to obtain pure sericin powder without impurities in a
cost effective manner by processing waste water obtained
from silk industries. The extracted sericin is subjected to
rotary drying to get concentrated sericin solution. The protein
content was estimated using Lowrys test. The sample was
then ultrasonicated to reduce the particle size and lyophilized
to obtain nano sericin powder. The characterization of nano
sericin was carried out with UV Visible Spectroscopy, Fourier
Transform Infrared Spectroscopy, Scanning Electron
Microscope, X-Ray Diffraction and Particle Size Analyzer.
Key words: Waste silk, Silk cocoons, sericin, nano particles, Characterization
1.0 Introduction
Silk is very important for wide range of applications. Silk is
composed of two important group of proteins, sericin and
fibroin. Fibroin is the fibrous component; it has great tensile
strength and property to withstand wear and tear. It finds great
application in textile industry. Sericin is the glue protein
which joins the fibroin fibrous components. The sericin
constitutes 20-30% of the silk cocoon. Sericin is a yellow,
brittle and inelastic substance which acts as an adhesive for
twin fibroin filaments concealing the unique luster for fibroin
[1]. The greatest sericin content is present in outer layer of
cocoon while the least sericin proportion is present in
innermost layer of cocoon [2]. Sericin contains 18 amino acids
including essential amino acids and is characterized by the
presence of 32 per cent of serine. The total amount of
hydroxyl amino acids in sericin is 45.8 per cent. There are
42.3 per cent of polar amino acid and 12.2 per cent of non-
polar amino acid residues. Sericin occurs mainly in an
amorphous random coil and to a lesser extent, in a -sheet
organized structure [3]. Their main role is to envelop the
fibroin. In presence of sericin the fibers are hard and tough
and become soft and lustrous after its removal [3]. Some of
the important properties of sericin are: it is soluble in hot
water, resists oxidation, antibacterial, UV resistant, absorbs
and releases moisture easily. One study has shown that sericin
is composed of three polypeptides, Sericin A, M, P together
with a practical method to isolate them. Sericin A was shown
to be a less -forming protein than sericin M and P, and
contributing more to its solubility. This method can be
extensively used to characterize each sericin molecule,
especially in its properties as a structural protein [4]. Nano
sericin finds application in medical and pharmaceutical
industry as it relieves constipation, cures bowel cancer,
accelerates absorption of materials and has been shown to act
as universal drug carrier; sulphonated sericin has anti-
thrombic effect, Sericin acts as anti-oxidant and offers
protection against oxidative stress and restores cell viability
[5]. Sericin has been found to have anti-tumor activity [6].
Sericin finds application in cosmetics, biomaterial, as
biopolymer [7], as anti-microbial agent and as hydrogel.
Sericin hydrogel formation can take place by cross linking
agents include N, N'-methylenebisacrylamide divinyl benzene,
and ethylene glycol dimethacrylate. It can also take place by
polymerization by irradiation.
2. BACKGROUND:
Sericin finds wide range of applications. The preparation of
nano particles can increase its applications to get more stable
biomaterial. Sericin as nano-particles has multiple
applications ranging from medical applications, skin care
application to culture of cells
application[14],[16],[17],[18],[22],[23]. Characterization is
the process of determination of the size or distribution of size
of a particle or substance30. Nano particle characterization is
an important step in understanding the size of particle and the
characteristic of the particle, so that it can be determined,
whether it have required properties of a nano particle
mandatory for it to be applied for its distinctive and specific
application. Characterization for the sericin nano particle is
done by Scanning Electron Microscope, Particle Size
Analyzer, Fourier Transform Infrared Spectroscopy, UV-
spectroscopy and X-Ray diffraction technique. A brief
description of the equipments and the process of
characterization are as below:
2.1 Scanning Electron Microscopy: A SEM is a type
of electron microscope that images a sample by scanning it
with a high-energy beam of electrons in a raster scan pattern.
2.2 Particle Size Analyzer: Particle size analyser is used to
characterize the size distribution of particles in a given
sample. It measures one-dimensional property of a particle
and relate this to the size of an "equivalent sphere". It
measures the surface area and volume of a particle and then
report the size of sphere.
2.3 Fourier Transform Infrared Spectroscopy: A preferred
method of infrared spectroscopy. In infrared spectroscopy, IR
radiation is passed through a sample. Some of the infrared
radiation is absorbed by the sample and some of it is passed
through (transmitted). The resulting spectrum represents the
molecular absorption and transmission, creating a molecular
fingerprint of the sample.
2.4 UV Visible Spectroscopy: A double beam
spectrophotometer is used. The absorbance is directly
proportional to the concentration and gives an estimate of the
concentration of the sample. Solution subjected to urea
method shows a peak of absorbance at 250nm. Solution not
subjected to urea method shows a peak of absorbance at 270
nm. The standard sericin protein has the absorption peaks
between 250 to 270 nm. The sericin obtained from various
 117

other methods also indicated the absorbance in the same

range. This represents the presence of protein in the sample.
1.0 X-Ray Diffraction: It measures the average spacings
between layers or rows of atoms. It determines the orientation
of a single crystal or grain finding the crystal structure of an
unknown material and measuring the size, shape and internal
stress of small crystalline regions[31],[32],[33],[34].

