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Sustainable Uses of Byproducts from Silk Processing
Sustainable Uses of Byproducts
from Silk Processing
Narendra Reddy and Pornanong Aramwit

Authors All books published by Wiley-VCH are carefully
produced. Nevertheless, authors, editors, and
Professor Narendra Reddy publisher do not warrant the information
Center for Incubation, Innovation, contained in these books, including this book, to be
Research, and Consultancy free of errors. Readers are advised to keep in mind
Jyothy Institute of Technology that statements, data, illustrations, procedural
Thataguni Post details or other items may inadvertently be
560109 Bengaluru inaccurate.
India
Library of Congress Card No.: applied for
Professor Pornanong Aramwit
Department of Pharmacy Practice British Library Cataloguing-in-Publication Data
Faculty of Pharmaceutical Sciences and A catalogue record for this book is available from
Center of Excellence in Bioactive the British Library.
Resources for Innovative Clinical Applications
Chulalongkorn University, Bangkok Bibliographic information published by the Deutsche
Thailand 10330 Nationalbibliothek
The Deutsche Nationalbibliothek lists this
The Academy of Science publication in the Deutsche Nationalbibliografie;
The Royal Society of Thailand detailed bibliographic data are available on the
Dusit, Bangkok Internet at <http://dnb.d-nb.de>.
Thailand 10330
© 2021 WILEY-VCH GmbH, Boschstraße 12,
Cover 69469 Weinheim, Germany
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Print ISBN: 978-3-527-34786-5

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Typesetting SPi Global, Chennai, India

Printing and Binding
Printed on acid-free paper
10 9 8 7 6 5 4 3 2 1
v
Contents
Preface xi
1 Sericin: Structure and Properties 1

1.1 Type of Silk Sericin 1
1.2 Localization of Silk Sericin 1
1.3 Molecular Mass of Sericin 2
1.3.1 Middle Silk Gland Sericin 2
1.3.2 Mulberry Cocoon sericin 2
1.3.3 Non‐mulberry Cocoon and Peduncle Sericin 5
1.4 Layers of Sericin 6
1.5 Sericin Amino Acid Components 6
1.5.1 Silk Gland of Mulberry Sericin 6
1.5.2 Sericin from Mulberry Cocoons 8
1.5.3 Sericin from Non‐mulberry Cocoons 12
1.6 Sericin Gene 14
1.7 Sericin Structure 16
1.8 Sericin Properties 19
1.8.1 Biophysical Properties 19
1.8.1.1 Water Solubility 19
1.8.1.2 Gelation 20
1.8.1.3 Thermal Stability 20
1.8.1.4 Ultraviolet (UV) protection 21
1.8.1.5 Adhesion Properties and Electrostatic Interaction 22
1.8.2 Biochemical Activity 22
1.8.2.1 Anti‐tyrosinase Activity 22
1.8.2.2 Anti‐elastase Activity 23
1.8.2.3 Antioxidant Activity 23
1.8.2.4 Anti‐lipid Peroxidation Activity 25
1.8.3 Biological Activity 25
1.8.3.1 Anti‐inflammatory Activity 25
1.8.3.2 Anti‐tumor Activity 27
1.8.3.3 Inducing Collagen Production 28
vi Contents
1.8.3.4 Antibacterial Activity 29

eferences 32
R
2 Processing Sericin 39
2.1 Effects of Source and Extraction Method of Sericin on Its Benefits
and Applications 39
2.1.1 Sericin Extraction 39
2.1.1.1 Water Extraction (WaterSS; HeatSS, AutoclaveSS) 39
2.1.1.2 Acid Extraction (AcidSS) 39
2.1.1.3 Alkali Extraction (AllkaliSS; Alkali‐L‐SS, Alkali‐H‐SS) 40
2.1.1.4 Urea Extraction (UreaSS) 40
2.1.1.5 Alcohol Extraction (AlcoholSS) 40
2.1.2 Effect of Source and Extraction on Sericin Properties 40
2.1.2.1 Molecular Weight of Sericin 40
2.1.2.2 Secondary Structure of the Sericin Protein 40
2.1.2.3 Phenolic contents 43
2.1.2.4 Antioxidant Activity 43
2.1.2.5 Anti‐tyrosinase Activity 44
2.1.2.6 Cytotoxicity 44
2.1.2.7 Cell Attachment, Cell Proliferation, and Collagen Production 44
2.1.2.8 Cell Protection 44
2.1.3 Benefit and Application of Extracted Mulberry Sericin and Non‐mulberry
Sericin 45
2.1.3.1 Pharmaceutics and Cosmetics 45
2.1.3.2 Wound Healing 45
2.1.3.3 Tissue Engineering 47
2.1.3.4 Drug Delivery 47
2.2 Modification of Sericin Structure 48
2.3 Chemical Modification 48
2.4 Glutaraldehyde Cross‐linking 50
2.5 Dehydrothermal (DHT) Cross‐linking 51
2.6 Carbodiimide Cross‐linking 51
2.7 Dimethylolurea (DMU) Cross‐linking 53
2.8 Enzymatic Cross‐linking 53
2.9 Physical Modification 54
2.9.1 Photo‐Cross‐linking 54
2.10 Forms of Sericin Processing 55
2.10.1 Two‐dimensional Structure of Sericin 55
2.10.1.1 Hydrogel 55
2.10.1.2 Film 56
2.10.2 Three‐dimensional Structure of Sericin 56
2.10.2.1 Electrospinning Silk Sericin and Its Blends 56
2.10.3 Pure Sericin Electrospun Fibers 57
2.10.4 Sericin Blends with Natural Polymers 59
2.10.5 Sericin Blends with Synthetic Polymers 60
Contents vii
2.10.6 Blends with Poly(caprolactone) 65

2.11 Sericin from Wild Silks 66
2.12 Fibroin–Sericin Blends 69
2.12.1 Freeze‐drying 71
2.12.2 Salt Leaching 71
2.12.3 Gas Foaming 73
2.13 Biomaterials from Sericin and Sericin–Protein Blends 74
2.13.1 Pure Sericin Biomaterials 74
2.13.1.1 Sericin–Fibroin Blends 77
2.13.1.2 Sericin and Non‐sericin Protein Blends 78
2.13.2 Sericin Blend with Polysaccharides 81
2.13.2.1 Sericin–Alginate Blends 81
2.13.2.2 Sericin–Chitosan Blends 83
2.13.2.3 Sericin–Carboxymethyl Cellulose Blends 83
2.13.2.4 Binary and Ternary Sericin Blends with Miscellaneous Polysaccharides 85
2.14 Blends with Synthetic Polymers 89
2.15 Sericin–Lignin Blends 90
References 91
3 Applications of Sericin 101

3.1 Medical Application 101
3.1.1 Antioxidant Properties 101
3.1.2 Antibacterial Activity 102
3.1.3 Wound Healing 104
3.1.4 Antitumor Effect 105
3.1.5 Antidiabetic Potential 106
3.1.6 Metabolic Effects 107
3.1.6.1 Gastrointestinal Tract 107
3.1.6.2 Circulatory and Immune Systems 108
3.1.6.3 Lipid Metabolism and Obesity 109
3.1.7 Vehicle for Drug Delivery 109
3.1.8 Supplement in Culture Media and Cryopreservation 112
3.2 Cosmetic Applications 113
3.3 Biotechnology Applications 117
3.3.1 Tissue Engineering 117
3.3.1.1 Skin Tissue Repair with Sericin‐Based Materials 117
3.3.1.2 Sericin‐Based Materials for Bone and Cartilage Tissue Engineering 119
3.3.1.3 Sericin‐Based Biomaterials for Other Tissues 120
3.4 Miscellaneous Application 121
3.4.1 Sericin‐Coated Material as an Air filter 121
3.5 Conclusion 121
References 123
4 Non-silk Applications of Mulberry Plants 131

4.1 Introduction 131
viii Contents
4.2 Medicinal Applications of Mulberry Plant Extracts 132

4.2.1 Polysaccharides 132
4.2.2 Phenols and Flavanoids 135
4.2.3 Pectins and Lignins 140
4.2.4 Production of Paper 142
4.2.5 Fermentation and Biogas Production 144
4.2.6 Synthesis of Nanomaterials 144
4.2.7 Environmental Remediation 148
4.2.8 Composites 150
4.2.9 Superabsorbents 151
References 153
5 Pupae and Its Applications 157

5.1 Applications of Pupae Oil 157
5.1.1 Extraction of Pupae Oil 157
5.1.2 Medical Applications of Pupae Oil 159
5.1.3 Biodiesel from Pupae Oil 163
5.2 Proteins from Silkworm Pupae 163
5.2.1 Medical Application of Pupae Peptides 169
5.2.2 Nanoparticles from Pupae Proteins 174
5.3 Pupae as Animal Feed 176
5.3.1 Chitin and Chitosan from Silkworm Pupae 181
5.4 Miscellaneous Applications 184
5.4.1 Carbonization of Pupae 184
5.4.2 Production of Lipids 185
5.4.3 Anti‐diabetic Drug 188
5.5 Environmental Remediation 189
References 189
6 Applications of Pupae Litter (excrement) 195

6.1 Chlorophylline and its Derivatives 195
6.2 Applications of Proteins in Litter 197
6.3 Extracts from Litter 198
6.4 Environmental Remediation Using Litter 199
6.5 Conversion into Carbon 203
6.6 Nanoparticles from Litter 209
References 211
7 Converting Silk Wastes into Composites, Energy Storage Devices, and Other
Value-Added Materials 213
7.1 Introduction 213
7.2 Composites from Silk Wastes 213
7.3 Reinforcement for Polypropylene (PP) Composites 214
7.4 Green Composites from Silk Wastes 219
7.5 Energy Applications 221
Contents ix
7.5.1 Carbonization of Waste Silk 221

7.6 Miscellaneous Applications 225
7.6.1 Extraction of Amino Acids and Bioactive Compounds 225
7.7 Fibers and Hydrogels 226
7.8 Environmental Remediation 227
7.9 Nutritious Food from Silkworm Eggs 229
References 229
Glossary 233
Index 237
xi
Preface
Silk and silk‐producing insects are one of the most fascinating creations found in nature.
Silk as a textile fiber has endured its prominence, elegance, and economic importance for
millenniums. Discovery of novel silks produced by spiders, weaver ants, mussels, and other
insects has further increased the interest, applications, and understanding of silk. However,
a majority of the commercial silk is produced by Bombyx mori and in two countries, China
and India. Even in these countries, the changing socioeconomic and environmental aspects
are increasing the costs and adding considerable constraints on silk production. Another
intriguing aspect of silk production and processing is the number and distinct by‐products
that are generated.
During commercial silk production, degumming is essential and results in removal of
about 25% of the protein sericin. After silk extraction, the worms (pupae) are considered
waste and disposed into the environment or used as low‐value feed. Similarly, the insects
generate considerable amounts of litter and leave parts of the leaf unconsumed during the
rearing cycle. Stems of the mulberry plants are pruned regularly and are a major source for
biomass. Sericin, pupae, litter, mulberry stems, and leaves are by‐products that are inevita-
bly generated during silk production and are available in large quantities. Many of these
by‐products have unique structure, properties, and applications.
The aim of this book is to provide detailed information on the sources, properties, and
potential applications of silk byproducts. Structure of sericin and its use in cosmetics and food
applications have been described in detail. Pupae, which consists of oil, proteins, and carbohy-
drates, has been used as a source for food, feed, energy, medical, biotechnology, and to develop
several other novel materials. Proteins extracted from pupae and litter have inherent activity
against cancer cells, bacteria, and even viruses. Similarly, chlorphyllin in litter and leaves has
pharmaceutical and neutraceutical significance. Mulberry plants show high potential for
removing toxic substances in the environment and phytochemicals in the mulberry stems and
leaves have medical value. The authors aim to elucidate these unique features of silk by‐prod-
ucts and hope to promote the use of silkworms beyond the production of silk.
1
Sericin: Structure and Properties
1.1 Type of Silk Sericin
Sericin is a natural product from the silkworm. Sericin is one of the major protein compo-
nents in the cocoons of Lepidopteron insects. Sericin is a glue-like coating protein sur-
rounded with filament protein, fibroin (Figure 1.1). In manufacturing silk, sericin is a
waste product from the degumming process. The silk sericin is classified into two types
based on the feeding source of the silkworms: mulberry and non-mulberry sericin. The
mulberry silkworm, Bombyx mori, is a well-known source of commercial silk production.
This worm is a completely domesticated species that feeds on mulberry leaves. B. mori had
long been developed for an indoor cultivation for the silk industry, whereas non-mulberry
silkworm or wild silkworm is the group that feeds on other leaves such as oak leaves and
castor oil leaves. Most of the non-mulberry silkworms cannot be reared indoors for their
whole life cycles. The well-known non-mulberry silkworms are Antheraea, Samia ricini (or
Philosamia ricini), and Cricula trifenestrata. Antheraea is a genus of silkworm that feeds on
oak leaves and produces “tasar” silk, such as Antheraea assamensis (producing muka silk),
Antheraea mylitta, Antheraea pernyi, and Antheraea yamamai. S. ricini produces the
famous “eri” silk. In the wild environment, S. ricini feeds on castor oil plant leaves. C. trifen-
estrata is a wild silkworm producing “cricula” silk. The diversity of silkworm sources
(genus, species, and diet) may produce distinct sericin characteristics.
1.2 Localization of Silk Sericin
Sericin is located at several sites of silkworms and cocoons. In the mulberry silkworm,
B. mori, it has been reported that sericin is present in three components including silk
gland, cocoon, and floss (Gamo et al. 1977; Kikkawa 1953; Yamada 1978). For non-mul-
berry silkworms, sericin is also secreted in the cocoon peduncle (Dash et al. 2006). The silk
gland is the site that produces sericin. In a histological study, sericin was found to be mainly
synthesized in the middle and posterior of the silk gland. Sericin protein is then sent to
anterior silk glands via the lumen for secretion and cocoon construction (Consortium 2008;
Kikkawa 1953; Yamanouchi 1922).
Sustainable Uses of Byproducts from Silk Processing, First Edition.

