Sustainable Uses of Byproducts from Silk Processing 3527347860, 9783527347865

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Sustainable Uses of Byproducts from Silk Processing

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

Authors Professor Narendra Reddy

Center for Incubation, Innovation, Research, and Consultancy Jyothy Institute of Technology Thataguni Post 560109 Bengaluru India Professor Pornanong Aramwit

Department of Pharmacy Practice Faculty of Pharmaceutical Sciences and Center of Excellence in Bioactive Resources for Innovative Clinical Applications Chulalongkorn University, Bangkok Thailand 10330 The Academy of Science The Royal Society of Thailand Dusit, Bangkok Thailand 10330 Cover

Cover Image: © Baona/iStock/Getty Images Plus/ Getty Images

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10  9  8  7  6  5  4  3  2  1

v

Contents

Preface  xi

1 1.1 1.2 1.3 1.3.1 1.3.2 1.3.3 1.4 1.5 1.5.1 1.5.2 1.5.3 1.6 1.7 1.8 1.8.1 1.8.1.1 1.8.1.2 1.8.1.3 1.8.1.4 1.8.1.5 1.8.2 1.8.2.1 1.8.2.2 1.8.2.3 1.8.2.4 1.8.3 1.8.3.1 1.8.3.2 1.8.3.3

Sericin: Structure and Properties  1 ­Type of Silk Sericin  1 ­Localization of Silk Sericin  1 ­Molecular Mass of Sericin  2 Middle Silk Gland Sericin  2 Mulberry Cocoon sericin  2 Non‐mulberry Cocoon and Peduncle Sericin  5 ­Layers of Sericin  6 ­Sericin Amino Acid Components  6 Silk Gland of Mulberry Sericin  6 Sericin from Mulberry Cocoons  8 Sericin from Non‐mulberry Cocoons  12 ­Sericin Gene  14 ­Sericin Structure  16 ­Sericin Properties  19 Biophysical Properties  19 Water Solubility  19 Gelation  20 Thermal Stability  20 Ultraviolet (UV) protection  21 Adhesion Properties and Electrostatic Interaction  22 Biochemical Activity  22 Anti‐tyrosinase Activity  22 Anti‐elastase Activity  23 Antioxidant Activity  23 Anti‐lipid Peroxidation Activity  25 Biological Activity  25 Anti‐inflammatory Activity  25 Anti‐tumor Activity  27 Inducing Collagen Production  28

vi

Contents

1.8.3.4 Antibacterial Activity  29 ­References  32 Processing Sericin  39 ­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 2 2.1

Contents

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 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.6.1 3.1.6.2 3.1.6.3 3.1.7 3.1.8 3.2 3.3 3.3.1 3.3.1.1 3.3.1.2 3.3.1.3 3.4 3.4.1 3.5

Applications of Sericin  101 ­Medical Application  101 Antioxidant Properties  101 Antibacterial Activity  102 Wound Healing  104 Antitumor Effect  105 Antidiabetic Potential  106 Metabolic Effects  107 Gastrointestinal Tract  107 Circulatory and Immune Systems  108 Lipid Metabolism and Obesity  109 Vehicle for Drug Delivery  109 Supplement in Culture Media and Cryopreservation  112 ­Cosmetic Applications  113 ­Biotechnology Applications  117 Tissue Engineering  117 Skin Tissue Repair with Sericin‐Based Materials  117 Sericin‐Based Materials for Bone and Cartilage Tissue Engineering  119 Sericin‐Based Biomaterials for Other Tissues  120 ­Miscellaneous Application  121 Sericin‐Coated Material as an Air filter  121 ­Conclusion  121 ­References  123

4 4.1

Non-silk Applications of Mulberry Plants  131 ­Introduction  131

vii

viii

Contents

4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.2.9

­Medicinal Applications of Mulberry Plant Extracts  132 Polysaccharides  132 Phenols and Flavanoids  135 Pectins and Lignins  140 Production of Paper  142 Fermentation and Biogas Production  144 Synthesis of Nanomaterials  144 Environmental Remediation  148 Composites  150 Superabsorbents  151 ­References  153

5 5.1 5.1.1 5.1.2 5.1.3 5.2 5.2.1 5.2.2 5.3 5.3.1 5.4 5.4.1 5.4.2 5.4.3 5.5

Pupae and Its Applications  157 ­Applications of Pupae Oil  157 Extraction of Pupae Oil  157 Medical Applications of Pupae Oil  159 Biodiesel from Pupae Oil  163 ­Proteins from Silkworm Pupae  163 Medical Application of Pupae Peptides  169 Nanoparticles from Pupae Proteins  174 ­Pupae as Animal Feed  176 Chitin and Chitosan from Silkworm Pupae  181 ­Miscellaneous Applications  184 Carbonization of Pupae  184 Production of Lipids  185 Anti‐diabetic Drug  188 ­Environmental Remediation  189 ­References  189

6 6.1 6.2 6.3 6.4 6.5 6.6

Applications of Pupae Litter (excrement)  195 ­Chlorophylline and its Derivatives  195 ­Applications of Proteins in Litter  197 ­Extracts from Litter  198 ­Environmental Remediation Using Litter  199 ­Conversion into Carbon  203 ­Nanoparticles from Litter  209 ­References  211

7

Converting Silk Wastes into Composites, Energy Storage Devices, and Other Value-Added Materials  213 ­Introduction  213 ­Composites from Silk Wastes  213 Reinforcement for Polypropylene (PP) Composites  214 ­Green Composites from Silk Wastes  219 ­Energy Applications  221

7.1 7.2 7.3 7.4 7.5

Contents

7.5.1 7.6 7.6.1 7.7 7.8 7.9

Carbonization of Waste Silk  221 ­Miscellaneous Applications  225 Extraction of Amino Acids and Bioactive Compounds  225 ­Fibers and Hydrogels  226 ­Environmental Remediation  227 ­Nutritious Food from Silkworm Eggs  229 ­References  229 Glossary  233 Index  237

ix

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 inevitably 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 carbohydrates, 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‐products and hope to promote the use of silkworms beyond the production of silk.

1

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 components in the cocoons of Lepidopteron insects. Sericin is a glue-like coating protein surrounded 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. trifenestrata 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-mulberry 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 polyacrylamide gel electrophoresis (SDS-PAGE). The diversity pattern of sericin was investigated 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 electrophoresis 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 sections, 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 temperature, pressure, urea, acid, and alkali solution. In 1980, Tokutake reported that sericin

Marker

Yellowish cocoon B

Yellowish cocoon A

Yellowish cocoon H

Yellowish cocoon U

Greenish cocoon B

Greenish cocoon A

Greenish cocoon H

Greenish cocoon U

White cocoon B

White cocoon A

White cocoon H

White cocoon U

Marker

1.3  ­Molecular Mass of Serici

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 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 gland. Source: Reprinted with permission from Gamo et al. (1977). © 1977 Elsevier.

MM2

A PM

P 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

3

4

1  Sericin: Structure and Properties Molecular

Origin Origin

weight

210 000–220 000 130 000 Molecular weight

75 000–78 000 67 000–68 000

220 000 210 000

53 000 36 000 34 000 32 000

130 000

22 000 Tracking dye (a)

Tracking dye

(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

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, respectively. 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. assamensis (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 nonmulberry cocoon sericin from S. ricini, A. assamensis, and A. mylitta. The autoclave technique 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 extraction 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).