3. EXPERIMENTAL

3.1 Materials and equipments used: Waste water of silk

industry, Hot Water bath, hot air oven, dialyzer, Tangential
flow filtration, UV spectrophotometer, Vacuum tray dryer,
Figure 1 Nano sericin powder
Rotary evaporator, Ultrasonicator , Lyophilizer, Particle size
analyzer, SEM, Micropipettes, Glass wares.
The characterization results are as shown below:
3.2 Methods:

3.2.1 Extraction:

The waste water was collected from the silk industry from
Ramnagram, Karnataka. The waste water was first filtered
from whatmann paper. The filtrates was centrifuged at 10000
rpm for the removal of the suspended particles and then
treated with activated charcoal, for removing the color and
odor. To the clear solution, Urea was added, such that the
concentration of the resultant solution was 8M. The reaction
mixture was incubated at room temperature with gentle
stirring for 30 minutes followed by heating at 85 C for an
hour on a hot plate. The solution was dialyzed against water
using a cellulose membrane with MWCO of 10kDa under
constant agitation. It was then ultrasonicated. The solution
wash then concentrated to one fifth of its volume using Rotary
drum evaporator and lyophilized to obtain sericin powder.

3.2.2 Characterization: The characterization of the nano

sericin powder was carried out with UV Spectroscopy, Fourier
Transform Infrared Spectroscopy, XRD, SEM and Particle
Size Analyzer [27][29].
Figure 2: UV Spectroscopy result, The absorbance result was
4. RESULTS highest for the wavelength peak of 205, 255 & 270 nm.

The results of the preparation of sericin nano particles and its

characterization by several techniques are shown in the figures
below. Fig. 1 shows the prepared nano particles in the
bottles. Figure 2, 3, 4, 5, 6, 7, 8 shows characterization of the
sericin nano particles by UV-Visible Spectroscopy, Lowrys
method, SDS-PAGE, Fourier Transform Infra Red
spectroscopy, Scanning Electron Microscopy, Particle Size
Analyzer, X-Ray Diffraction respectively. The result of the
characterization is represented as caption of the figures.

4.1 Protein estimation

Protein estimation by Lowrys method.

4.2 FTIR
Fourrier Transmission Infrared Spectroscopy Figure 3: Lowrys Method, The concentration of protein was
360ug/ml at optical density of 660nm.
Figure 4: SDS PAGE, The SDS PAGE showed light bands with
molecular weights of sericin in the range of 20-300kDa.
 International Journal of Science Research December 2012
Figure 5. Fourier Transform Infrared Spectroscopy
The graph shows many peaks between 1250 and 1700 cm of
sericin group of proteins.
4.3. SEM-Scanning electron microscope
Figure 6. Scanning Electron Microscopy picture of sericin from
non-urea treated and urea treated method. (Scale 200um)
The particles in the sample which is not urea subjected are
very close to each other and they tend to get agglomerated and
they are not as stable as urea subjected sericin. The result
shows that the sericin particles in the urea subjected sample
looks very stable and are arranged very loosely.
5. DISCUSSION
The waste water of silk industry after boiling the cocoons is
thrown away without any specific use. In this paper the
successful attempt has been made to prepare the nano particle
of the major protein, sericin, from this wasted silk water. The
technique has been developed to prepare the nano particles
successfully (Fig.1). The following characterization
techniques have been used to confirm protein and size of
particles. Figure 2, shows characterization of the sericin nano
particles by UV-Visible Spectroscopy, which is showing the
absorption maxima peak between 250 nm and 270 nm which
confirms the presence of sericin protein. The protein
estimation using Lowrys method, (fig. 3) also confirms the
presence of protein. The further confirmation of sericin
protein has been done by SDS-PAGE, Fig. 4, which
indicates the band of sericin group of proteins between 20 and
300 kDa.
The Fourier Transform Infra Red Spectroscopy results further
confirms the presence of many peaks between 1250 and 1700
cm of sericin group of proteins.
The synthesis of nano particle has been confirmed Scanning
Electron Microscopy, Particle Size Analyzer, X-Ray
Diffraction and results have been shown in figures 5, 6, 7, and
8 respectively. This procured sericin nano particle can find
great applications ranging from pharmaceutical, cosmetic to
biotech industry.
6. CONCLUSION
The handsome recovery of the silk sericin group of proteins
and synthesis of nano particle from silk waste which can
readily find wide range of applications and also save the
environment hazard caused by the silk waste.
The protocol for the synthesis and characterization of nano
sericin particles from waste water of silk industry was
devised. This is a relatively simple cost effective method.
The extracted pure sericin was subjected to urea treatment to
get the nano size. The particles exhibit least mean size in
comparison to other methods. Some of highlights are: There
was successful
Figure 7. Particle Size Analyzer result, Sericin solution upon
urea treatment showed relatively smaller particles with the
minimum size of 2.6 nm, Sericin without urea treatment has 10%
of particle with minimum size of 260nm.