Narendra Reddy and Pornanong Aramwit.
© 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.
2 1 Sericin: Structure and Properties
Sericin
Fibroin
15k V X2, 000 10 μ m 080705
Figure 1.1 Scanning electron microscope (SEM) of a silk filament that contains fibroin and sericin.
1.3 Molecular Mass of Sericin
The molecular mass of sericin has been observed using sodium dodecyl sulfate and poly-
acrylamide gel electrophoresis (SDS-PAGE). The diversity pattern of sericin was investi-
gated from various extraction sites, extraction methods, species, and strains. Figure 1.2
shows the molecular mass of sericin from different extraction methods using SDS-PAGE.
1.3.1 Middle Silk Gland Sericin

The middle silk gland (MSG, Figure 1.3) is a synthesis site for sericin in silkworm. Sericin
obtained from MSG is known as native sericin. The MSG sericin measured by gel electro-
phoresis was found in intact bands and various protein sizes. Sericin extracted from the silk
gland of mulberry silkworm was identified with three sizes of sericin including 130, 210,
and 220 kDa as shown in Figure 1.4 (Sprague 1975). The study of sericin extracted from
four MSG sections, including the anterior, middle to anterior, middle, and posterior sec-
tions, found five different sizes of sericin polypeptides. Two polypeptides, 177 and 134 kDa,
were isolated from the anterior MSG. The middle section of the MSG had two polypeptides
(309 and 145 kDa). One polypeptide (80 kDa) was found in the posterior section of the MSG
(Gamo et al. 1977). Therefore, various sericin polypeptides were observed in mulberry
sericin extracted from the MSG in the range between 80 and 309 kDa.
1.3.2 Mulberry Cocoon sericin

Cocoon sericin has been isolated, and the molecular mass of the protein was studied.
Multiple sericin polypeptides have been extracted by several approaches including tem-
perature, pressure, urea, acid, and alkali solution. In 1980, Tokutake reported that sericin
1.3 Molecular Mass of Serici 3
Yellowish cocoon U
Yellowish cocoon H
Yellowish cocoon A
Yellowish cocoon B
Greenish cocoon U
Greenish cocoon H
Greenish cocoon A
Greenish cocoon B
White cocoon U
White cocoon H
White cocoon A
White cocoon B
Marker
Marker
225
150
100
75
50
35
25
15
10
Figure 1.2 Sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE) of sericin
extracted from white, green, and yellow cocoons using the following methods: urea solution (U),
high temperature–high pressure (H), citric acid solution (A), and sodium carbonate solution (B).
Different silk strains with various extraction methods show different molecular weights ranging
from 10 to >225 kDa. Source: Reprinted with permission from Aramwit et al. (2010a).
Figure 1.3 Diagrammatic representation of the silk gland in the mature MM2
larvae of silkworms. Shadowed parts in the figure indicate the section
used for the extraction of silk proteins, fibroin, and sericin. A, anterior
gland; AM, anterior section in the middle gland; MM, middle section in
the middle gland; PM, posterior section in the middle gland; P, posterior
A
gland. Source: Reprinted with permission from Gamo et al. (1977). © 1977
Elsevier. PM
AM
MV1
and fibroin protein could be separated by the precipitation of sericin in acidic conditions
(pH 5.5). Later, the gel filtration approach was used to separate the precipitated sericin into
five fractions (Tokutake 1980). This result suggests that sericin comprises a variety of forms.
In 1982, Gamo and colleagues reported the molecular size of sericin proteins obtained by
boiling with an alkali solution for extracting the protein from the cocoon. SDS-PAGE
Molecular weight
Origin
Origin
210 000–220 000
130 000
Molecular weight 75 000–78 000

220 000 67 000–68 000
210 000 53 000
36 000
34 000
130 000 32 000
22 000
Tracking
dye Tracking
dye
(a) (b)
Figure 1.4 Separation of the protein components of sericin by sodium dodecyl sulfate and
polyacrylamide gel electrophoresis on slabs of (a) 4% acrylamide and 0.1% bisacrylamide;
(b) 8% acrylamide and 0.2% bisacrylamide. Source: Reprinted with permission from Aramwit et al.
(2010a).
analysis revealed the molecular size of sericin in three different bands including the sizes
>226.5, 226.5, and 218.8 kDa (Gamo 1982). In 2002, Takasu et al. compared the molecular
mass between cocoon sericin and sections of the MSG. Cocoon proteins were extracted by
saturated aqueous lithium thiocyanate solution. The four cocoon proteins were identified
from SDS-PAGE analysis and named after the similar sizes found in subparts of the
MSG. The two close bands around 250 kDa were named sericin A, as a reference to the
specific size found in the anterior of the MSG. The fraction of 400 kDa was named sericin
M, which was abundantly found in the middle of the MSG. The molecular size 150 kDa was
named sericin P; this protein was the lowest expressed and found only in the posterior
subpart of the MSG (Takasu et al. 2002). Moreover, the observation of sericin patterns was
different depending on the extraction methods. In 2010, Aramwit et al. compared the
extracted sericin protein pattern from four different extraction methods, including urea,
autoclave (high heat and high pressure), acidic solution, and alkaline solution. Clear
bands were observed with urea extraction, which found sericin in various sizes from 10
to >225 kDa. Sericin extracted by autoclave showed smear patterns ranging from 20 to
150 kDa. Acid and alkaline extraction solutions revealed band patterns mixing between
clear bands and smears between 50–150 kDa and 15–75 kDa, respectively. In addition, both
acid and alkaline extractions shared a clear band pattern at 50–70 kDa (Aramwit et al.
2010b). From all this information, it is evident that the extraction method affected the size
and pattern of cocoon sericin proteins and is related to its biological properties.
Additionally, the different silkworm strains gave different patterns of sericin proteins. The
study of B. mori from three strains based on the pigment color (different concentrations of
1.3 Molecular Mass of Serici 5
flavonoids and carotenoids in cocoons), white shell, greenish shell, and yellow shell
(Figure 1.5), had variations in sericin molecular mass. Urea-extracted sericin revealed the
clear bands in all strains, but different size ranges for the white shell and yellow shell strains
with proteins ranging from 10 to >225 kDa, while the greenish shell strain had mass ranging
from 10 to 150 kDa. The autoclave method showed smear bands ranging from 50–150,
35–100, and 35–75 kDa for the white shell, greenish shell, and yellow shell strains, respec-
tively. For acid and alkali extraction methods, all strains also displayed different bands
within the range of 35–150 and 15–75 kDa, respectively (Aramwit et al. 2010b). This suggests
that not only the sericin extraction method but also the strain of silkworms affected the
molecular mass structure of extracted-sericin proteins.
1.3.3 Non-mulberry Cocoon and Peduncle Sericin

Non-mulberry cocoon sericin has been studied in S. ricini, A. assamensis, and A. mylitta. In
2004, Ahmad et al. observed non-mulberry sericin at 66 kDa from S. ricini and A. assamen-
sis (Ahmad et al. 2004). In 2007, Dash et al. reported a 70 kDa sericin extracted from
A. mylitta (Dash et al. 2007). Isolated sericin from non-mulberry cocoons’ molecular mass
revealed a range between 66 and 70 kDa, which is smaller than observations for mulberry
sericin.
The differences in the sericin extraction methods had dissimilar protein patterns in non-
mulberry cocoon sericin from S. ricini, A. assamensis, and A. mylitta. The autoclave tech-
nique showed smear bands in all sericin species. The high temperature and high pressure
from this extraction method might degrade all types of sericin protein. For the urea extrac-
tion method, there appeared a diversity of sericin protein sizes. S. ricini sericin was detected
at the size higher than 300 kDa and in the range of 200–250 kDa. A. assamensis was observed
in two bands with a size >250 kDa and at approximately 90 kDa. For A. mylitta, three bands
were revealed with sizes of 250, 200, and 70 kDa (Sahu et al. 2016). These non-mulberry
sericins detected by this study are composed of a high molecular mass in between 200 and
300 kDa, which was close to the high molecular mass of mulberry sericin. A similar study
was performed comparing five extraction methods: urea, autoclave, conventional, acidic
solutions, and alkaline solutions in A. assamensis and S. ricini. A. assamensis showed
smeared bands in urea, autoclave, and conventional methods, whereas acidic and alkali
solutions displayed a clear band at 75 kDa. For S. ricini, a clear band at 75 kDa was revealed
(a) (b) (c)
Figure 1.5 Physical appearance of silk cocoons, while shell (a), green shell (b), and yellow shell (c).
along with smeared protein in urea, conventional, and alkali solutions. Smears with no
intact bands of sericin proteins were observed from the acid and autoclave extraction meth-
ods (Kumar and Mandal 2017). These data showed that cocoon sericin protein patterns
were different depending on their species and extraction methods. Therefore, the cocoon
sericin protein might have a diversity of structures.
The peduncle is a strong filament in a ring form for attaching the non-mulberry cocoon
to the branches of a tree. Sericin extracted from the peduncle of A. mylitta had a single band
detected at 200 kDa (Dash et al. 2006). The size of this protein is similar to that observed for
the MSG sericin extraction of A. mylitta (Dash et al. 2009). This sericin protein might have
a major role in the action of the A. mylitta peduncle.
1.4 Layers of Sericin
The microstructure of silk gland sericin has been observed in silkworm. Histological stud-
ies have shown that sericin can be clearly divided into three distinct parts. Sericin I is the
inner layer connected to fibroin. Sericin II is the middle layer and is the most abundant
type. Sericin III is the outer layer, which covers the outside and is mostly mucous
(Kikkawa 1953).
Cocoon sericin could be separated into three layers based on its solubility properties from
extraction methods such as temperature, pressure, urea, acid, or alkali solution. Three lay-
ers have been divided into outer, middle, and inner layers, which are connected to fibroin.
The amino acid composition of each layer has been defined differently. The pattern of
amino acid residues (mol%) was used to identify the type of sericin protein. Fifteen amino
acids have been identified. Four residues, including serine, threonine, glycine, and aspartic
acid, were present at higher levels in all three layers (Shaw and Smith 1951).
1.5 Sericin Amino Acid Components
The amino acid composition of sericin has been reported from parts of silkworms and
cocoons, including the silk gland, cocoon, floss, and peduncle. The percentage of amino
acid contents in the total amino acid component (mol%) could indicate the individual
structure of each sericin. The application of percent amino acid components was used as a
reference for the distinction between sericin and fibroin silk protein (Gamo et al. 1977).
1.5.1 Silk Gland of Mulberry Sericin