5

6

1  Sericin: Structure and Properties

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 methods (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 studies 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 layers 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%, respectively (Sprague 1975). In 1977, Gamo et al. reported the amino acid composition of sericin

8

1  Sericin: Structure and Properties

from different sections of the MSG, including the middle (fraction: s-1), anterior (fraction: 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 exception is the anterior MSG fraction s-5, which secreted serine content of 16 mol%. This fraction (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 aspartic 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 component, on average 10%, instead of threonine residue. This data meant that sericin at different 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 methods, 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 commonly 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 methods. 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 extraction 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 different amino acid components from alkali extraction methods (Table 1.2). The alkaline extractions 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

Reference

Species (strain)

Yamada (1978)

B. mori (mandarina)

Tokutake (1980)

Gamo (1982)

Takasu (2002)

B. mori (shugetsu ×  Hosho)

B. mori

B. mori

Teramoto et al. 2004

Aramwit (2009)

B. mori

B. mori (chul 1/1)

B. mori (chul 3/2)

B. mori (chul 4/2)

Cocoon

Cocoon

Cocoon

Cocoon Autoclave

Source

Floss

Cocoon

Cocoon Cocoon

Detail

Floss sericin

Inner cocoon

Outer cocoon

Acid S×2 Sericin A precipitation (227 kDa) (250 kDa)

Sericin M Sericin P (400 kDa) (150 kDa)

Sericin

Autoclave

Autoclave

Name

mol%

mol%

mol%

mol%

mol%

mol%

mol%

mol%

Cocoon

mol%

Cocoon

mol%

Cocoon

Cocoon

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

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 asparagine

18.61

18.30

12.90

14.90

13.30

15.70

11.30

16.30

15.64

15.62

15.97

Trace

0.42

0.64

Trace

—

0.10

0.00

—

—

0.44

0.43

0.27

4.31

4.90

4.78

4.25

11.10

12.80

3.10

3.10

4.70

4.61

4.76

4.86

Cysteine Glutamic acid +  glutamine Glycine

4.98

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

Yamada (1978)

Reference

Species (strain)

B. mori (mandarina)

Tokutake (1980)

Gamo (1982)

Takasu (2002)

B. mori (shugetsu ×  Hosho)

B. mori

B. mori

Cocoon

Cocoon Cocoon Cocoon

Cocoon

Cocoon

Teramoto et al. 2004

Cocoon

Source

Floss

Detail

Sericin A Sericin M Sericin P S×2 Outer Acid Floss Inner sericin cocoon cocoon precipitation (227 kDa) (250 kDa) (400 kDa) (150 kDa) mol% mol% mol% mol% mol% mol% mol% mol%

Name

Aramwit (2009)

B. mori

B. mori B. mori (chul 1/1) (chul 3/2)

Cocoon

Cocoon

Sericin

Autoclave Autoclave

mol%

mol%

B. mori (chul 4/2) Cocoon

Cocoon

Autoclave mol%

mol%

1.30

1.00

1.15

1.11

1.99

1.40

0.50

0.90

1.60

2.89

2.07

6.00

5.40

1.80

1.00

2.70

2.35

2.51

2.78

0.11

0.11

—

—

—

—

—

3.39

0.57

0.18

0.42

0.47

0.69

0.40

0.40

0.20

0.70

0.40

0.28

0.39

0.36

0.51

0.56

0.69

—

—

0.60

1.30

0.70

0.54

0.62

0.71

36.80

39.00

35.40

33.20

34.20

33.63

34.50

33.84 8.34

0.90

Leucine

0.85

Lysine

1.89

2.26

Methionine

0.12

0.10

Phenylalanine

0.43

Proline

0.66

1.17

40.28

28.12

29.05

18.90

6.29

11.39

8.44

5.21

4.00

3.30

9.70

12.20

8.00

8.16

8.43

—

—

—

—

—

—

—

—

—

—

—

—

Tyrosine

4.09

3.28

3.39

4.10

0.10

0.70

4.00

4.60

2.90

3.45

3.64

3.47

Valine

3.46

2.67

2.91

3.34

1.20

0.70

3.20

3.90

3.30

2.88

3.04

2.92

Serine Threonine Tryptophan

Table 1.2  (Continued) Reference

Aramwit et al. (2010)

Species (strain)

B. mori (Chul 1/1)

Source

Cocoon

Detail (extraction method)

Autoclave

Urea

Acid

Alkali

Autoclave

Urea

Acid

Alkali

Autoclave

Urea

Acid

Alkali

mol%

mol%

mol%

mol%

mol%

mol%

mol%

mol%

mol%

mol%

mol%

mol%

Name Alanine Arginine Aspartic acid + asparagine

Cocoon

4.1

4.33

B. mori (Chul 3/2) Cocoon

3.72

Cocoon

4.21

Cocoon

4.45

Cocoon

B. mori (Chul 4/2) Cocoon

Cocoon

Cocoon

Cocoon

Cocoon

Cocoon

3.8

3.57

2.87

5.41

4.92

4.92

2.95

5.21

4.87

3.79

3.09

5.71

5.24

4.83

15.64

18.31

15.93

19.88

15.62

17.93

16

21.58

15.97

17.69

16.61

19.92

3.96

4.98

4.63

3.56

4.4

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 acid + glutamine

4.61

5.27

5.75

5.93

4.76

6.02

5.4

7.66

4.86

5.97

5.88

7.03 12.58

15.03

11.23

10.49

11.01

15.09

10.75

10.38

11.16

15.14

10.96

10.69

Histidine

Glycine

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

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

Lysine

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

8.3

5.56

Threonine Tryptophan

8.16

8.36

8.51

6.49

8.43

8.78

8.78

6.09

8.34

9.04

—

—

—

—

—

—

—

—

—

—

—

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

—

12

1  Sericin: Structure and Properties

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 threonine (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 (2006)

Dash (2006)

Dash (2007)

Dash (2008)

Yamada and Tsubouchi (2001)

Species (strain)

A. mylitta

A. mylitta

A. mylitta

A. mylitta

Cricula trifenestrata

Source

Peduncle

Cocoon

Cocoon

Cocoon

Cocoon

Detail

Peduncle sericin

Cocoon sericin

Sericin (70 kDa)

Cocoon sericin

Crude 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

—

—

—

—

2.60

Aspartic acid + asparagine

—

—

—

—

—

Glutamic acid + glutamine

Cysteine

11.16

5.70

5.98

5.70

1.50

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

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 different 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).

A. mylitta

A. assama

S. ricini

Degumming

Lyophil icorion

Sericin solution Degummed cocoon pieces

Urea method

Authoc time method

Cut cocoons

Cocoons

B. mori

Lyophilized 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).

13

1  Sericin: Structure and Properties Urea degummed

(b)

(c)

(e)

(f)

(h)

(g)

Autoclave degummed

Urea degummed

s. rkini

s. rkini

Fresh (no degumming)

s. rkini

(d)

Autoclave degummed

Urea degummed A. anama

A. anama

Fresh (no degumming)

A. anama

(a)

Autoclave degummed

A. mytitta

A. mytitta

A. mytitta

Fresh (no degumming)

Fresh (no degumming)

(i) Autoclave degummed

B. mori

B. mori

Urea degummed

B. mori

14

(j)

(k)

(l)

Figure 1.7  Scanning electron microscope (SEM) images of the cocoons of mulberry and nonmulberry 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

(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 alternative 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 silkworm 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 silkworm species appear to have different effects on their functions.