Figure 8. X-Ray Diffraction, XRD characterization result of the
peak obtained for sericin powder, The sericin nano particle size
observed was 56.99 and 3.98 at 2 and 22.5 -2.
procurement of sericin proteins from the waste water of silk
industry. The characterization result as depicted by the UV
Visible spectroscopy, Fourier transform infrared spectroscopy,
Protein estimation by Lowrys method, and SDS page result
confirms the pure preparation of sericin group of proteins. The
Scanning electron microscopy and Particle size analysis result
show the synthesis of good quality nano sericin particles.
This method of extraction is effective and simple. The pure
sericin powder and nano sericin thus obtained from silk waste
may be tested for various applications such as production of
hydrogel, biopolymers, biomaterials, and antioxidant and for
other antimicrobial applications which have to be exploited in
future studies.
1) Sericin extracted shows antimicrobial properties and this
property can be exploited for various applications.
2) The inherent gelling property of sericin to prepare
hydrogels showed better gelation on addition of cross linkers
like Glutaraldehyde which can be used for wound dressing
and as soil conditioner.
3) We succeed in obtaining membrane having sufficient
strength to be used as artificial skin. Toxicology tests are
further to be carried out on the obtained membrane taking its
mechanical strength and diffusion coefficient into
consideration for practical use. This displays potential
utilization of the waste for wide range potential applications.
FUTURE PROSPECTS:
The sericin in nano form can be used for various applications:
Cosmetics [14][18][23], Replacing binding material in
perfume stick, Scaffolds in medical applications[16],[17],
Anti microbial products[22], as hydro gels in diapers and
napkins.
ACKNOWLEDGEMENT: We are thankful to Department
of Biotechnology, New Delhi, for funding the project and RV
College of Engineering for providing the infrastructure to
conduct the experiments.
REFERENCES:
1. Apichart sonthisombat PhD and Peter T Speakman PhD. Silk-
Queen of fibres-The concise story.Assistant professor (2004).
2. Mondal*, K. Trivedy and S. Nirmal Kumar. The silk proteins,
sericin and fibroin in silkworm, Bombyx mori Linn., - a review
Caspian J. Env. Sci. 2007, Vol. 5 No. 2 pp. 63~76
119
3. M N Padamwar and A P Pawar. Silk sericin and its applications:A
review jol of scientific and industrial research vol.63 April
2004,pp.323-329.
4. Takasu,Y.Yamada, H., and Tsubouchi,K. Isolation of three main
sericin components from the coccon of the silkworm, Bombyx
mori. Biosci.Biotechnol.Biochem., 66, (2002) 2715-2718
5. Rupesh Dash#, Chitrangada Acharya#, PC Bindu# & SC Kundu
Antioxidant potential of silk protein sericin against hydrogen
peroxide-induced oxidative stress in skin fibroblasts BMB reports
vol 41 march 2008 pp 236-241.
6. Siqin zhaorigetu, Noriyuki yanaka, Masahiro sasaki, Hiromitsu
watanabe and Norihisa kato Silk protein, sericin, suppresses
DMBA-TPA-induced mouse skin tumorigenesis by reducing
oxidative stress, inflammatory responses and endogenous tumor
promoter TNF- oncology reports 10: 2003,pp 537-543.
7. Yoshii, F., Kume, N., Makuuchi, K. and Sato, F. (2000) Hydrogel
composition containing silk protein. Japan Patent 06-017373A.
8. Miyairi, S. and Sugiura, M. (1978) Properties of b-glucosidase
immobilized in sericin membrane. J. Fermen. Tech. 56, 303308.
9. Hunt TK. Disorder of Wound Healing. World J Surg. 1980;4:271-
7.
10. Tsubouchi K, Igarashi Y and Takasu Y. Sericin Enhances
Attachment of cultured Human Shin Fibroblasts. Biosci
Biotechnol Biochem. 2005;69(2):403-05.
11. Nagai N et al. Enhancing Effects of Sericin on Corneal Wound