The amino acid residue composition in the silk gland of mulberry silkworm is revealed
in Table 1.1. Most of the common amino acid composition (>10 mol%), which was found
in all extraction methods, is serine and aspartic acid (including asparagine). In 1975,
Sprague reported the amino acid content of sericin with three different molecular masses,
including 220, 210, and 130 kDa. The four major amino acid residues, including aspartic
acid, glutamic acid, lysine, and serine, had averages of 27, 22, 19, and 14 mol%, respec-
tively (Sprague 1975). In 1977, Gamo et al. reported the amino acid composition of sericin
from different sections of the MSG, including the middle (fraction: s-1), anterior (frac-
tion: s-2 and s-5), middle to anterior (fraction: s-3), and posterior (fraction: s-4) sections
(Gamo et al. 1977). Serine residues had an average of 30 mol% in all sections. The excep-
tion is the anterior MSG fraction s-5, which secreted serine content of 16 mol%. This frac-
tion (s-5) contains serine in amounts that are half that of the other fractions. The minor
component amino acids of all fractions were glutamic acid (average 15 mol%) and aspar-
tic acid (average 13%). Threonine was found as a high percentage residue in fractions s-1,
s-2, and s-4, on average 9%, while fraction s-3 and s-5 had a glutamic acid-rich compo-
nent, on average 10%, instead of threonine residue. This data meant that sericin at differ-
ent sections of the silk gland presented unique characteristics of sericin structure.
1.5.2 Sericin from Mulberry Cocoons

The amino acid composition of cocoon sericin was studied from various extraction meth-
ods, species, and strains. Amino acid components from mulberry silk sericin were different
(Table 1.2). In mulberry cocoon sericin, the highest three amino acid components com-
monly found were serine (average 32 mol%), glycine (average 17 mol%), and aspartic acid
(average 16 mol%). The less abundant sericin amino acid is threonine (average 8 mol%).
However, the amino acid composition of sericin is different based on the extraction meth-
ods. Sericin from the acid precipitation method was found to have alanine-rich residues
(15 mol%) instead of threonine. Alanine richness is not commonly found in other extrac-
tion methods (Tokutake 1980). Different sericin fractions showed differences in amino acid
contents, such as the fraction sericin A (13 mol%) having glutamic acid richness. Meanwhile,
the other fractions, sericin M and sericin P, showed high threonine contents at an average
of 11 mol% (Takasu et al. 2002). The strain of silkworm did not show differences in the ratio
of the amino acid residues by the autoclave extraction method (Aramwit et al. 2009).
Cocoon sericin containing high amounts of serine, glycine, and aspartic acid is common.
However, the different extraction methods may affect some amino acid contents, such as
glutamic acid, threonine, and alanine. In addition, strains of silkworms revealed the differ-
ent amino acid components from alkali extraction methods (Table 1.2). The alkaline extrac-
tions from three different silkworm strains found different components. The fourth top
amino acid composition was found to be glutamic acid rich in Chul 3/2 and Chul 4/2 strains
(average 7 mol%), whereas Chul 1/1 had threonine (7 mol%) (Aramwit et al. 2010a). The
major amino acid residues of cocoon sericin were similar. Only some less common amino
acid components such as threonine, glutamic acid, and alanine were different among
extraction methods and strains. The modification of sericin structure by chemical solution
was supported by the study of the properties of sericin being improved (Teramoto et al.
2004). Therefore, the extraction method interferes with the sericin structure.
Floss is a soft filament that covers the mulberry silk cocoon of B. mori. Its function is to
protect and hang the cocoon on a tree branch. Floss sericin has been extracted and tested
for the amino acid content (Table 1.2) (Yamada 1978). Three major amino acid residues
were serine (40 mol%), glycine (18 mol%), and aspartic acid (10 mol%). The serine of floss
sericin revealed around twofold higher percentage content when compared to cocoons.
The differences in amino acid composition in floss and cocoons may reflect their specific
properties and functions.
Table 1.2 Amino acid composition of sericin obtained from the cocoons of mulberry silkworms.
Group of
silkworm
sericin Mulberry silkworm sericin
Tokutake Gamo Teramoto

Reference Yamada (1978) (1980) (1982) Takasu (2002) et al. 2004 Aramwit (2009)
B. mori
(shugetsu × B. mori B. mori B. mori
Species (strain) B. mori (mandarina) Hosho) B. mori B. mori B. mori (chul 1/1) (chul 3/2) (chul 4/2)
Source Floss Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon
Floss Inner Outer Acid S×2 Sericin A Sericin M Sericin P
Detail sericin cocoon cocoon precipitation (227 kDa) (250 kDa) (400 kDa) (150 kDa) Sericin Autoclave Autoclave Autoclave
Name mol% mol% mol% mol% mol% mol% mol% mol% mol% mol% mol% mol%
Alanine 4.43 4.84 5.15 15.20 4.40 5.50 4.10 8.10 5.00 4.10 4.45 4.98
Arginine 3.30 3.03 3.41 3.02 3.20 2.90 3.40 4.00 3.20 2.87 2.95 3.09
Aspartic acid + 10.20 18.61 18.30 12.90 14.90 13.30 15.70 11.30 16.30 15.64 15.62 15.97
asparagine
Cysteine Trace 0.42 0.64 Trace — 0.10 0.00 — — 0.44 0.43 0.27
Glutamic 4.31 4.90 4.78 4.25 11.10 12.80 3.10 3.10 4.70 4.61 4.76 4.86
acid +
glutamine
Glycine 18.17 16.90 16.70 24.20 14.90 14.30 16.00 14.10 15.30 15.03 15.09 15.14
Histidine 0.68 0.86 0.99 0.98 1.00 1.00 1.30 — 1.40 1.06 1.22 1.37
Isoleucine 0.67 0.60 0.67 1.82 0.60 0.20 0.50 0.80 0.60 0.56 0.65 0.61
(Continued)
Table 1.2 (Continued)
Group of
silkworm
sericin Mulberry silkworm sericin
Tokutake Gamo Teramoto

Yamada (1978) (1980) (1982) Takasu (2002) et al. 2004 Aramwit (2009)
Reference
B. mori
(shugetsu × B. mori B. mori B. mori
Hosho) B. mori B. mori B. mori (chul 1/1) (chul 3/2) (chul 4/2)
Species (strain) B. mori (mandarina)
Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon
Source Floss
Floss Inner Outer Acid S×2 Sericin A Sericin M Sericin P
Sericin Autoclave Autoclave Autoclave
Detail sericin cocoon cocoon precipitation (227 kDa) (250 kDa) (400 kDa) (150 kDa)
mol% mol% mol% mol% mol% mol% mol% mol%
Name mol% mol% mol% mol%
0.85 0.90 1.17 1.99 1.40 0.50 0.90 1.60 1.30 1.00 1.15 1.11
Leucine
1.89 2.26 2.89 2.07 6.00 5.40 1.80 1.00 2.70 2.35 2.51 2.78
Lysine
0.12 0.10 0.11 0.11 — — — — — 3.39 0.57 0.18
Methionine
0.43 0.42 0.47 0.69 0.40 0.40 0.20 0.70 0.40 0.28 0.39 0.36
Phenylalanine
0.66 0.51 0.56 0.69 — — 0.60 1.30 0.70 0.54 0.62 0.71
Proline
40.28 28.12 29.05 18.90 36.80 39.00 35.40 33.20 34.20 33.63 34.50 33.84
Serine
6.29 11.39 8.44 5.21 4.00 3.30 9.70 12.20 8.00 8.16 8.43 8.34
Threonine
— — — — — — — — — — — —
Tryptophan
4.09 3.28 3.39 4.10 0.10 0.70 4.00 4.60 2.90 3.45 3.64 3.47
Tyrosine
3.46 2.67 2.91 3.34 1.20 0.70 3.20 3.90 3.30 2.88 3.04 2.92
Valine
Table 1.2 (Continued)
Reference Aramwit et al. (2010)
Species (strain) B. mori (Chul 1/1) B. mori (Chul 3/2) B. mori (Chul 4/2)
Source Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon Cocoon
Detail
(extraction method) Autoclave Urea Acid Alkali Autoclave Urea Acid Alkali Autoclave Urea Acid Alkali
Name mol% mol% mol% mol% mol% mol% mol% mol% mol% mol% mol% mol%
Alanine 4.1 4.33 3.72 4.21 4.45 3.8 3.57 3.96 4.98 4.63 3.56 4.4
Arginine 2.87 5.41 4.92 4.92 2.95 5.21 4.87 3.79 3.09 5.71 5.24 4.83
Aspartic 15.64 18.31 15.93 19.88 15.62 17.93 16 21.58 15.97 17.69 16.61 19.92
acid + asparagine
Cysteine 0.44 0.39 0.53 0.23 0.43 0.33 0.5 0.19 0.27 0.42 0.52 0.16
Glutamic 4.61 5.27 5.75 5.93 4.76 6.02 5.4 7.66 4.86 5.97 5.88 7.03
acid + glutamine
Glycine 15.03 11.23 10.49 11.01 15.09 10.75 10.38 11.16 15.14 10.96 10.69 12.58
Histidine 1.06 3.26 2.47 1.72 1.22 2.82 2.83 2.38 1.37 2.5 2.29 2.15
Isoleucine 0.56 0.96 0.87 0.75 0.65 0.95 0.9 0.87 0.61 0.74 0.66 1.03
Leucine 1 1.58 1.43 1.56 1.15 1.58 1.44 1.51 1.11 1.63 1.37 1.81
Lysine 2.35 3.14 3.48 2.89 2.51 3.55 3.03 2.71 2.78 2.5 3.16 3.08
Methionine 3.39 0.12 0.06 0.15 0.57 0.08 0.06 0.13 0.18 0.06 0.05 0.15
Phenylalanine 0.28 0.6 0.71 0.81 0.39 0.66 0.67 0.72 0.36 0.63 0.57 0.81
Proline 0.54 1.46 0.78 1.24 0.62 0.79 0.73 0.92 0.71 1.16 0.79 1.01
Serine 33.63 31.27 31.86 30.01 34.5 32.24 32.01 28.41 33.84 30.69 31.95 27.59
Threonine 8.16 8.36 8.51 6.49 8.43 8.78 8.78 6.09 8.34 9.04 8.3 5.56
Tryptophan — — — — — — — — — — — —
Tyrosine 3.45 0.36 5.56 5.24 3.64 1.24 5.81 4.92 3.47 2.67 5.59 4.9
Valine 2.88 2.96 2.95 2.94 3.04 3.28 3.03 3.03 2.92 2.98 2.76 2.99
1.5.3 Sericin from Non-mulberry Cocoons

The cocoon sericin from non-mulberry silkworm has been reported from two species,
A. mylitta (Dash et al. 2007, 2008) and C. trifenestrata (Yamada and Tsubouchi 2001). The
amino acid analysis revealed three major components, including serine, glycine, and threo-
nine (Table 1.3). Interestingly, glutamic acid, which has a high content in mulberry sericin,
is less observed in non-mulberry sericin. The serine component in cocoon sericin of
A. mylitta averages 19 mol%, which is lower than that in mulberry sericin (average 32 mol%).
However, C. trifenestrata serine showed 40 mol%, which is higher than mulberry cocoon
sericin but similar to the floss of mulberry sericin. Non-mulberry cocoon sericin showed a
Table 1.3 Amino acid composition of sericin obtained from non-mulberry silkworms.
Group of
silkworm sericin Non-mulberry
Reference Dash Dash Dash Dash Yamada and

(2006) (2006) (2007) (2008) Tsubouchi (2001)
Species (strain) A. mylitta A. mylitta A. mylitta A. mylitta Cricula trifenestrata
Source Peduncle Cocoon Cocoon Cocoon Cocoon
Detail Peduncle Cocoon Sericin Cocoon Crude sericin

sericin sericin (70 kDa) sericin (400 kDa)
Name mol% mol% mol% mol% mol%
Alanine 4.80 6.01 2.95 6.01 4.90