15

1  Sericin: Structure and Properties

The ser genes transcribe differently depending on the silkworm larval stages. The silkworm larval (instar) is developed in five stages, and then the mature stage is started to create 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 transcripts 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 purpose 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 spectroscopy (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

16

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

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 possibly 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 alkaline) has been studied by Kumar et al. (Figure 1.9) (Kumar and Mandal 2017). The secondary 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 compared to the sericin structure from the urea extraction method. Meanwhile, the conventional 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

220 kDa 160 kDa 120 kDa 100 kDa 90 kDa 80 kDa 70 kDa 60 kDa

220 kDa 160 kDa 120 kDa 100 kDa 90 kDa 80 kDa 70 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

C

D

E

F

(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).

17

1  Sericin: Structure and Properties

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 variable structures of sericin from different extraction methods may cause the different properties and biological activities of sericin proteins.

100

100 % Transmittance

80

1241

1241

1624 1650

60

40 1800

(a)

1600 1400 1200 Wavelength (cm–1)

100 80 60

1506–1520 1624

60

(b)

1600 1400 1200 Wavelength (cm–1)

1000

100

1241

1244

1650

20

(c)

1552–1515 1624 1650

40 1800

1000

40

0 1800

80

% Transmittance

% Transmittance

1549–1515

% Transmittance

1600 1400 1200 Wavelength (cm–1)

80 1544 60

40 1800

1000

(d)

1652

1600 1400 1200 Wavelength (cm–1)

1000

100 1241 % Transmittance

18

80

1544 1652

60

40 1800

(e)

B. mori P. ncini A. assamensis

1600 1400 1200 Wavelength (cm–1)

1000

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

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 silkworm 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 composition. 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 watersoluble properties. Additionally, temperature is also a factor that changes the sericin structure. 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

19

20

1  Sericin: Structure and Properties

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 evaluate 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 solution. 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; cellulose) (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 capable 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 polymers. 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 aluminum 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 temperature 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 temperature. 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.

1.8  ­Sericin Propertie Amide I 1656.6 Amide II 1532

191

192.754

(B)

191

(C) (D)

195.303 204

Amide III 1248

Absorb arce

CD (mde g)

(A)

(A) (B)

194

(C)

204.307 200.948 200.555 200 (a)

(D)

220

240

260

280

Wavelength (nm)

900

1200

1500

1800

–1 Wavelength (cm )

(b)

100

Mass (96)

90

80 70

(A)

60

(C)

(B)

(D) 50

50

100 150

(c)

200

250 300

350

Temperature (°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 photoprotective 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.

21

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1  Sericin: Structure and Properties

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 adherence 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

1.8  ­Sericin Propertie

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 consequently 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 antielastase 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 measured 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

Nistari FL Nistari Verde Ovale FL Verde Ovale AP

Arancio Oro 208 FL

Rosa

Oro 208 201 A FL

Oro Gigante ADPR FL ADPR

R3G

Coin Sejaku Green BG Treotto Rosa SA48LB

Han-Han

PK12FL

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.

23

St. dev. Vmax

80

201 A ADPR

13.109

4.326

1.275

0.11

16.252

8.796

1.201

0.206

70

ADPR (FL)

13.032

3.655

1.162

0.107

AP

17.313

7.891

1.229

0.225

Arancio

20.903

6.211

1.527

0.249

Daizo

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

Nistari (FL)

24.522

14.403

1.463

0.318

Orgosolo

19.149

7.174

1.005

0.13

Oro 208

20.455

6.23

1.307

0.188

Oro gigante

27.084

18.583

1.514

0.386

PK12 (FL)

38.495

32.269

1.681

0.627

R3G

20.382

11.089

1.417

0.277

Romagna

43.763

25.109

1.536

0.527

SA48LB

11.542

3.478

1.17

0.091

23.411

12.909

1.404

0.228

Treotto rosa (FL)

47.178

47.702

1.765

0.862

Verde ovale

11.992 19.431

3.525 11.688

1.148 1.261

0.088

Verde ovale (FL)

0.248

Mean activity (%)

50 40 30 20 10 0 A A AD DP PR R (F L) A Ar P an cio Da ito G Ha 133 nNi Ha st ar n i( F or L) go t O olo r O o2 ro gi 08 ga PK nte 12 (F L) R Ro 3G m ag Se ja SA4 na k Tr u g BLB r ea e tto en ro BG V sa Ve erd (F e L) rd o e ov val e al e (F L)

Sejaku green BG

60

1

Mean KCL St. dev. KCL Mean Vmax

20

Sample

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.

1.8  ­Sericin Propertie

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 isolated 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 similar 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 peroxidation. 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 treatment 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

25

1  Sericin: Structure and Properties

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). 120 100

% Inhibition

26

1h

2h

5h

6h

3h

4h

80 60 40 20 0 Water

Sericin 0.080 mg/ml

Sericin 0.040 mg/ml

Sericin Sericin 0.020 0.010 mg/ml mg/ml

Sericin 0.004 mg/ml

Acetone 1% IND

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) indomethacintreated 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).

1.8  ­Sericin Propertie

Moreover, sericin was reported to be effective for suppressing the inflammatory mediators, cyclooxygenase-2 enzyme and nitric oxide production (Aramwit et  al. 2013). Additionally, the effect of sericin increased anti-inflammatory cytokine expression, including IL-4, IL-10, and transforming growth factor-β, and reduced the production of the allergic 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

Collagen (mcg/ml)

500 400 300 200 100 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.

27

1  Sericin: Structure and Properties

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 addition, sericin downregulated the expression of a melanogenesis regulatory gene, microphthalmia-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 mulberry 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

100 80 60 40 20 0

(a)

Negative 12.5 control

100 chlorhexidine

60 40 20 25 50 Sericin mg ml

60 40 20 0

Biofilm formation(%)

80

Negative 12.5 control

80

Negative 12.5 control

(d)

100 chlorhexidine

100 80 60 40 20 0

100 chlorhexidine

25 50 Sericin mg ml

Urea

120

100

0

100

(b)

Alkali

120

(c)

25 50 Sericin mg ml

Acid

120 Biofilm formation(%)

Biofilm formation(%)

120

Biofilm formation(%)

28

Negative 12.5 control

25 50 Sericin mg ml

100 chlorhexidine

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.)

1.8  ­Sericin Propertie

Sericin isolated from four extraction methods (heat, urea, acid, and alkali) induced collagen 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 promoting 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 property, sericin was used for biomaterial and cosmetic applications, especially skin treatment (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 sericin

Acid-extracted sericin

Alkali-extracted sericin

Urea-extracted sericin

Negative control

12.5 mg/ml sericin

25 mg/ml sericin

50 mg/ml sericin

N/A

100 mg/ml sericin

1.2 mg/ml chlorhexidine

N/A

N/A

N/A

N/A

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 ureaextracted 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).