Healing in Rat Debrided Corneal Epithelium. Biol Pharm Bull.
2009;32(5):933-936.
12. Kato et al. 1998. Masakazu, Kazuhisa, Hideyuki, Hiroshi, and
Shigeru. 2003.
13. M. Mondal*, K. Trivedy and S. Nirmal Kumar, The silk proteins,
sericin and fibroin in silkworm, Bombyx mori Linn., - a review,
Caspian J. Env. Sci, Vol. 5 No. 2 (2007) pp. 63~76.
14. Padanwar, M.N. Pawar, A.P., Daithankar. And Mahadik, K.R .
Silk sericin as a Moisturizer an in vivo study. J.Cosmet. Dermat. 4,
(2005) 250-257.
15. Yamada H, Fuwa N & Nomura M, Use of sericin as antioxidants
and tyrosinase inhibitors, Eur Pat Appl, EP 841065 A2 (to Seiren
Co Ltd Japan) 13 May 1998, pp 9; Chem Abstr, 129(1) (1998)
8425.
16. Zhang, Y. Tao, M.L, Shen.,W.D., Zhou, Y., Yue, Ding and Zhou,
W.L., Immobilization of L-asparaginase on the microparticles of
the natural silk sericin protein and its characters. Biomaterials
(2004) 25: 17, 3751-3759.
17. Komatsu, K. Studies on dissolution behaviors and structural
characteristic of silk sericin (1975).
18. Kato N & Sasaki M New physiological function of sericin and its
application for cosmetic and food, Fragrance J, (2000), 28(4) 28-
33.
19. Zhu L J, Arai M & Hirabayashi K, Relationship between adhesive
properties and structure of sericin in cocoon filament, J Sericult
Sci Jap, 64(5) (1995) 420-426.
20. Padamwar M.N. and A P Pawar, Silk sericin and its applications:
A review. Journal of Scientific and Industrial Research, Vol. 63,
April 2004, pp 323-329
21. Hu G & Zhu L J Characteristics and structure of gel with fibroin
and sericin, Gongxueyuan Xuebao, (1997) 14(3) 154- 158.
22. . Sarovart S, Boonya Sudatis, Pratep Meesilpa, Brian P Grady and
Rathnawan Magarphan The use of sericin as an antioxidant and
antimicrobial for polluted air treatment. Rev. Adv. Mater. Sci. 5:
(2003) 193 198.
23. Hidetoshi Teramoto, Ken-ichi Nakajima and Chiyuki Takabayashi
Preparation of elastic silk sericin hydrogel. Biosci. Biotechnol.
Biochem. 69, 4: (2005) 845 847.
24. . Aramwit, P.; Sangcakul, A. The effects of sericin cream on
wound healing in rats. Biosci Biotechnol. Biochem. 2007, 71,
2473-2477.
25. Takahashi, M.; Tsujimoto, K.; Yamada, H.; Takagi, H.; Nakamori,
S. The silk protein, sericin, protects against cell death caused by
acute serum deprivation in insect cell culture. Biotechnol. Lett.
2003, 25, 1805-1809.
26. Kurioka, A.; Kurioka, F.; Yamazaki, M. Characterization of
sericin powder prepared from citric acid-degraded sericin
polypeptides of the silkworm, Bombyx Mori. Biosci. Biotechnol.
Biochem. 2004, 68, 774-780.
27. Sprague, K.U. The Bombyx mori silk proteins: Characterization of
large polypeptides.Biochemistry 1975, 14, 925-931.
28. Zurovec, M.; Yang, C.; Kodrik, D.; Sehnal, F. Identification of a
novel type of silk protein and regulation of its expression. J. Biol.
Chem. 1998, 273, 15423-15428.
 International Journal of Science Research December 2012

29. . Takasu, Y.; Yamada, H.; Tsubouchi, K. Isolation of three main

sericin components from the
cocoon of the silkworm, Bombyx mori. Biosci. Biotechnol.
Biochem. 2002, 66, 2715-2718.
30. Juh Tzeng Lue, A review of characterization and physical
properties studies of metallic nano-particles, 2000.
31. Jyh Ping Chen, Preparation and characterization of biomimetic silk
fibroin/Chitosan composite nanofibers by electrospinning for
osteoblasts culture, 2012.
32. Juh-Tzeng Lue, A review of characterization and physical property
study of metallic nano particles, 2001.
33. zge Malay, formation and characterization of silk fibroin /
hyaluronic acid complexes and their use in ionotrophic drug
delivery, 2005,
34. Bivash Mandal, Preparation and Physicochemical Characterization
of Eudragit RL100 Nanosuspension with potential for Ocular
Delivery of Sulfacetamide.