Arginine 4.01 3.36 2.87 3.36 2.90
Aspartic — — — — 2.60
acid + asparagine
Cysteine — — — — —
Glutamic 11.16 5.70 5.98 5.70 1.50
acid + glutamine
Glycine 24.40 18.11 19.20 16.11 20.80
Histidine 6.30 11.15 13.51 10.15 —
Isoleucine 2.10 1.56 1.11 1.56 0.80
Leucine 0.10 1.76 1.25 1.76 1.10
Lysine 0.90 2.95 2.20 2.95 0.70
Methionine 0.80 — — — —
Phenylalanine — — — — —
Proline 1.20 1.28 0.98 1.28 2.50
Serine 21.42 17.78 19.40 19.78 39.80
Threonine 10.20 12.22 12.32 13.22 13.10
Tryptophan — — — — —
Tyrosine 0.70 2.38 1.94 2.38 7.10
Valine 2.40 1.29 1.01 1.29 —
1.5 Sericin Amino Acid Component 13
very low percent molecular content of aspartic acid, but a high component of threonine
(average 12 mol%). The minor amino acid component in A. mylitta is histidine (average
12 mol%), and it is tyrosine in C. trifenestrata (7 mol%). Cysteine and phenylalanine were
not found in non-mulberry sericin. From the amino acid composition of non-mulberry
cocoon sericin and mulberry cocoon sericin, it seems that the structure of sericin is differ-
ent and may lead to different structures and biological properties.
The peduncle, which has a function in hanging A. mylitta cocoons on the tree, had an amino
acid composition that was high in glycine (24 mol%), serine (21 mol%), glutamic acid (11 mol%),
and threonine (10 mol%) (Dash et al. 2006). The amino acid content is similar to the cocoon
sericin of A. mylitta, which has no aspartic acid residue. A major component of glutamic acid
was observed, which was different from the cocoon part that had a low mol% component. The
amino acid composition of the peduncle sericin was different from the cocoon sericin.
Therefore, the peduncle sericin may have a special property specific to its function.
Overall, a high serine content (>10 mol%) was found in all parts of the silkworm for all
species and extraction methods. The other major amino acids, glycine, threonine, aspartic
acid (including asparagine), glutamic acid (including glutamine), lysine, histidine, and
tyrosine, were found to vary in each part. The different amino acid components of sericin
might be related to its structure, property, and function (Figures 1.6 and 1.7).
B. mori A. mylitta A. assama S. ricini

Cocoons
Cut cocoons
Degumming
Sericin
Authoc time method
solution
Lyophil icorion
Degummed cocoon pieces
Lyophilized
Urea method
sericin powder
Figure 1.6 Physical appearance of mulberry and non-mulberry silk cocoons before and after
degumming with different methods of sericin isolation. Source: Reprinted with permission from
Sahu et al. (2016).
Fresh (no degumming) Urea degummed Autoclave degummed

A. mytitta
A. mytitta
A. mytitta
(a) (b) (c)
A. anama
A. anama
A. anama
(d) (e) (f)
s. rkini
s. rkini
s. rkini
(g) (h) (i)

B. mori
B. mori
B. mori
(j) (k) (l)
Figure 1.7 Scanning electron microscope (SEM) images of the cocoons of mulberry and non-
mulberry silkworms. The cocoons are observed before (50×) and after degumming (100×) using urea
and autoclave degumming methods. Scale bar represents 100 μm. Source: Reprinted with
permission from Sahu et al. (2016).
1.6 Sericin Gene
Sericin proteins are translated from sericin genes, which are produced from MSG cells.
Several studies have identified and characterized the sericin genes from MSG cells. The
sericin genes were differently expressed in the MSG subparts (including anterior, middle,
and posterior) and the stages of the silkworm larvae. Based on sericin transcripts, B. mori
sericin genes could be separated into three types: sericin 1 (ser1), sericin 2 (ser2), and sericin
3 (ser3). These genes have a specific purpose for their localization as described in Chapter 11
under the B. mori genome (Dong et al. 2015).
The ser1 gene was first identified by Okamoto et al., in 1982. Two sericin mRNAs extracted
from the MSG were discovered at lengths of 11.0 and 9.6 kb. The mRNA complementary
sequences represent similar genomic diriboxynucleic acid (DNA), which determined five
exons with 114 bp internal in the repetitive region consisting of approximately 60 repeats.
The repetitive region was composed of 38 amino acids which had high residues of serine
1.6 Sericin Gen 15
(40 mol%), aspartic acid (17 mol%), glycine (15 mol%), and threonine (10 mol%) (Okamoto
et al. 1982). The composition of this region was similar to the amino acid component from
the crude sericin protein extracted from the MSG. In 1986, Michaille et al. identified four
variable sizes of sericin mRNAs that were compatible with 24 kb genomic DNA, including
10.5, 9.0, 4.0, and 2.8 kb (Michaille et al. 1986). The two sericin mRNAs had similar lengths
between 10.5 and 9.0 kb and 11.0 and 9.6 kb, respectively, as reported by Okamoto (1982). In
1997, Garal et al. analyzed 4.0 kb of ser1 transcripts and combined the ser1 gene sequence
from mRNA and genome sequences, which were previously reported by Okamoto and
Michaille (Garel et al. 1997; Michaille et al. 1986; Okamoto et al.1982). The summarized ser1
gene sequence revealed 23 kb with nine exons and eight introns that were elucidated (Garel
et al. 1997). The four major transcripts from the ser1 gene: 10.5, 9.0, 4.0, and 2.8 kb, were
different by the absence of different exons (Michaille et al. 1986; Michaille et al. 1990). The
variation of MSG sericin mRNA splicing was reported due to the production from alterna-
tive splicing of the sericin gene. The transcription of sericin was expressed in various types
depending on the silkworm larval development stage (Ishikawa and Suzuki 1985). The silk-
worm larval stage was detected in various types of sericin transcripts in MSG cells (Couble
et al. 1987). This suggests that the developmental stage induced different splicing of sericin
genes in the cells. In addition, the amino acid compositions revealed in sericin M (middle of
MSG) (Takasu et al. 2002) were similar to the ser1 protein (Tsubouchi et al. 2005).
The ser2 gene, another sericin gene discovered in MSG, was first identified with two
encoding matured mRNAs at lengths 5.4 and 3.1 kb by Couble et al. in 1987 (Couble et al.
1987). In 1990, Michaille et al. reported two groups of ser2 transcripts, one of 3.1 kb and a
variable-sized one between 5.0 and 6.4 kb (Michaille et al. 1990). In 2009, Kludkiewicz et al.
reported two ser2 mRNAs containing 5.7 and 3.1 kb (Kludkiewicz et al. 2009). The ser2
proteins were predicted to have 1740 and 882 amino acid residues, which were identified as
230 and 120 kDa. The entire ser2 gene from the genomic DNA sequence database (Accession
number GQ381286) is composed of 13.54 kb with 13 exons and 12 introns. The different
mature mRNA sequences were observed by the deletion of the repetitive region at the exon
9a position. This evidence confirmed its generation by alternative splicing (Kludkiewicz
et al. 2009). From these results, the ser2 transcript revealed the identical sizing at the small
length of 3.1 kb and the polymorphism of the long transcript gene in the range of 5.0–6.4 kb
from alternative splicing at a gene exon.
The ser3 gene was mainly obtained in the floss and outer layer of a silkworm cocoon.
Takasu and colleagues identified 4.9 kb of ser3 gene transcript. The genomic length of the
ser3 gene revealed 6.575 kb with three exons and two introns observed. Therefore, the ser3
protein consists of 1271 amino acid residues with two regions of motif sequences, 86 amino
acid residues with 10 repeats, and 18 amino acid residues with 18.5 repeats. The estimated
size of ser3 protein was 120 kDa (Takasu et al. 2007).
Non-mulberry A. yamamai had the sericin genes identified from the transcriptomic
study of the last instar silk gland. Five sericin genes were discovered including AySrn1,
AySrn2, AySrn3, AySrn4, and AySrn, with transcription size variation between 3.8 and
5.5 kb. The AySrn gene characters are different from B. mori sericins. All of the AySrn genes
have short two exons with 22–28% of serine residues (Zurovec et al. 2016). The lower
amount of exons in the sericin gene and its amino acid components are different from
sericin genes in B. mori (3–12 exons). Therefore, the sericin proteins from different silk-
worm species appear to have different effects on their functions.
The ser genes transcribe differently depending on the silkworm larval stages. The silk-
worm larval (instar) is developed in five stages, and then the mature stage is started to cre-
ate a cocoon. The ser genes were differentially expressed by the specific instar stage. The
development of B. mori instar stage has been influenced by the sericin gene expressions. In
the last silkworm stage (the fifth instar), the transcripts of ser1 and ser3 started increasing
at day 4 of the fifth instar, whereas ser2 transcripts were highly expressed since the third
instar and decreased rapidly on day 4 of the fifth instar. This suggests that the expression of
sericin genes is specific and dependent on the silkworm development stage. Moreover,
sericin transcripts have investigated the expression in the fifth instar of each MSG subpart,
including the anterior, middle, and posterior sections. The ser1 transcripts were highly
expressed in the middle and posterior sections of MSG cells. The ser3 transcript was
expressed at the anterior and middle MSG cells (Takasu et al. 2010, 2007). The ser2 tran-
scripts were detected faintly in middle MSG and highly expressed at anterior MSG cells
(Takasu et al. 2010). Therefore, these three sericin genes might contain some specific pur-
pose which are beneficial for the different developmental stages of the silkworm.
1.7 Sericin Structure
The secondary structure of sericin has been observed by Fourier transform infrared spec-
troscopy (FTIR). It has been reported that the silkworm species and isolation method
affected to the sericin structure. Aramwit et al. reported B. mori cocoon sericins from four
isolation methods, including autoclaving, urea, acidic, and alkaline solutions, and mainly
found random coil and β-sheet structures (Figure 1.8). However, the urea extraction
Amide I Amide II Amide III
urea process
Absorbance
Heat process
Base process
Acid process
1800 1700 1600 1500 1400 1300 1200 1100 1000 900
–1
Wave number (cm )
Figure 1.8 FTIR spectra of SS obtained from various extraction methods: acidic, alkaline, urea, and heat
processes. Source: Reprinted with permission from Aramwit et al. (2010a). © 2010 John Wiley & Sons.
1.7 Sericin Structur 17
method found different peaks related to the urea solution, which suggests that urea might
be integrated into the sericin structure. Therefore, the urea used in sericin extraction pos-
sibly affected the sericin protein structure and function (Aramwit et al. 2010a).
The structure of sericin from different silkworm species (B. mori, A. assamensis, and
S. ricini) and cocoon extraction methods (urea, conventional, autoclaved, acidic, and alka-
line) has been studied by Kumar et al. (Figure 1.9) (Kumar and Mandal 2017). The second-
ary structure of sericin protein was classified based on the percentage of α-helix, β-sheet,
turns, and random coils. The sericin structures were variable among species and extraction
methods. All three species had a similarly high percentage of β-sheet and random coil
when obtained using the urea extraction method. The mulberry silkworms, B. mori, sericin
extraction with an acidic solution showed a higher percentage of α-helix and turns com-
pared to the sericin structure from the urea extraction method. Meanwhile, the conven-
tional and alkali methods resulted in the absence of the β-sheet, whereas the autoclaved
method resulted in an absence of α-helix. In contrast, non-mulberry silkworm sericins of
A B C D E F A B C D E F
220 kDa 220 kDa
160 kDa 160 kDa
120 kDa 120 kDa
100 kDa 100 kDa
90 kDa 90 kDa
80 kDa 80 kDa
70 kDa 70 kDa
60 kDa 60 kDa
50 kDa 50 kDa
40 kDa 40 kDa
30 kDa 30 kDa
20 kDa 20 kDa
10 kDa 10 kDa
(a) (b)
A B C D E F
220 kDa
160 kDa
120 kDa
100 kDa
90 kDa
80 kDa
70 kDa
60 kDa
50 kDa
40 kDa
30 kDa
20 kDa
10 kDa
(c)
Figure 1.9 Molecular weight distribution of sericin extracted from cocoons using different
extraction methods in sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE).
10% SDS-PAGE gel showing bands of (A) protein ladder and sericin extracted from (a) Bombyx mori,
(b) Antheraea assamensis, and (c) Philosamia ricini using (B) urea degradation, (C) acid degradation,
(D) autoclaved, (E) conventional method, and (F) alkali degradation. Source: Reprinted with
permission from Kumar and Mandal (2017).
A. assamensis and S. ricini from the urea extraction method did not result in α-helix in the
structure. The β-sheet structure of sericin extracted from A. assamensis was absent in all
extraction methods. However, S. ricini sericin had a variety of percentages (Figure 1.10)
(Kumar and Mandal 2017). The differences in the α-helix structure between mulberry and
non-mulberry sericins might be from the different gene sequences of each species. This
evidence may lead to a different function of each sericin protein among species. The vari-
able structures of sericin from different extraction methods may cause the different proper-
ties and biological activities of sericin proteins.
100 100
1549–1515 1241
% Transmittance 1241
% Transmittance
1624 1552–1515
80 80
1650
1624
1650
60 60
40 40
1800 1600 1400 1200 1000 1800 1600 1400 1200 1000
Wavelength (cm–1) Wavelength (cm–1)
(a) (b)
100 100
1241
1506–1520
80 1244
1624
% Transmittance
% Transmittance
80
60 1650
1544
40 1652
60
20
0 40
1800 1600 1400 1200 1000 1800 1600 1400 1200 1000
Wavelength (cm–1) Wavelength (cm–1)
(c) (d)
100
1241
% Transmittance
80 1544
1652
60
B. mori
P. ncini
A. assamensis
40
1800 1600 1400 1200 1000
Wavelength (cm–1)
(e)
Figure 1.10 FTIR spectra of sericin extracted from the cocoons of Bombyx mori, Antheraea
assamensis, and Philosamia ricini using (a) urea degradation, (b) autoclaving, (c) acid degradation,
(d) conventional method, and (e) alkali degradation. Source: Reprinted with permission from Kumar
and Mandal (2017). © 2017 Elsevier.
1.8 Sericin Propertie 19
The predicted structures of three B. mori sericin gene sequences from ser1, ser2, and ser3
have been observed to have a repetitive region of the sericin protein. The repetitive region
of the ser1 protein is composed of 38 amino acids with approximately 60 repeats, and a 40%
serine residue component was revealed in the high β-sheet content (Garel et al. 1997). The
repetitive sequence of the ser2 gene consists of 15 amino acids rich in charged residues,
including lysine, aspartic acid, glutamic acid, and arginine at this region that formed the
β-sheet structure of ser2 proteins (Michaille et al. 1990). The amino acid components
appearing in MSG ser2 proteins were different from those found in cocoon ser1 and ser3
proteins. The ser2 proteins produced from the MSG stopped expression before the silk-
worm contracted the cocoon. This showed that there was very little ser2 protein left in the
cocoon. Therefore, ser2 proteins were not observed in the cocoon isolate (Kludkiewicz
et al. 2009; Takasu et al. 2010). The repetitive region of the ser3 gene is composed of 86
repeating amino acids and another 8 repeating amino acids with 45% sericin residue com-
position. The ser3 repetitive regions were predicted to contain lower formations of the
β-sheet structure than the ser1 structure (Takasu et al. 2007). The different amino acid
components of each repeated sequence were formed by different structures and properties
of each ser protein. This data suggests that the different ser genes produced the specific
protein properties appropriate for each sericin layer.
1.8 Sericin Properties
1.8.1 Biophysical Properties