29

1  Sericin: Structure and Properties Heat

300 250 200 150 100 50 0

(a)

Negative 12.5 control

Bacterial viability (%)

200 150 100 50 25 50 Sericin mg ml

200 150 100 50 Negative 12.5 control

(d)

100 chlorhexidine

300 250 200 150 100 50 0

100 chlorhexidine

25 50 Sericin mg ml

Urea

350

250

Negative 12.5 control

250

(b)

300

0

300

0

100 chlorhexidine

Alkali

350

(c)

25 50 Sericin mg ml

Acid

350 Bacterial viability (%)

Bacterial viability (%)

350

Bacterial viability (%)

30

Negative 12.5 control

25 50 Sericin mg ml

100 chlorhexidine

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 autoclaving preparation slightly affected S. aureus but did not affect both E. coli and P. aeruginosa (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 antibacterial 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 antibiofilm 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

1.8  ­Sericin Propertie Heat-extracted sericin

Acid-extracted sericin

Alkali-extracted sericin

Urea-extracted 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, alkalineextracted 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 heatextracted and acid-extracted sericins were found to reduce the biofilm formation dosedependently, 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 bacteria 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 varies 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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­References Ahmad, R., Kamra, A., and Hasnain, S.E. (2004). Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori. DNA and Cell Biology 23 (3): 149–154. Akturk, O., Tezcaner, A., Bilgili, H. et al. (2011). Evaluation of sericin/collagen membranes as prospective wound dressing biomaterial. Journal of Bioscience and Bioengineering 112 (3): 279–288. Aramwit, P., Kanokpanont, S., De-Eknamkul, W. et al. (2009). The effect of sericin with variable amino-acid content from different silk strains on the production of collagen and nitric oxide. Journal of Biomaterials Science, Polymer Edition 20 (9): 1295–1306. Aramwit, P., Damrongsakkul, S., Kanokpanont, S., and Srichana, T. (2010a). Properties and anti-tyrosinase activity of sericin from various extraction methods. Biotechnology and Applied Biochemistry 55 (2): 91–98. Aramwit, P., Kanokpanont, S., Nakpheng, T., and Srichana, T. (2010b). The effect of sericin from various extraction methods on cell viability and collagen production. International Journal of Molecular Sciences 11: 2200–2211. Aramwit, P., Towiwat, P., and Srichana, T. (2013). Anti-inflammatory potential of silk sericin. Natural Product Communications 8 (4): 501–504. Aramwit, P., Ratanavaraporn, J., Ekgasit, S. et al. (2015). A green salt-leaching technique to produce sericin/PVA/glycerin scaffolds with distinguished characteristics for wounddressing applications. Journal of Biomedical Materials Research Part B: Applied Biomaterials 103 (4): 915–924. Aramwit, P., Luplertlop, N., Kanjanapruthipong, T., and Ampawong, S. (2018). Effect of urea-extracted sericin on melanogenesis: potential applications in post-inflammatory hyperpigmentation. Biological Research 51 (1): 54. Aramwit, P., Napavichayanun, S., Pienpinijtham, P. et al. (2020). Antibiofilm activity and cytotoxicity of silk sericin against Streptococcus mutans bacteria in biofilm: An in vitro study. Journal of Wound Care 28 (4): S25–S35. Bakhsheshi-Rad, H.R., Ismail, A.F., Aziz, M. et al. (2020). Development of the PVA/CS nanofibers containing silk protein sericin as a wound dressing: In vitro and in vivo assessment. International Journal of Biological Macromolecules 149: 513–521. Banagozar Mohammadi, A., Torbati, M., Farajdokht, F. et al. (2019). Sericin alleviates restraint stress induced depressive- and anxiety-like behaviors via modulation of oxidative stress, neuroinflammation and apoptosis in the prefrontal cortex and hippocampus. Brain Research 1715: 47–56. Chaisabai, W., Khamhaengpol, A., and Siri, S. (2018). Sericins of mulberry and non-mulberry silkworms for eco-friendly synthesis of silver nanoparticles. Artificial Cells, Nanomedicine, and Biotechnology 46 (3): 536–543. Chlapanidas, T., Farago, S., Lucconi, G. et al. (2013). Sericins exhibit ROS-scavenging, antityrosinase, anti-elastase, and in vitro immunomodulatory activities. International Journal of Biological Macromolecules. 58: 47–56. Cichorek, M., Wachulska, M., Stasiewicz, A., and Tymińska, A. (2013). Skin melanocytes: biology and development. Postepy dermatologii i alergologii 30 (1): 30–41.

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Couble, P., Michaille, J.J., Garel, A. et al. (1987). Developmental switches of sericin mRNA splicing in individual cells of Bombyx mori silkgland. Developmental Biology 124 (2): 431–440. Dash, R., Mukherjee, S., and Kundu, S.C. (2006). Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta. International Journal of Biological Macromolecules 38 (3-5): 255–258. Dash, R., Ghosh, S.K., Kaplan, D.L., and Kundu, S.C. (2007). Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta. Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology 147 (1): 129–134. Dash, R., Acharya, C., Bindu, P.C., and Kundu, S.C. (2008). Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts. BMB Reports 41 (3): 236–241. Dash, B.C., Mandal, B.B., and Kundu, S.C. (2009). Silk gland sericin protein membranes: fabrication and characterization for potential biotechnological applications. Journal of Biotechnology 144 (4): 321–329. Deng, L., Guo, W., Li, G. et al. (2019). Hydrophobic IR780 loaded sericin nanomicelles for phototherapy with enhanced antitumor efficiency. International Journal of Pharmaceutics 566: 549–556. Dong, Y., Dai, F., Ren, Y. et al. (2015). Comparative transcriptome analyses on silk glands of six silkmoths imply the genetic basis of silk structure and coloration. BMC Genomics 16: 203. Gamo, T. (1982). Genetic variants of the Bombyx mori silkworn encoding sericin proteins of different lengths. Biochemical Genetics 20 (1–2): 165–177. Gamo, T., Inokuchi, T., and Laufer, H. (1977). Polypeptides of fibroin and sericin secreted from the different sections of the silk gland in Bombyx mori. Insect Biochemistry 7: 285–295. Garel, A., Deleage, G., and Prudhomme, J.-C. (1997). Structure and organization of the Bombyx mori sericin 1 gene and of the sericins 1 deduced from the sequence of the Ser 1B cDNA. Insect Biochemistry and Molecular Biology 27 (5): 469–477. Ghensi, P., Bettio, E., Maniglio, D. et al. (2019). Dental implants with anti-biofilm properties: a pilot study for developing a new sericin-based coating. Materials (Basel) 12 (15): 2429. He, H., Cai, R., Wang, Y. et al. (2017). Preparation and characterization of silk sericin/PVA blend film with silver nanoparticles for potential antimicrobial application. International Journal of Biological Macromolecules 104 (Pt A): 457–464. Heim, K.E., Tagliaferro, A.R., and Bobilya, D.J. (2002). Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. Journal of Nutritional Biochemistry 13 (10): 572–584. Ishikawa, E. and Suzuki, Y. (1985). Tissue- and stage-specific expression of sericin genes in the middle silk gland of Bombyx mori. Development, Growth & Differentiation 27 (1): 73–82. Isobe, T., Ikebata, Y., Onitsuka, T. et al. (2013). Cryopreservation for bovine embryos in serum-free freezing medium containing silk protein sericin. Cryobiology 67 (2): 184–187. Jena, K., Pandey, J.P., Kumari, R. et al. (2018). Free radical scavenging potential of sericin obtained from various ecoraces of tasar cocoons and its cosmeceuticals implication. International Journal of Biological Macromolecules 120 (Pt A): 255–262. Kato, N., Sato, S., Yamanaka, A. et al. (1998). Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Bioscience, Biotechnology, and Biochemistry 62 (1): 145–147.

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Kikkawa, H. (1953). Biochemical genetics of Bombyx mori (silkworm). In: Advances in Genetics (ed. M. Demerec), 107–140. Academic Press. Kiro, A., Bajpai, J., and Bajpai, A.K. (2017). Designing of silk and ZnO based antibacterial and noncytotoxic bionanocomposite films and study of their mechanical and UV absorption behavior. Journal of the Mechanical Behavior of Biomedical Materials 65: 281–294. Kludkiewicz, B., Takasu, Y., Fedic, R. et al. (2009). Structure and expression of the silk adhesive protein Ser2 in Bombyx mori. Insect Biochemistry and Molecular Biology 39 (12): 938–946. Kumar, J.P. and Mandal, B.B. (2017). Antioxidant potential of mulberry and non-mulberry silk sericin and its implications in biomedicine. Free Radical Biology and Medicine 108: 803–818. Kumar, J.P. and Mandal, B.B. (2019). The inhibitory effect of silk sericin against ultravioletinduced melanogenesis and its potential use in cosmeceutics as an anti-hyperpigmentation compound. Photochemical and Photobiological Sciences 18 (10): 2497–2508. Kumar, P., Kumar, D., Sikka, P., and Singh, P. (2015). Sericin supplementation improves semen freezability of buffalo bulls by minimizing oxidative stress during cryopreservation. Animal Reproduction Science 152: 26–31. Kumar, J.P., Alam, S., Jain, A.K. et al. (2018). Protective activity of silk sericin against UV radiation-induced skin damage by downregulating oxidative stress. ACS Applied Bio Materials 1 (6): 2120–2132. Ma, M., Hussain, M., Dong, S., and Zhou, W. (2016). Characterization of the pigment in naturally yellow-colored domestic silk. Dyes and Pigments 124: 6–11. Mandal, B.B., Ghosh, B., and Kundu, S.C. (2011). Non-mulberry silk sericin/poly (vinyl alcohol) hydrogel matrices for potential biotechnological applications. International Journal of Biological Macromolecules 49 (2): 125–133. Manosroi, A., Boonpisuttinant, K., Winitchai, S. et al. (2010). Free radical scavenging and tyrosinase inhibition activity of oils and sericin extracted from Thai native silkworms (Bombyx mori). Pharmaceutical Biology 48 (8): 855–860. Michaille, J.-J., Couble, P., Prudhomme, J.-C., and Garel, A. (1986). A single gene produces multiple sericin messenger RNAS in the silk gland of Bombyx mori. Biochimie 68 (10): 1165–1173. Michaille, J.-J., Garel, A., and Prudhomme, J.-C. (1990). Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori. Gene 86 (2): 177–184. Miyamoto, Y., Teramoto, N., Hayashi, S., and Enosawa, S. (2010). An improvement in the attaching capability of cryopreserved human hepatocytes by a proteinaceous high molecule, sericin, in the serum-free solution. Cell Transplantation 19 (6): 701–706. Miyamoto, Y., Oishi, K., Yukawa, H. et al. (2012). Cryopreservation of human adipose tissuederived stem/progenitor cells using the silk protein sericin. Cell Transplantation 21 (2–3): 617–622. Muhammad Tahir, H., Saleem, F., Ali, S. et al. (2020). Synthesis of sericin-conjugated silver nanoparticles and their potential antimicrobial activity. Journal of Basic Microbiology 60 (5): 458–467. Nayak, S., Talukdar, S., and Kundu, S.C. (2012). Potential of 2D crosslinked sericin membranes with improved biostability for skin tissue engineering. Cell and Tissue Research 347 (3): 783–794.

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Nayak, S., Dey, S., and Kundu, S.C. (2014). Silk sericin-alginate-chitosan microcapsules: hepatocytes encapsulation for enhanced cellular functions. International Journal of Biological Macromolecules 65: 258–266. Ohnishi, K., Murakami, M., Morikawa, M., and Yamaguchi, A. (2012). Effect of the silk protein sericin on cryopreserved rat islets. Journal of Hepato-Biliary-Pancreatic Sciences 19 (4): 354–360. Okamoto, H., Ishikawa, E., and Suzuki, Y. (1982). Structural analysis of sericin genes. Homologies with fibroin gene in the 5’ flanking nucleotide sequences. Journal of Biological Chemistry 257 (24): 15192–15199. Padamwar, M.N. and Pawar, A.P. (2004). Silk sericin and its applications: a review. Journal of Scientific & Industrial Research 63 (4): 323–329. Qi, C., Xu, L., Deng, Y. et al. (2018). Sericin hydrogels promote skin wound healing with effective regeneration of hair follicles and sebaceous glands after complete loss of epidermis and dermis. Biomaterials Science 6 (11): 2859–2870. Rocha, L.K.H., Favaro, L.I.L., Rios, A.C. et al. (2017). Sericin from Bombyx mori cocoons. Part I: extraction and physicochemical-biological characterization for biopharmaceutical applications. Process Biochemistry 61: 163–177. Sahu, N., Pal, S., Sapru, S. et al. (2016). Non-mulberry and mulberry silk protein sericins as potential media supplements for animal cell culture. BioMed Research International 2016: 7461041. Sapru, S., Ghosh, A.K., and Kundu, S.C. (2017). Non-immunogenic, porous and antibacterial chitosan and Antheraea mylitta silk sericin hydrogels as potential dermal substitute. Carbohydrate Polymers 167: 196–209. Schallreuter, K.U., Kothari, S., Chavan, B., and Spencer, J.D. (2008). Regulation of melanogenesis–controversies and new concepts. Experimental Dermatology 17 (5): 395–404. Shah, A., Ali Buabeid, M., Arafa, E.A. et al. (2019). The wound healing and antibacterial potential of triple-component nanocomposite (chitosan-silver-sericin) films loaded with moxifloxacin. International Journal of Pharmaceutics 564: 22–38. Shaw, J.T.B. and Smith, S.G. (1951). Amino-acids of silk sericin. Nature 168 (4278): 745–745. Sprague, K.U. (1975). The Bombyx mori silk proteins: characterization of large polypeptides. Biochemistry 14 (5): 925–931. Suganuma, K., Nakajima, H., Ohtsuki, M., and Imokawa, G. (2010). Astaxanthin attenuates the UVA-induced up-regulation of matrix-metalloproteinase-1 and skin fibroblast elastase in human dermal fibroblasts. Journal of Dermatological Science 58 (2): 136–142. Suktham, K., Koobkokkruad, T., Wutikhun, T., and Surassmo, S. (2018). Efficiency of resveratrol-loaded sericin nanoparticles: promising bionanocarriers for drug delivery. International Journal of Pharmaceutics 537 (1–2): 48–56. Takasu, Y., Yamada, H., and Tsubouchi, K. (2002). Isolation of three main sericin components from the cocoon of the silkworm, Bombyx mori. Bioscience, Biotechnology, and Biochemistry 66 (12): 2715–2718. Takasu, Y., Yamada, H., Tamura, T. et al. (2007). Identification and characterization of a novel sericin gene expressed in the anterior middle silk gland of the silkworm Bombyx mori. Insect Biochemistry and Molecular Biology 37 (11): 1234–1240. Takasu, Y., Hata, T., Uchino, K., and Zhang, Q. (2010). Identification of Ser2 proteins as major sericin components in the non-cocoon silk of Bombyx mori. Insect Biochemistry and Molecular Biology 40 (4): 339–344.