1.8.1.1 Water Solubility
Sericin can be dissolved in hot water and could be precipitated after exposure to cold water
(Sprague 1975). The water solubility of sericin was explained by the correlation with its
amino acid content. Amino acid compositions of both mulberry and non-mulberry sericin
have relatively high levels of serine, glycine, and threonine. The polar hydroxyl side chain
residue of serine and threonine accounting over 30% in its total amino acid profile resulted
in sericin presenting strong hydrophilicity (Padamwar and Pawar 2004). The correlation
between sericin structure and its water-soluble properties has been studied in mulberry
(B. mori) and non-mulberry (A. mylitta, A. assamensis, and S. ricini) sericins by circular
dichroism spectroscopy. The secondary structures of sericin were determined to be random
coils, β-sheet, and low α-helix content. In aqueous solution, sericin rapidly changed from
random coil to β-sheet. However, an FTIR study showed that the sericin powder was mostly
in the random coil and α-helix conformation (Sahu et al. 2016). This evidence shows that
the β-sheet conformation, which is largely seen in aqueous sericin, is the solubilized form
in water. Therefore, β-sheet formation is one of the factors of sericin imparting its water-
soluble properties. Additionally, temperature is also a factor that changes the sericin struc-
ture. Sericin forms an insoluble structure at high water temperatures. At low water
temperatures, sericin converts from random coil to β-sheet. This property is beneficial for
gel formation and can be useful for biomaterial applications (Zhu et al. 1998).
As previously discussed in the gene sequence information, the repetitive regions of the
ser genes (ser1, ser2, and ser3) from B. mori sequences have been shown to have high
contents of β-sheet formation. The high number of repetitive regions makes it possible to
increase the hydrophilic property of sericin protein. However, ser3 protein, which has a
lower content of β-sheet formation than ser1 protein, was reported to be more hydrophilic
than ser1 protein in a hydrophobicity prediction study (Garel et al. 1997; Takasu et al. 2007).
This data suggests that the prediction method used may not be directly applicable to evalu-
ate sericin protein properties. Therefore, to characterize sericin property, several techniques
may be needed to collect the information that would be required for finding new applications.
1.8.1.2 Gelation
The gelation property of sericin has been observed under various conditions, depending on
sericin solution concentration, temperature, and pH. The study of mulberry sericin from
B. mori has revealed that gelation formed rapidly at a high concentration of sericin solu-
tion. The gelation rate was elevated at a high temperature (40 °C) and decreased at a lower
temperature. Gel setting time was faster at pH 6 and became slower at a higher pH. During
the gelation process, the strength of sericin increased, whereas the surface tension
decreased. The secondary structure of sericin in the gelation process changed from random
coil to β-sheet structure (Zhu et al. 1998; Zhu et al. 1995). This evidence suggests that
sericin gelation is a thermoreversible process.
The property of sericin gelation was applied to the sericin protein as a biomaterial
crosslinked with various types of polymers, such as biopolymers (polysaccharides; cellu-
lose) (Wang et al. 2017), synthetic polymer (polyvinyl alcohol) (Aramwit et al. 2010b), or
self-assembled (hydrogel) (Zhang et al. 2019). The network crosslinking between sericin
and polymers was generally formed by covalent bonding at the polar functional groups of
sericin amino acids (hydroxyl, carboxyl, and amino). The crosslinking could be produced
by chemical and physical techniques. Chemical crosslinks were performed using agents
such as glutaraldehyde (Nayak et al. 2012; Wang et al. 2017) and genipin (Aramwit et al.
2013, 2010; Wang et al. 2015). For physical crosslinks, ultraviolet (UV) light was used for
photo-crosslinking bonds (Qi et al. 2018). These results demonstrated that sericin is capa-
ble of forming a gel with various polymers and several crosslinking techniques.
Gelation was also reported in non-mulberry sericin, A. mylitta. Similar to mulberry
sericin, gel formation was observed in the crosslinking between A. mylitta sericin and poly-
mers. This non-mulberry sericin was bonded with polyvinyl alcohol via a glutaraldehyde
crosslinking agent (Mandal et al. 2011). Likewise, the natural polymer (cellulose) was
reported to crosslink with sericin by the dual crosslinking agents, glutaraldehyde, and alu-
minum chloride (Nayak et al. 2014). Therefore, sericin from all sources could be efficiently
used as a biomaterial application for future medicines and cosmetics.
1.8.1.3 Thermal Stability

Thermogravimetric analysis tests the mass stability of sericin according to time and tem-
perature change. Sericins extracted from mulberry (B. mori) and non-mulberry (A. mylitta,
A. assamensis, and S. ricini) silkworms have been studied for its stability as related to tem-
perature. The non-mulberry sericins were reported to present more stability than mulberry
sericin. The highest stability was for sericin from S. ricini (Figure 1.11) (Sahu et al. 2016).
This property suggests that sericins from different species have different structures and
properties.
191 Amide I
1656.6
Amide II
(A) 1532
Amide III
192.754 (B)
1248
Absorb arce
CD (mde g)
191 (C) (A)

(D)
195.303 (B)
204
194
(C)
204.307
200.948
(D)
200.555
200 220 240 260 280 900 1200 1500 1800
–1
Wavelength (nm) Wavelength (cm )
(a) (b)
100
90
Mass (96)
80
70 (A)
(B)
60 (C)
(D)
50
50 100 150 200 250 300 350
Temperature (°C)
(c)
Figure 1.11 (a) Circular dichroism (CD) spectra of 0.1% w/v sericin solution from cocoons of different
species: (A) A. mylitta, (B) A. assamensis, (C) S. ricini, (D) Bombyx mori; (b) FTIR spectrum of sericin
powders from the various species: (A) A. mylitta, (B) A. assamensis, (C) S. ricini, (D) B. mori; (c)
thermogravimetric analysis (TGA) curves of lyophilized sericin powders of (A) A. mylitta, (B) A. assma, (C)
S. ricini, (D) B. mori. Source: Reprinted with permission from Sahu et al. (2016). Licensed under CC BY 4.0.
1.8.1.4 Ultraviolet (UV) protection

Sericin has shown the ability to protect cells from UV radiation. The study of the photopro-
tective properties of B.mori reported that sericin was effective in reducing skin oxidative
stress (Zhaorigetu et al. 2003), inhibiting UVB-induced apoptosis (Dash et al. 2008), and
absorbing UVC radiation (Kiro et al. 2017). Non-mulberry sericin from A. assamensis and
S. ricini was reported to increase cell viability against both UVA and UVB more than B. mori
sericin (Kumar et al. 2018). The A. assamensis sericin enhanced collagen production from
both UVA and UVB radiation, while the B. mori sericin enhanced protection only from UVA
(Kumar and Mandal 2019). This information seems to indicate that non-mulberry sericins
have photoprotective properties that are better than those of mulberry sericin.
1.8.1.5 Adhesion Properties and Electrostatic Interaction

The adhesion property is important for silkworm in its developmental stages, especially
during the cocoon stage. The adhesion property of sericin is beneficial in cementing cocoon
scaffolding and attaching the cocoon to the tree branch by floss or peduncle. The study of
crude ser2 proteins extracted from the anterior MSG showed that the tensile strength
needed to detach the adhesive from wooden surfaces was about 120 ± 30 N/cm2. The adher-
ence strength was higher than starch glue (42 ± 20 N/cm2) but less than bone glue
(502 ± 132 N/cm2). The adhesion property was facilitated by the high contents of charged
amino acid in ser2 proteins, which provide the electrostatic interactions between the
wooden surface and ser2 proteins (Kludkiewicz et al. 2009).
1.8.2 Biochemical Activity

Sericin has had several biochemical activities beneficial for medical applications as
discussed below.
1.8.2.1 Anti-tyrosinase Activity