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Takechi, T., Wada, R., Fukuda, T. et al. (2014). Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods. Biomedical Reports 2 (3): 364–369. Teramoto, H., Nakajima, K., and Takabayashi, C. (2004). Chemical modification of silk sericin in lithium chloride/dimethyl sulfoxide solvent with 4-cyanophenyl isocyanate. Biomacromolecules 5 (4): 1392–1398. The International Silkworm Genome Consortium (2008). The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochemistry and Molecular Biology 38 (12): 1036–1045. Tokutake, S. (1980). Isolation of the smallest component of silk protein. Biochemical Journal 187 (2): 413–417. Tsubouchi, K., Igarashi, Y., Takasu, Y., and Yamada, H. (2005). Sericin enhances attachment of cultured human skin fibroblasts. Bioscience, Biotechnology, and Biochemistry 69 (2): 403–405. Tyeb, S., Shiekh, P.A., Verma, V., and Kumar, A. (2020). Adipose-derived stem cells (ADSCs) loaded gelatin-sericin-laminin cryogels for tissue regeneration in diabetic wounds. Biomacromolecules 21 (2): 294–304. Wang, J., Zhang, S., Xing, T. et al. (2015). Ion-induced fabrication of silk fibroin nanoparticles from Chinese oak tasar Antheraea pernyi. International Journal of Biological Macromolecules 79: 316–325. Wang, W., Wang, N., Liu, C., and Jin, J. (2017). Effect of silkworm pupae peptide on the fermentation and quality of yogurt. Journal of Food Processing and Preservation 41 (3): e12893. Wu, J.-H., Wang, Z., and Xu, S.-Y. (2008). Enzymatic production of bioactive peptides from sericin recovered from silk industry wastewater. Process Biochemistry 43 (5): 480–487. Xue, R., Liu, Y., Zhang, Q. et al. (2016). Shape changes and interaction mechanism of Escherichia coli cells treated with sericin and use of a sericin-based hydrogel for wound healing. Applied and Environmental Microbiology 82 (15): 4663–4672. Yalcin, E., Kara, G., Celik, E. et al. (2019). Preparation and characterization of novel albuminsericin nanoparticles as siRNA delivery vehicle for laryngeal cancer treatment. Preparative Biochemistry & Biotechnology 49 (7): 659–670. Yamada, M. (1978). Amino acid composition of the sericin extracted from cocoon of the mulberry wild silkworm, Bombyx mori mandarina Moore, and its species specificity. The Journal of Sericultural Science of Japan 47 (2): 108–112. Yamada, H. and Tsubouchi, K. (2001). Characterization of silk proteins in the cocoon fibers of Cricula trifenestrata. International Journal of Wild Silkmoth and Silk (Japan) 6: 47–51 Yamanouchi, M. (1922). Morphologische Beobachtung über die Seidensekretion bei der Seidenraupe. Journal of the College of Agriculture, Hokkaido Imperial University, Sapporo, Japan 10 (4): 1–49. Zhang, Y., Jiang, R., Fang, A. et al. (2019). A highly transparent, elastic, injectable sericin hydrogel induced by ultrasound. Polymer Testing 77: 105890. Zhao, R., Li, X., Sun, B. et al. (2014). Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings. International Journal of Biological Macromolecules 68: 92–97. Zhaorigetu, S., Sasaki, M., Watanabe, H., and Kato, N. (2001). Supplemental silk protein, sericin, suppresses colon tumorigenesis in 1,2-dimethylhydrazine-treated mice by reducing oxidative stress and cell proliferation. Bioscience, Biotechnology, and Biochemistry 65 (10): 2181–2186.

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Zhaorigetu, S., Yanaka, N., Sasaki, M. et al. (2003). Inhibitory effects of silk protein, sericin on UVB-induced acute damage and tumor promotion by reducing oxidative stress in the skin of hairless mouse. Journal of Photochemistry and Photobiology B: Biology 71 (1-3): 11–17. Zhu, L.J., Arai, M., and Hirabayashi, K. (1995). Gelation of silk sericin and physical properties of the gel. The Journal of Sericultural Science of Japan 64 (5): 415–419. Zhu, L., Yao, J., and Li, Y. (1998). Structure transformation of sericin protein dissolved from cocoon layer in hot water. Journal of Zhejiang Agricultural University 3: S881–S883. Zurovec, M., Yonemura, N., Kludkiewicz, B. et al. (2016). Sericin composition in the silk of Antheraea yamamai. Biomacromolecules 17 (5): 1776–87.

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2 Processing Sericin 2.1  ­Effects of Source and Extraction Method of Sericin on Its Benefits and Applications Methods for the extraction of sericin from mulberry and non-mulberry cocoons should be intensely investigated because they have effects on the physical, chemical, and biological properties of sericin. Five different methods that are often used in sericin extraction are described in this chapter.

2.1.1  Sericin Extraction 2.1.1.1  Water Extraction (WaterSS; HeatSS, AutoclaveSS)

Water extraction is a nonchemical extraction resulting in nontoxicity to the environment and no impurities in the extract. It is a major extraction method of mulberry silk sericin (Bombyx mori). Distilled water is used as the solvent in this method, while heat and an autoclave are the major processes of water extraction. For heat extraction (HeatSS), small pieces of cocoons were soaked in hot distilled water at 80–120 °C. For autoclave extraction (AutoclaveSS), the cocoons were immersed in distilled water, then autoclaved at various conditions: 110–121 °C, 15 lbf/in2 for 20–60 minutes (Aramwit et al. 2010a, 2010b; Butkhup et  al. 2012; Kumar and Mandal 2017; Kurioka et  al. 2004; Sahu et  al. 2016). Then, the extract was filtered to separate silk fibroin and other solid residues. 2.1.1.2  Acid Extraction (AcidSS)

Citric acid was mostly used as the solvent in acid extraction. Small pieces of cocoons were soaked in 1.25% citric acid and then boiled for 30 minutes. After that, the extract was filtered to remove insoluble fibers. The filtered solution was dialyzed in distilled water for at least three days to remove citric acid. The pH of that solution may be measured to verify the removal of citric acid (Aramwit et  al. 2010a, 2010b; Kumar and Mandal 2017; Kurioka et al. 2004).

Sustainable Uses of Byproducts from Silk Processing, First Edition. Narendra Reddy and Pornanong Aramwit. © 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.

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2.1.1.3  Alkali Extraction (AllkaliSS; Alkali-L-SS, Alkali-H-SS)

Two concentrations of sodium carbonate were mostly used in alkali extraction: 0.02 M (Alkali-L-SS) and 0.5 M (Alkali-H-SS). Small pieces of cocoons were immersed in 0.02 M or 0.5 M sodium carbonate. After that, they were boiled for 30 minutes. The extract was filtered to eliminate the solid residue and then dialyzed in distilled water for three days to remove sodium carbonate. The pH of the extract was measured to confirm the removal of sodium carbonate (Aramwit et al. 2010a, 2010b; Dash et al. 2008; Kumar and Mandal 2017; Kurioka et al. 2004). This method was suggested to be the optimal extraction method for non-mulberry SS (Antheraea mylitta) (Yun et al. 2013). 2.1.1.4  Urea Extraction (UreaSS)

Small pieces of cocoons were soaked in 8 M of urea for 30 minutes. Then, they were boiled at 80–85 °C for 5–30 minutes. The extract was centrifuged and filtered to remove all insoluble residues. Finally, it was dialyzed in distilled water for three days to remove urea (Aramwit et al. 2010a, 2010b; Kumar and Mandal 2017; Sahu et al. 2016). 2.1.1.5  Alcohol Extraction (AlcoholSS)

Ethanol was mostly used as the solvent in alcohol extraction. Small pieces of cocoons were immersed in 70% ethanol. The extract was autoclaved at 121 °C, 15 lbf/in2 for 60 minutes (Butkhup et al. 2012).

2.1.2  Effect of Source and Extraction on Sericin Properties 2.1.2.1  Molecular Weight of Sericin

Table 2.1 shows the molecular weight (MW) of sericin that was extracted using five different methods: WaterSS, AcidSS, AlkaliSS, UreaSS, and AlcoholSS. Sodium dodecyl sulfate and polyacrylamide gel electrophoresis (SDS-PAGE) assay was used for molecular weight analysis. In comparison between mulberry and non-mulberry sericin, the patterns of sodium dodecyl sulfate (SDS) bands in WaterSS and AlkaliSS were almost the same. A smear band was observed in WaterSS, while a smear with clear bands was noticed in AlkaliSS. However, in UreaSS, clear bands were found in mulberry sericin while a smear with clear bands was detected in non-mulberry sericin. In AcidSS, the patterns of SDS bands were also different depending on the source of sericin. For molecular weight ranges of sericin, the highest molecular weight range was shown in UreaSS for both mulberry and non-mulberry sericins. The lowest range of molecular weight was detected in AlkaliSS for both sericin types, but especially mulberry sericin. Consequently, the extraction method had effects on the different patterns of SDS bands and molecular weight ranges while the source of sericin had a slight affect. 2.1.2.2  Secondary Structure of the Sericin Protein

The secondary structure of sericin was identified by Fourier-transformation infrared spectrophotometry (FTIR) and circular dichroism (CD). The major secondary structures of sericin were alpha-helix and beta-sheet structures. However, these structures were different depending on the extraction method and source of sericin (Table 2.2). The secondary structure was related to the stability of sericin, with the structure of alpha-helix being

2.1  ­Effects of Source and Extraction

Method of Sericin on Its Benefits and Application

stabilized by intrachain hydrogen bonds between the NH and CO groups of the main chain (Berg et al. 2002). These bonds were considered slightly weaker than those found in betasheets and were readily disrupted by a hydrophilic surrounding. However, it was more stable in more hydrophobic environments. The beta-sheets were stabilized by hydrogen bonding between polypeptide strands (Berg et al. 2002). This had an advantage on sericin stabilization in water (Sahu et al. 2016) and also influenced gel forming of sericin. Moreover, stable beta-sheets were preferred in the dehydrated state, such as in the scaffold application, so it might be cross-linked by cross-linking agents including glutaraldehyde. The major secondary structures of mulberry and non-mulberry sericin in each extraction were similar. However, in the comparison of extraction methods, beta-sheets were the major secondary structure of AutoclaveSS and UreaSS, while alpha-helix was mostly found in AlkaliSS. In AcidSS, both alpha-helix and beta-sheet structures were detected. Accordingly, the extraction methods affected the secondary structure of sericin. A stable sericin seemed to be generated by autoclave extraction and urea extraction. Also, the secondary structure of sericin from two sources might not be different. 2.1.2.3  Phenolic contents

The phenolic content of sericin was measured using Folin–Ciocalteu reagent. The total phenolic content was calculated in comparison to the standard curve of gallic acid (GAE). The phenolic content of non-mulberry sericin (Antheraea assamensis and Philosamia ricini) in AutoclaveSS, Alkali-H-SS, Alkali-L-SS, AcidSS, and UreaSS was higher than that of mulberry SS (B. mori) (Kumar and Mandal 2017). However, Butkhup et al. (2012) found that AutoclaveSS and AlcoholSS of mulberry SS (B. mori) contained a higher phenolic content than non-mulberry SS (Samia ricini). Therefore, strains of silk cocoon seem to have an effect on the phenolic content of sericin more than the type of sericin. In the comparison of extraction methods, a descending order of phenolic content was detected in the AutoclaveSS, Alkali-H-SS, Alkali-L-SS, AcidSS, and UreaSS of both sericin types (Kumar and Mandal 2017). Moreover, the phenolic content of sericin in AutoclaveSS was also higher than that in AlcoholSS (Butkhup et  al. 2012). These contents were in range of 50–500 mg GAE/100 g. The major phenolic presented in all samples was catechin (Butkhup et al. 2012). The phenolic content has effect on the biological properties of sericin, especially antioxidant activity. 2.1.2.4  Antioxidant Activity

Antioxidant activity was tested by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, the ferric ion reducing antioxidant power (FRAP) assay, and the 2,2′-azinobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) cation radical scavenging assay. For AutoclaveSS, Alkali-H-SS, Alkali-L-SS, AcidSS, and UreaSS, higher antioxidant activity was shown in non-mulberry sericin extract (A. assamensis and P. ricini) compared to mulberry SS extract (B. mori) (Kumar and Mandal 2017). However, AutoclaveSS and AlcoholSS of mulberry sericin (B. mori) showed higher antioxidant activity than non-mulberry sericin (S. ricini) (Butkhup et al. 2012). These results agree with the phenolic contents of sericin in which the strain of silk cocoon affected phenolic content and antioxidant activity. Comparing AutoclaveSS, Alkali-H-SS, Alkali-L-SS, AcidSS, and UreaSS, the maximum antioxidant activity of non-mulberry sericin was detected in Alkali-L-SS while the highest

43

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2  Processing Sericin

antioxidant activity of mulberry sericin was found in AutoclaveSS (Kumar and Mandal 2017). Moreover, AutoclaveSS of both non-mulberry SS and mulberry SS had a higher ability for free radical scavenging activity than AlcoholSS (Butkhup et al. 2012). The 50% DPPH reduction (IC50) of both sericin types was around 1–2 mg/ml. Total phenolic content had a positive correlation with the antioxidant activity. Moreover, its hydroxyl (serine) and carboxyl (aspartic acid) groups may be the major parts of chelation with oxidative stress (Kato et al. 1998). 2.1.2.5  Anti-tyrosinase Activity

The descending order of anti-tyrosinase activity was UreaSS, AutoclaveSS, AcidSS, and AlkaliSS of mulberry sericin, respectively. Furthermore, the pigmentation of color cocoon had an effect on anti-tyrosinase activity. A cocoon with pigmentation could inhibit tyrosinase activity more than a cocoon without pigmentation (Aramwit et al. 2010a). 2.1.2.6  Cytotoxicity

Two major influences of sericin on cytotoxicity were the extraction method and concentration of sericin. At 8 mcg/ml of mulberry sericin, no cytotoxicity was found in AutoclaveSS, Alkali-H-SS, AcidSS, and UreaSS. However, there was slight toxicity in UreaSS at 60 mcg/ ml of mulberry sericin and significant toxicity in UreaSS at 100 mcg/ml of mulberry sericin, whereas the lowest toxicity was shown for AutoclaveSS, Alkali-H-SS, and AcidSS at concentrations up to 100 mcg/ml of mulberry sericin (Aramwit et al. 2010b). 2.1.2.7  Cell Attachment, Cell Proliferation, and Collagen Production

The time of maximum attachment of L929 cells in DMEM supplemented with 0.05% sericin of mulberry sericin and non-mulberry sericin was evaluated. Non-mulberry sericin induced the attachment of cells faster than mulberry sericin (10 vs. 12 hours) (Sahu et al. 2016). Both sericin types generated by AutoclaveSS could promote cell proliferation better than serum-free medium (Sahu et  al. 2016). Moreover, mulberry sericin extracted by AutoclaveSS fully proliferated after 72 hours (Aramwit et al. 2010b). All extraction methods (AutoclaveSS, Alkali-H-SS, AcidSS, and UreaSS) could induce collagen type I production. Nonetheless, the lowest amount of collagen was found in UreaSS, while the highest amount of collagen was observed in AutoclaveSS at 8–200 mcg/ml (Aramwit et al. 2010b). 2.1.2.8  Cell Protection

The cell protection activity of sericin was evaluated by the determination of viable cells after treatment with sericin and hydrogen peroxide. The fibroblast cells were incubated with Alkaline-L-SS for 24 hours. Then, they were treated with hydrogen peroxide to induce oxidative stress. The results showed that Alkaline-L-SS at concentration of 35, 50, and 100 ng/ml of both mulberry sericin and non-mulberry sericin significantly increased cell viability. Therefore, both sericin types provided a protective effect on fibroblast cells. Their antioxidant property might be the cause of this cell protection activity (Dash et al. 2008). However, there were no available data on the cell protection activity of sericin extracted by other methods.