Tyrosinase is an enzyme that plays a major role in melanin synthesis, which plays a key
role in cell protection from UV damage (Cichorek et al. 2013), and it is also a key enzyme
in melanogenesis on the skin (Schallreuter et al. 2008). Sericin anti-tyrosinase activity was
investigated in various studies by in vitro assay using mushroom tyrosinase. The mulberry
silkworm, B. mori, was subjected to isolate sericin protein for anti-tyrosinase activity assay.
The sericin collected by the heat isolation method had a 50% inhibitory concentration
(IC50) of tyrosine activity observed at a concentration of 10 mg/ml (Kato et al. 1998; Wu
et al. 2008). Sericin from urea extraction revealed the highest anti-tyrosinase activity
among other methods (including autoclave, acidic, and alkaline extraction methods)
(Aramwit et al. 2010a). The sericin isolated by the autoclave method (high heat and high
pressure) revealed an IC50 of 1–7 mg/ml depending on the silkworm strain in the study
(Aramwit et al. 2010a; Manosroi et al. 2010). Sericin from the acidic extraction method
showed some activity against tyrosinase (Aramwit et al. 2010a). Two reports revealed no
inhibitory activity from sericin extracted by the alkaline method (Aramwit et al. 2010a;
Kumar and Mandal 2019). In contrast, there is a report that revealed that the sericin from
the alkaline extraction method had IC50 values of 3–19 mg/ml from various silkworm
strains (Manosroi et al. 2010). This suggests that different factors of sericin extraction
methods and silkworm strains affect sericin protein in tyrosinase inhibitory activity. The
alkali extraction method might interfere with the B. mori sericin anti-tyrosinase activity.
However, a recent study of sericin isolated from the cocoon of non-mulberry silkworms
reported that the sericin isolated by an alkali extraction method from A. assamensis and
S. ricini had anti-tyrosinase activity with IC50 values of 6 and 10 mg/ml, respectively (Kumar
and Mandal 2019). This information shows that the sericin proteins are different in their
biological properties with each species. The evidence of this is supported by a report of
B. mori sericin extracted from several strains with different silkworm diets. The results
showed that the diet influenced the sericin property related to anti-tyrosinase activity
(Chlapanidas et al. 2013). Additionally, the B. mori sericin extracted from different colors of
the cocoon (flavonoids and carotenoids) showed that the color of the cocoon is associated
with an increase of the inhibitory effect of tyrosinase. The elimination of cocoon color
from sericin extraction reduced the anti-tyrosinase activity (Aramwit et al. 2010a). There
are many factors of sericin anti-tyrosinase activity including genes, species, extraction
methods, and cocoon colors.
1.8.2.2 Anti-elastase Activity

Elastase is a proteolytic enzyme that functions in the degradation of the elastin and conse-
quently in the skin losing elasticity. The expression of elastase protein can be induced by
UV radiation (Suganuma et al. 2010). Sericin isolated from various strains of B. mori
cocoons first had its anti-elastase activity discovered by Chlapanidas et al. in 2013
(Figures 1.12 and 1.13) (Chlapanidas et al. 2013). Non-mulberry, Antheraea spp. (tasar),
sericin revealed anti-elastase activity. Interestingly, tasar sericin extracted from the waste
products of the silk industry also retained anti-elastase activity (Jena et al. 2018). This anti-
elastase property of sericin is beneficial in sun protection. Therefore, sericin was proposed
to be used for application in cosmetic products.
1.8.2.3 Antioxidant Activity

Sericin isolated from the cocoon of B. mori has shown antioxidant properties when meas-
ured using 1,1-diphenyl-2-picrylhydrazyl, chemiluminescence, oxygen radical absorbance
capacity, electron spin resonance, and other techniques. The sericin extracted from high
pigment cocoon strains (yellow–green cocoon) revealed higher anti-oxidative activity than
low pigment cocoons (white cocoon) (Takechi et al. 2014). Previous information has shown
that the sericin protein extracts have different sizes for the cocoons of various color strains
(Aramwit et al. 2010b). The chemical is accumulated in the sericin layer (Ma et al. 2016).
Moreover, chemicals such as flavonoids have been reported to have antioxidant properties
(Heim et al. 2002).
An antioxidant assay using a skin fibroblast cell line (AH927) by pre-incubated sericin
before hydrogen peroxide-stimulated oxidative stress showed antioxidant properties in
sericins extracted from the cocoons of both mulberry (B. mori) and non-mulberry
Nistari
mod. Daizo
Arancio Coin
Nistari FL Oro 208
FL
Nistari Rosa Sejaku
Oro 208 Green BG
Verde Ovale 201 A FL Treotto
FL Rosa
Verde Oro Gigante
R3G SA48LB
Ovale
ADPR FL Han-Han PK12FL

AP
ADPR Romagna G133

Orgosolo
Figure 1.12 Image of 24 Bombyx mori cocoon strains fed with artificial and/or mulberry leaf diets.
Source: Reprinted with permission from Chlapanidas et al. (2013). © 2013 Elsevier.
80
Sample Mean KCL St. dev. KCL Mean Vmax St. dev. Vmax
201 A 13.109 4.326 1.275 0.11 70

ADPR 16.252 8.796 1.201 0.206
ADPR (FL) 13.032 3.655 1.162 0.107
60
AP 17.313 7.891 1.229 0.225
Arancio 20.903 6.211 1.527 0.249
Daizo 50
Mean activity (%)
25.21 9.282 1.376 0.244

G133 14.011 4.397 1.179 0.113
Han-Han 15.159 4.237 1.314 0.108 40
Nistari (FL) 24.522 14.403 1.463 0.318
Orgosolo 19.149 7.174 1.005 0.13 30
Oro 208 20.455 6.23 1.307 0.188
Oro gigante 27.084 18.583 1.514 0.386 20
PK12 (FL) 38.495 32.269 1.681 0.627
R3G 20.382 11.089 1.417 0.277
10
Romagna 43.763 25.109 1.536 0.527
SA48LB 11.542 3.478 1.17 0.091
23.411 0
Sejaku green BG 12.909 1.404 0.228
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Treotto rosa (FL) 47.178 L)

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47.702 1.765 0.862

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AD DP
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3.525 1.148 0.088

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Verde ovale (FL) 19.431 11.688 1.261 0.248

e
k
rd
ea
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Figure 1.13 Anti-elastase activity of sericin samples as a function of the strain and diet. Source: Reprinted with permission
from Chlapanidas et al. (2013). © 2013 Elsevier.
silkworms (A. mylitta) (Dash et al. 2008). The sericin extraction method also affected the
antioxidant activity. In B. mori sericin, the highest antioxidant activity was for sericin iso-
lated by the autoclaving method and the lowest activity was for sericin isolated by the acidic
method (Kumar and Mandal 2017). Unlike mulberry sericin, A. assamensis and S. ricini
showed that the conventional method resulted in the highest observed antioxidant activity
(Kumar and Mandal 2017). In addition, the sericin obtained from waste products from the
Antheraea spp. (tasar) silk industry also retained its antioxidant activity (Jena et al. 2018).
Not only the in vitro assay but also the in vivo experiment with rats orally treated with
sericin showed antioxidant activity in the rat brain homogenate (Banagozar Mohammadi
et al. 2019). These results might be beneficial for sericin applications in pharmaceuticals in
the future. Moreover, the advantageous sericin anti-oxidative stress property was used for
the cryoprotection of several cell types, such as human hepatocytes (Miyamoto et al. 2010),
adipose tissue-derived stem cells (Miyamoto et al. 2012), islet cells (Ohnishi et al. 2012),
bovine embryonic cells (Isobe et al. 2013), and buffalo spermatozoa (Kumar et al. 2015).
1.8.2.4 Anti-lipid Peroxidation Activity

Sericin protein was first investigated for anti-lipid peroxidation activity in 1998 by Kato
et al. In B. mori sericin, cocoon sericin extracted by heating showed inhibitory activity of
lipid peroxidation by thiobarbituric acid reactive substances (TBARS) and conjugated
diene assays during an in vitro test with rat brain homogenization (Kato et al. 1998). A simi-
lar observation of anti-lipid peroxidation has been observed from rats with oral treatment
with sericin. The rat brain homogenization showed that sericin reduced the activity of lipid
peroxidation by TBARS assay (Banagozar Mohammadi et al. 2019). Non-mulberry sericins
from A. assamensis and S. ricini have also been reported to have activity against lipid per-
oxidation. The activity against lipid peroxidation has been found to be 75–90% depending
on the sericin concentration. However, the extraction methods, including autoclaving and
alkali, did not significantly affect the anti-lipid peroxidation activity (Kumar and Mandal
2017). Not only the sericin from cocoon has the anti-lipid peroxidation activity, but also the
sericins obtained from waste products from the Antheraea spp. (tasar) silk industry also
retain their anti-lipid peroxidation activity (Jena et al. 2018). The anti-lipid peroxidation
activity by sericin could be found in both mulberry and non-mulberry sericin. The activity
is maintained in several sources of sericin, such as cocoon and waste products from the silk
industry. However, the extraction method affected its activity.
1.8.3 Biological Activity

1.8.3.1 Anti-inflammatory Activity
The anti-inflammatory activity by B. mori sericin has been reported in several studies. In an
experiment of sericin treatment in rat wounds, it was revealed that sericin initially induced
the activity of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), and
interleukin-1β (IL-1β). However, after long-term treatment for seven days, the inflammation
did not progress. In this study, sericin induced inflammation at the starting point of treat-
ment but it did not accelerate the progression of wound inflammation (Aramwit et al. 2009).
An in vivo acute inflammation model of carrageenan-induced paw edema showed that
sericin at a high concentration (0.080 mg/ml) significantly inhibited the inflammation
induced by carrageenan (Figure 1.14) (Aramwit et al. 2013). The histopathological changes
of the rat tissues indicated that there was less cellular infiltration in the dermal layer of
indomethacin-treated (positive control) and sericin-treated rats, while water-treated tissues
showed a massive cellular infiltration in the dermal layer (Figure 1.15) (Aramwit et al. 2013).
120
1h 2h 3h 4h
100 5h 6h
80
% Inhibition
60
40
20
0
Water Sericin Sericin Sericin Sericin Sericin Acetone 1% IND
0.080 0.040 0.020 0.010 0.004
mg/ml mg/ml mg/ml mg/ml mg/ml
Figure 1.14 Percentage of edema inhibition, induced by carrageenan injection, from sericin at
different concentrations at various time points. Source: Reprinted with permission from Aramwit
et al. (2013). © 2013 SAGE Publications.
(a) (b)
(c) (d)
Figure 1.15 Histological changes in rat tissue (×64) (a) normal histological structure of the
epidermal and dermal layers with no obvious cellular infiltration, (b) water-treated tissues followed
by carrageenan injection with massive cellular infiltration in the dermal layer, (c) indomethacin-
treated tissues followed by carrageenan injection show less cellular infiltration in the dermal layer
compared with the water-treated tissues, (d) sericin-treated (0.080 mg/ml) tissues followed by
carrageenan injection shows almost intact dermis with little cellular infiltration. Source: Reprinted
with permission from Aramwit et al. (2013).
Moreover, sericin was reported to be effective for suppressing the inflammatory media-
tors, cyclooxygenase-2 enzyme and nitric oxide production (Aramwit et al. 2013).
Additionally, the effect of sericin increased anti-inflammatory cytokine expression, includ-
ing IL-4, IL-10, and transforming growth factor-β, and reduced the production of the aller-
gic chemokine ligands 8 (CCL8) and CCL18 (Aramwit et al. 2018). This information
suggests that the effect of sericin interferes with several mechanisms related to reducing
inflammation. The effect of sericin on the inflammation of the neurological systems in
animal models showed that sericin decreased cytokine expression, including the nuclear
factor kappa-light-chain-enhancer of activated B cells, TNF-α, and IL-1β proteins in the
brains of the mouse model (Banagozar Mohammadi et al. 2019). This anti-inflammatory
activity of sericin could be used for several applications such as wound healing (Aramwit
et al. 2013), nanomicelles for tumor treatment (Deng et al. 2019), or nanoparticles for drug
delivery (Suktham et al. 2018; Yalcin et al. 2019).
1.8.3.2 Anti-tumor Activity

Sericin has been found to be active against several anti-tumor cells. The B. mori sericin
suppressed several carcinogeneses such as colon and skin tumors. The inhibitory activity
varies depending on the type of cancer cells. In colon tumors, sericin was reported to have
the ability to reduce colon tumors by reducing colonic 8-hydroxydeoxyguanosine (oxidative
stress in colon cancer) and 4-hydroxynonenal (inhibits cell proliferation). Furthermore,
sericin was induced by nitric oxide synthase protein to kill tumor cells (Zhaorigetu et al.
2001). In skin cancer, sericin has a strong anti-tyrosinase activity, which is a key enzyme in
600
500
400
Collagen (mcg/ml)
300
200
100
0
8 16 24 32 40 60 80 100 200 400 600 800 1000
SS concentration (µg/ml)
Figure 1.16 Collagen type I production in fibroblast cell line L929 when various sericin
concentrations were added into the culture medium for 24 h to make the final concentration of
sericin in each well 8–1000 μg/ml. Δ is acid-degraded sericin, ο is alkali-degraded sericin, • is
heat-degraded sericin, and ▴ is urea-extracted sericin. Source: Reprinted with permission from
Aramwit et al. (2010b). Licensed under CC BY 3.0.
melanogenesis (Aramwit et al. 2018; Kumar and Mandal 2019). Sericin reduced skin
tumors from UV radiation by antioxidant activity to reduce oxidative stress, decrease
cyclooxygenase-2, and lower cell proliferation on the skin (Zhaorigetu et al. 2003). In addi-
tion, sericin downregulated the expression of a melanogenesis regulatory gene, microph-
thalmia-associated transcription factor, in melanocytes (Aramwit et al. 2018). By this
information, it is shown that sericin is effective against tumor cells by attacking several
melanogenesis involvement proteins.
Non-mulberry sericins from A. assamensis sericin and S. ricini have also shown anti-tumor
activity via anti-tyrosinase secretion. Interestingly, their activity is more effective than mul-
berry sericin (Kumar and Mandal 2019). In in vitro anti-tumor testing, non-mulberry sericin
actively destroyed human squamous carcinoma (A431) and human tongue carcinoma (SAS)
cells. It has been reported that sericin from both mulberry and non-mulberry extractions killed
the cancer cells by upregulating the tumor suppressor gene, p53 (Kumar and Mandal 2019).
1.8.3.3 Inducing Collagen Production

Sericin has been reported to induce fibroblast cell proliferation and collagen production.
It has been reported that sericin induces fibroblast cell proliferation (Aramwit et al. 2009).
Heat Acid
120 120
100 100
Biofilm formation(%)
80 80
60 60
40 40
20 20
0 0
Negative 12.5 25 50 100 chlorhexidine Negative 12.5 25 50 100 chlorhexidine
(a) control Sericin mg ml (b) control Sericin mg ml
Alkali Urea
120 120
100 100
80 80
60 60
40 40
20 20
0 0
control Sericin mg ml control Sericin mg ml
(c) (d)
Figure 1.17 Biofilm formation percentage of Streptococcus mutans strains (ATCC25175 (black bar)
and UA159 (grey bar)) when grown in the presence of (a) heat-extracted sericin, (b) acid-extracted
sericin, (c) alkali-extracted sericin, and (d) urea-extracted sericin (12.5, 25, 50, and 100 mg/ml),
brain–heart infusion medium in the absence of sericin (negative control), and 1.2 mg/ml
chlorhexidine (positive control) at 37 °C for 24 h. Source: Reprinted with permission from Aramwit
et al. (2020). © 2020 MA Healthcare Ltd.)
Sericin isolated from four extraction methods (heat, urea, acid, and alkali) induced col-
lagen production depending on its concentration (Figure 1.16). However, the heat
extraction method had the highest activation of collagen synthesis (Aramwit et al.
2010b). The effectiveness of collagen production is related to the amino acid composition
of sericin. The various strains of silkworms revealed different amino acid compositions.
The high contents of methionine and cysteine residues in sericin protein are the promot-
ing factor for collagen production (Aramwit et al. 2009). On the other hand, non-mulberry
sericins from A. assamensis and S. ricini reportedly had a protective effect from collagen
degradation induced by UV radiation (Kumar and Mandal 2019). Because of this prop-
erty, sericin was used for biomaterial and cosmetic applications, especially skin treat-
ment (Akturk et al. 2011; Aramwit et al. 2015; Bakhsheshi-Rad et al. 2020; Tyeb
et al. 2020).
1.8.3.4 Antibacterial Activity

Sericin has been tested for its antibacterial activity against Escherichia coli,
Staphylococcus aureus, and Pseudomonas aeruginosa. Mulberry, (B. Mori), sericin
Heat-extracted Acid-extracted Alkali-extracted Urea-extracted

sericin sericin sericin sericin
Negative
control
12.5 mg/ml
sericin
25 mg/ml
sericin
50 mg/ml
sericin
100 mg/ml N/A

sericin
1.2 mg/ml N/A N/A N/A N/A

chlorhexidine
Figure 1.18 SEM images of ATCC25175 of Streptococcus mutans strains when grown in the
presence of heat-extracted sericin, acid-extracted sericin, alkali-extracted sericin, and urea-
extracted sericin (12.5, 25, 50, and 100 mg/ml), brain–heart infusion medium (negative control), and
1.2 mg/ml. Source: Reprinted with permission from Aramwit et al. (2020).
Heat Acid
350 350
300 300
Bacterial viability (%)

250 250
200 200
150 150
100 100
50 50
0 0
(a) (b)
Alkali Urea
350 350
300 300
250 Bacterial viability (%) 250
200 200
150 150
100 100
50 50
0 0
(c) (d)
Figure 1.19 Viability percentage of Streptococcus mutans strains (ATCC25175 [black bar] and
UA159 [grey bar]) in the biofilms after treatment with (a) heat-extracted sericin, (b) acid-extracted
sericin, (c) alkaline-extracted sericin, and (d) urea-extracted sericin (12.5, 25, 50, and 100 mg/ml),
brain–heart infusion medium in the absence of sericin (negative control), and 1.2 mg/ml
chlorhexidine (positive control) at 37 °C for 24 h. Source: Reprinted with permission from Aramwit
et al. (2020). © 2020 MA Healthcare Ltd.
caused E. coli cell membrane damage (Xue et al. 2016). The purity and extraction
method of sericin affected its antibacterial properties. The commercially available pure
sericin is active against S. aureus similar to antibiotic (penicillin/streptomycin) while
having very low activity against P. aeruginosa and S. aureus. Cocoon sericin from auto-
claving preparation slightly affected S. aureus but did not affect both E. coli and P. aer-
uginosa (Rocha et al. 2017). Antibacterial activity may not be the main property in both
mulberry and non-mulberry sericin, as sericin has many other bioactivity properties
for medical applications. Therefore, sericin is useful in combination with other anti-
bacterial bioactive molecules for enhancing its activity and advancing biomaterial
properties. The biomaterials developed from sericin obtained from both mulberry
(B. mori) and non-mulberry (A. mylitta and S. ricini) sericins have been combined with
other biopolymers such as chitosan nanofiber or film (Sapru et al. 2017; Shah et al.
2019; Zhao et al. 2014), or chemical agents such as silver nanoparticles (He et al. 2017;
Muhammad Tahir et al. 2020; Chaisabai et al. 2018), zinc oxide nanoparticles, and anti-
biofilm titanium (Ghensi et al. 2019) to further enhance the antibacterial and other
biological properties.
Besides antibacterial activity, sericin has been found to inhibit biofilm formation
(Aramwit et al. 2020). However, the extraction method affects this activity. It was found
that urea-extracted sericin showed the highest potential anti-biofilm activity for
Heat-extracted Acid-extracted Alkali-extracted Urea-extracted

sericin sericin sericin sericin
Negative
control
12.5 mg/ml
sericin
25 mg/ml
sericin
50 mg/ml
sericin
100 mg/ml
sericin N/A
1.2 mg/ml
chlorhexidine
Figure 1.20 Scanning electron microscope (SEM) images of ATCC25175 of Streptococcus mutans
strains in the biofilms after treatment with heat-extracted sericin, acid-extracted sericin, alkaline-
extracted sericin, and urea-extracted sericin (12.5, 25, 50, and 100 mg/ml), brain–heart infusion
medium (negative control), and 1.2 mg/ml chlorhexidine (positive control) at 37 °C for 24 h.
Source: Reprinted with permission from Aramwit et al. (2020).
Streptococcus mutans in terms of both inhibition and disruption effects, compared with
sericins extracted by heat, acids, or alkaline solutions (Figures 1.17–1.20). The heat-
extracted and acid-extracted sericins were found to reduce the biofilm formation dose-
dependently, while the alkaline-extracted sericin did not show either an inhibition or a
disruption effect on the bacterial biofilm. The urea-extracted sericin also killed the bac-
teria residing within the biofilm, possibly due to its modified structure, which may
destabilize the bacterial cell wall, leading to membrane disintegration and, finally,
cell death.
From these data, it can be inferred that the sericin structure is quite complicated and var-
ies from the nature of the silk strain and extraction methods, among other factors, which
results in various biological properties. Due to these variations, the type of silkworm and
processing method are the key factors for sericin selection.
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of the body, and then to introduce a tube, or resort to some similar
expedient for preventing cicatricial contraction, and perhaps even
subsequent closure. Silver tubes were formerly used, whose
openings were corked and kept closed when the tube was not in
use. In consequence of this foreign body with the irritation it
produced there was always more or less leakage and discomfort.
The more recent methods have been devised with an intent of
making a tunnel rather than a direct opening, through which, as
needed, a soft rubber tube may be introduced, whose walls shall
collapse at other times and close themselves, if necessary, with a
little assistance, by pressure, thus preventing leakage. Sometimes it
is possible to attain this ideal. At other times a rubber tube is worn a
greater part at least of the twenty-four hours.
Fig. 529 Fig. 530
Gastrostomy: Witzel’s method. Tube in Gastrostomy: Witzel’s method. Tube in

position; sutures ready to tie. position; sutures ready to close
(Richardson.) abdominal wall. (Richardson.)
Fig. 531
Gastrostomy by Frank’s method: cone of stomach stitched into the peritoneal

wound. (Richardson.)
All operative methods include fixation and consequent adhesion of

the anterior stomach wall to the parietal peritoneum, just below the
border of the ribs. Of the many methods employed the following will
be described, most of which can be easily appreciated in diagram:
Figs. 529 and 530 illustrate, for instance, Witzel’s method, where a
sterile, soft rubber catheter is infolded in the stomach wall, and finally
passed into its cavity through the smallest opening that may suffice
for the purpose, after which the outer layer of the stomach is
completely closed over it. The stomach itself is stitched to the deep
margins of the external wound, and these are then closed without
drainage. If everything has been neatly done feeding may be begun
within a few hours. Care should be exercised about passing into a
stomach which has long been without much food a quantity which
may disturb it, or of a quality which may distress it. A procedure very
much like Witzel’s is that described by Marwedel, who first sews the
stomach to the abdominal wound after drawing it partly into the
wound, in order to afford sufficient working material, and then infolds
the tube and inserts its lower end through a small opening. This is
perhaps preferable, since the stomach being so fastened up at once
there is no possibility of leakage into the abdomen.
Figs. 531, 532 and 533 illustrate Frank’s method, where the
stomach is pulled up through a sufficiently long incision and drawn
out into a cone, whose apex is then brought out through a second
small incision, parallel to the first and at a distance of an inch or so
from it. Here an actual opening is made into the stomach, while the
cone is fastened to the skin here and to the peritoneum through the
other opening, which is then completely closed. This method cannot
be applied to a contracted stomach.
Fig. 532 Fig. 533
Gastrostomy by Frank’s method: cone of Gastrostomy by Frank’s method: suture

stomach pushed through the second of abdominal wound; stomach stitched in
skin incision. (Richardson.) the skin incision. (Richardson.)
Cardiospasm.—Operation for this condition consists essentially in

a gastrotomy as above, the opening being made
sufficiently near to the cardia in order that either with finger or with
suitable dilating instrument passed upward from below, the
contracted cardiac orifice may be stretched, or, if necessary, nicked
at several points, and then forcibly dilated, in this latter procedure
great care should be given that stress be distributed as much as
possible. If it be practicable to introduce any dilating instrument a
four-bladed uterine dilator would probably be ideal for the purpose.
Operations for Pyloric Stenosis.—Among the earliest
suggestions of a method of
pylorodiosis was that of Loreta, who opened the stomach near the
pyloric end and deliberately introduced through the constricted
pyloric ring a dilating instrument, fashioned much after the shape of
the ordinary glove stretcher, which, in fact, might be used for such a
purpose should emergency require. The operation is simple and but
slightly dangerous, but it was found that strictures here as elsewhere
tend to contract, even after forcible dilatation, and that the method,
while temporarily successful, was but seldom permanently so. It was
applicable only to the cicatricial, i. e., the non-malignant cases.
A plastic method was then suggested independently by Heinecke
and Mikulicz, with which their names are often connected and which
is referred to as pyloroplasty. It consists essentially in making a
buttonhole incision in one direction and then closing it in the
opposite, as illustrated in Figs. 534, 535 and 536.
Fig. 534
Linear pyloroplasty. Seat and length of cut. (Richardson.)

Fig. 535
Linear pyloroplasty. Appearance of cut sutured transversely. Two more sutures to

be applied. (Richardson.)
Fig. 536
Pyloroplasty. Shape of cut when more than a linear incision is desirable.

(Richardson.)
When cicatricial tissue is not too dense, and the parts not
infiltrated, it has given satisfactory results. Even here it has been
found to be frequently reduced in size by subsequent contraction,
and the method suggested by Finney is more serviceable.
Fig. 537 Fig. 538
Finney’s pyloroplasty: posterior suture. Finney’s pyloroplasty: anterior sutures

(Bergmann.) drawn aside; incision made.
(Bergmann.)
Fig. 539 Fig. 540
Finney’s pyloroplasty: posterior suture of Finney’s pyloroplasty: anterior stitches

mucous membrane. (Bergmann.) inserted but not tied. (Bergmann.)
Finney’s pyloroplasty consists in making an anastomotic opening

between the pyloric end of the stomach and the first part of the
duodenum, and will be best appreciated from the accompanying
illustrations (Figs. 537, 538, 539, 540 and 541).
Fig. 541
Finney’s pyloroplasty: anterior suture completed. (Bergmann.)
The opening can be made as extensively as desired, and it is not

easy to see how it can be subsequently reduced to a degree
disadvantageous to the patient.
Gastro-enterostomy may be needed in non-malignant cases,
because of fixation and the impossibility of bringing the pyloric end of
the stomach out sufficiently to make operation feasible. It will be
required in cases of cancer when pylorectomy is not indicated. The
method of making gastro-enterostomy will be described later.
Operations for Dilatation of the Stomach.—Gastroplication
consists of taking a
number of “tucks” in the stomach wall and thus reducing its capacity.
The purpose and the method of the operation will be appreciated by
the accompanying illustrations. These operations are mainly
indicated, however, in the absence of pyloric stenosis, for if a free
opening be afforded from the dilated stomach into the upper bowel
the gastric enlargement will usually be spontaneously reduced (Figs.
542 and 547).
Gastropexy is a term applied to fixation of the stomach to the
anterior abdominal wall. It has been thus stitched up in a few cases
when greatly dilated or depressed into the lower abdomen. Fig. 548
illustrates the method. The stomach has also been suspended by
shortening the gastrohepatic and gastrophrenic ligaments, as
illustrated in Fig. 549.
Fig. 542 Fig. 543
Gastroplication. When the threads a a´, b b´ are drawn Sectional view to show
up a fold is formed. (Bircher.) result of operation.
Operations for Gastric Ulcer.—In dealing surgically with an ulcer
of the stomach the selection has to
be made between anastomosis and direct exposure of the stomach
wall with the performance of a gastrotomy (i. e., opening the
stomach) and then discovering the site of the ulcer, either treating it
with the actual cautery, the curette, or, preferably, when this general
method is adopted, completely excising the involved area and
bringing the margins of the wound thus made together with sutures,
which over the mucosa only may be of chromic gut. Should it seem
advisable to excise the entire thickness of the stomach wall it would
be better to suture in two layers, making the external one of thread
or silk, while the inner one may be made of reliable chromic catgut. If
this operation be attempted the incision into the stomach should be
made sufficiently large to permit of thorough exploration. Nothing
being found in the anterior wall, the gastrocolic omentum should be
opened and the entire stomach palpated between the operator’s
hands. Any suspiciously indurated spot on the posterior wall may
then be so manipulated as to be brought into view through the
anterior opening. Other surgeons besides myself have noted the
occurrence of serious hemorrhage, which, upon exposure, must
have come from small fissures or cracks in the mucous membrane.
In fact the lesion which may furnish a considerable amount of blood
may thus be so small and concealed as to be really difficult of
exposure. However, exploration should be made as thoroughly as
possible. The stomach having been opened and the ulcer found, it
should be treated by one of the above methods. If, on the other
hand, nothing be found the surgeon still has the measure of gastro-
enterostomy. Any ulcer, however, which is threatening perforation
can usually be recognized by the sense of touch alone, corroboration
being afforded by inspection. An ulcer which is recognized and found
to be favorably situated may be completely excised. It has been
found, however, that this ideal measure of local attack gives but little
better results than does the general procedure of gastro-
enterostomy, while, on the other hand, it is less satisfactory in some
respects and seems to be an equally if not more dangerous
procedure.
Fig. 544 Fig. 545
Surface view of the result. Sectional view of the

result when two folds
are turned in.
Fig. 546 Fig. 547
Gastroplication. (Brandt.) Sectional view of the

result.
The rationale of making an anastomotic opening between the

stomach and the upper end of the bowel is simply this: that thereby
the stomach is given a degree of physiological rest to which it has
long been a stranger, and that food may pass easily from the
stomach into the upper bowel without irritating or aggravating the
ulcerated portion, which is usually at the pyloric end. It should be
understood, then, that gastro-enterostomy, done for this purpose, is
simply a means of carrying out the universally applicable canon of
physiological rest for diseased organs or surfaces. The operation of
making this anastomosis will be described below.
Pylorectomy and Gastrectomy.—A complete removal of the
pyloric end of the stomach is
usually referred to as pylorectomy, while still more extensive
extirpation of portions of the stomach proper are spoken of as
gastrectomies. In a few instances it has been possible to practically
remove the entire stomach, this having first been done by Schlatter.
Such an operation would be spoken of as total gastrectomy. These
operations are done almost exclusively for removal of areas involved
in cancerous growth. Obviously the more extensive the growth the
greater the amount of stomach which should be removed. For some
reason as yet unknown cancer of the stomach rarely transgresses
the pyloric ring, and thus the first part of the duodenum usually
escapes involvement, even though the stomach be extensively
diseased. All these operations, therefore, include simply the removal
of a part terminating with the pyloric ring proper. It is seldom
necessary to take away any of the duodenum. Removal of the
pylorus may be also applicable in certain cases of benign strictures,
where the mere plastic operations would seem insufficient, as well
as in the cases of ulcers encroaching upon the pyloric ring itself.
For all of these operations the stomach is exposed through a
median incision, or, if a tumor presents distinctly upon the right side,
the incision may be made even far to the right and near the
semilunar line. Through an opening sufficiently liberal the stomach
and the movable part of the duodenum are withdrawn and carefully
examined. When the pylorus is so fastened by dense adhesions
within the abdomen that it cannot be withdrawn it is best to abstain
from this particular procedure, as the mechanical difficulties too
greatly enhance its dangers. Suitable clamps, whose blades are
protected with soft rubber, are essential in order that the duodenum
may be clamped beyond the line of its division, and that the stomach
as well may be fixed between their blades, for the double purpose of
controlling hemorrhage and preventing escape of contents. The
omentum along the involved part of the stomach should then be
carefully tied off, in a series of loops, before its vessels are cut, and
one should take great pains to hunt out enlarged lymph nodes and
include them in the area to be removed, or else make a separate
incision for those that cannot be thus extirpated. To leave lymph
nodes which are perceptibly involved in the cancerous process is to
invite the speediest possible return of the disease, even though the
operation should be successful. The upper and lower borders of the
stomach being thus freed, the surgeon is then at liberty to cut away
all the diseased portion, going at least an inch beyond the apparent
limit of the disease. There will result from any such operation two
divided ends of the alimentary canal, i. e., one, that of the divided
stomach, much larger than the other, which is the upper end of the
duodenum.
Fig. 548
Rovsing’s operation for gastroptosis: V, stomach; V₁, position of the stomach
before operation; U, urinary bladder; N, right kidney; A, B, C, silk sutures; x, x,
scarifications. (Bergmann.)
Two procedures are now open to the surgeon: He may entirely

close each of these openings with sutures and then make a posterior
gastro-enterostomy, making new openings for this purpose, and by
the common method described below, or he may reduce the size of
the stomach opening and endeavor to fit it to that of the duodenum in
such a way as to bring the two openings opposite each other, where
they are then approximated as in ordinary end-to-end resection of
the intestine. The earlier operation of Billroth and his followers was
made according to the latter plan. It has been found usually easier
and more successful to adopt the former method, as it is easier thus
to prevent leakage and consequent infection; that is, the majority of
operators would today probably completely close the stomach and
the duodenum, and proceed at once to make a posterior
gastrojejunostomy.
Fig. 549
Suspension of stomach by three rows of interrupted stitches through the

gastrohepatic and gastrophrenic ligaments: 1, 2, 3, single stitches of the three
rows. (Beyea.)
Fig. 550
Resection of the pylorus. Suture completed. (Richardson.)
Figs. 550, 551 and 552 give a fair idea of the procedure of end-to-
end reunion. The edges of the mucosa should be united with
chromic gut, the stitches being close to each other, to prevent
leakage and to control hemorrhage from small vessels. The external
sutures of silk or thread should be placed sufficiently deep to afford a
strong bond of union, and, at the same time, to escape the mucosa.
Some difficulty is met here, for the thin wall of the duodenum should
be attached to the thick wall of the stomach, but with care it can be
done. When the divided stomach end has been reduced or trimmed
off in such a way as to leave only a portion to be matched with the

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Sustainable Uses of Byproducts from

Silk Processing Narendra Reddy

Sustainable Food Systems from Agriculture to Industry

Sustainable Nanoscale Engineering: From Materials

Handbook of Grape Processing By-Products: Sustainable

The Uses of Idolatry Cavanaugh

Data Sovereignty: From the Digital Silk Road to the

Hydrocarbon Biorefinery: Sustainable Processing of

Sustainable Metal Extraction from Waste Streams Chauhan

Instrumentation And Control Systems Reddy

Narendra Reddy and Pornanong Aramwit

Print ISBN: 978-3-527-34786-5

Typesetting SPi Global, Chennai, India

1 Sericin: Structure and Properties 1

1.8.3.4 Antibacterial Activity 29

2.10.6 Blends with Poly(caprolactone) 65

3 Applications of Sericin 101

4 Non-silk Applications of Mulberry Plants 131

4.2 ­Medicinal Applications of Mulberry Plant Extracts 132

5 Pupae and Its Applications 157

6 Applications of Pupae Litter (excrement) 195

7.5.1 Carbonization of Waste Silk 221

Sericin: Structure and Properties

1.1 ­Type of Silk Sericin

1.2 ­Localization of Silk Sericin

Sustainable Uses of Byproducts from Silk Processing, First Edition.

15k V X2, 000 10 μ m 080705

1.3 ­Molecular Mass of Sericin

1.3.1 Middle Silk Gland Sericin

1.3.2 Mulberry Cocoon sericin

Molecular weight 75 000–78 000

1.3.3 Non-mulberry Cocoon and Peduncle Sericin

(a) (b) (c)

1.4 ­Layers of Sericin

1.5 ­Sericin Amino Acid Components

1.5.1 Silk Gland of Mulberry Sericin

1.5.2 Sericin from Mulberry Cocoons

Tokutake Gamo Teramoto

Tokutake Gamo Teramoto

Reference Aramwit et al. (2010)

1.5.3 Sericin from Non-mulberry Cocoons

Reference Dash Dash Dash Dash Yamada and

Species (strain) A. mylitta A. mylitta A. mylitta A. mylitta Cricula trifenestrata

Source Peduncle Cocoon Cocoon Cocoon Cocoon

Detail Peduncle Cocoon Sericin Cocoon Crude sericin

Name mol% mol% mol% mol% mol%

Alanine 4.80 6.01 2.95 6.01 4.90

B. mori A. mylitta A. assama S. ricini

Fresh (no degumming) Urea degummed Autoclave degummed

(g) (h) (i)

(j) (k) (l)

1.6 ­Sericin Gene

1.7 ­Sericin Structure

Amide I Amide II Amide III

1.8 ­Sericin Properties

1.8.1 Biophysical Properties

1.8.1.3 Thermal Stability

191 (C) (A)

1.8.1.4 Ultraviolet (UV) protection

4.2 Medicinal Applications of Mulberry Plant Extracts 132

1.1 Type of Silk Sericin

1.2 Localization of Silk Sericin

1.3 Molecular Mass of Sericin

1.4 Layers of Sericin

1.5 Sericin Amino Acid Components

1.6 Sericin Gene

1.7 Sericin Structure

1.8 Sericin Properties