2.1  ­Effects of Source and Extraction

Method of Sericin on Its Benefits and Application

2.1.3  Benefit and Application of Extracted Mulberry Sericin and Non-mulberry Sericin Because of the physical, chemical, and biological properties of sericin, it was used in pharmaceutical, cosmetic, wound healing, tissue engineering, and drug delivery applications (Table 2.3). It was prepared in several forms including solution, gel, cream, hydrogel, and a scaffold. However, the source and extraction method of sericin mostly affected the properties and benefits of sericin. 2.1.3.1  Pharmaceutics and Cosmetics

Sericin was used as an active ingredient in natural pharmaceutics and cosmetic because of its antioxidant activity, anti-tyrosinase activity, and moisturizing effect. The most common sericin source for pharmaceutics and cosmetics was mulberry sericin, especially B. mori. The extraction methods for these preparations were WaterSS and AlkaliSS. Padamwar et al. (2005) evaluated the effects of sericin gel on the skin in healthy volunteers. The results showed that the hydration of sericin gel–treated skin was significantly higher than that of normal skin, while there was no significant difference between gel base–treated skin and normal skin. The transepidermal water loss (TEWL) of sericin gel–treated skin was also lower than that of normal skin. Accordingly, the moisturizing effect of sericin might be attributed to the increased restoration of amino acids in the skin, resulting in greater water retention and less water evaporation. This moisturizing effect also positively affected dry skin and pruritus. After six weeks of uremic pruritus treatment, the skin irritation of sericin cream–treated sites was significantly lower than the cream base–treated site while skin hydration was significantly higher than that of cream base–treated sites (Aramwit et al. 2012a). In addition to the topical application of sericin, sericin was also evaluated in terms of dietary sericin. Dietary sericin had a positive effect on the skin. Kim et al. (2012) showed that after 10 weeks of sericin feeding, epidermal hydration of atopic dermatitis mice treated with dietary sericin was significantly higher than that treated with the control diet, resulting in improved skin dryness in atopic dermatitis. Furthermore, dietary sericin had potential as a chemoprotective agent for colon carcinogenesis. The incidence and number of colon tumors of mice supplemented with 3% sericin for 115 days were decreased (Sasaki et al. 2000a). These results agreed with Okazaki et al. (2011), who found that the amount of fecal immunoglobulin A (IgA) in the colon of rats supplemented with sericin increased after three weeks of treatment resulting in a lower risk of colon cancer. The consumption of 3% sericin powder also increased the absorption of Zn (41%), Fe (41%), Mg (21%), and Ca (17%) in rats after 12 days of feeding (Sasaki et al. 2000b). In addition, dietary sericin was reported to reduce serum lipids. Rats fed with a 4% sericin diet had significantly lower serum levels of triglycerides (33%), cholesterol (16%), phospholipids (18%), and free fatty acids (27%) than rats fed without sericin after five weeks of feeding (Okazaki et al. 2010). 2.1.3.2  Wound Healing

Sericin could activate fibroblasts to promote collagen type I synthesis resulting in the promotion of wound healing. Mulberry sericin extracted by AutoclaveSS was the most used sericin preparation for wound healing. In the in vivo test by Aramwit et  al. (2009), the

45

2.1  ­Effects of Source and Extraction

Method of Sericin on Its Benefits and Application

sericin cream–treated wound showed a significantly higher percentage of wound size reduction than the cream base– and normal saline–treated wounds. Moreover, the level of inflammatory mediator (tumor necrosis factor-α [TNF-alpha] and interleukin-1β [IL-1beta]) in sericin cream–treated wounds was lower than that of the cream base– and normal saline–treated wounds. Therefore, sericin cream promoted wound healing without exacerbating the inflammatory process. In a clinical study, a total of 65 second-degree burn wound patients showed a wound healing time for the sericin and silver sulfadiazine cream– treated wounds to be significantly shorter than that of the silver sulfadiazine cream–treated wounds (22.42 ± 6.33 vs. 29.28 ± 9.27, p = 0.001). In addition, sericin with silver sulfadiazine cream showed a significantly higher percentage of wound reduction than silver sulfadiazine cream after 14 days of treatment (Aramwit et al. 2013). Sericin scaffolds were also developed as wound dressings in the study by Siritientong et al. (2014). The scaffolds composed of sericin (3%), poly(vinyl alcohol) (PVA) (2%), and glycerin (1%) were prepared by freeze-drying methods and ethyl alcohol precipitation. The sericin scaffold could induce fibroblast cell migration to 100% migration at 72 hours in the scratch test. The healing time of wounds treated with the sericin scaffold was significantly shorter than for wounds treated with Bactigras®, which is a fine-mesh gauze impregnated with paraffin and chlorhexidine acetate. No significant difference in skin irritation was found between the sericin scaffold and Bactigras in 110 healthy volunteers. 2.1.3.3  Tissue Engineering

Sericin was prepared in many forms for tissue engineering including solution, film, and scaffolds. Non-mulberry sericin extracted by AlkaliSS seems to be popular in tissue engineering products. Nayak et al. (2013) studied the effect of sericin on the promotion of osseointegration. The NaOH-activated titanium foil (1 × 1 cm) was coated with 1% w/v of sericin at 37 °C for two days using glutaraldehyde as a cross-linker. Coated sericin samples showed increased osteoblast cell adhesion and cell proliferation. The mRNA expression of bone sialoprotein, osteocalcin, and alkaline phosphatase was upregulated in osteoblast cells cultured on sericin-coated samples. Therefore, coating sericin on titanium samples might have the potential to increase the osseointegration in medical applications. Mandal et al. (2009) found that a sericin/gelatin scaffold was cytocompatible for cell attachment and proliferation and could be used in tissue engineering and biomedical applications. In their study, non-mulberry sericin extract was blended with gelatin and cross-linked with glutaraldehyde. Adding sericin to gelatin scaffolds increased the pore size of the scaffold compared to pure gelatin scaffold. 2.1.3.4  Drug Delivery

Sericin was one of the biocompatible agents so it was developed to be a carrier in drug delivery systems in terms of nanoparticles, hydrogels, or scaffolds. Non-mulberry sericin was blended with pluronic surfactant for the formation of nanoparticles. The sericin nanoparticles could deliver both hydrophilic and hydrophobic drugs to target sites (Mandal 2009c). Furthermore, sericin hydrogels were fabricated by cross-linking pure mulberry sericin extract with glutaraldehyde. The sericin hydrogel could be used as a drug delivery vehicle that provided a sustained release (Wang et al. 2014).

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2  Processing Sericin

The major extraction method of mulberry sericin (B. mori) was WaterSS because there were no impurities and the cost was low. However, the most popular extraction method for non-mulberry sericin (A. mylitta) was AlkaliSS. Mulberry sericin extracted by WaterSS and non-mulberry sericin extracted by AlkaliSS showed high phenolic compound levels and antioxidant activity. Moreover, they could promote cell attachment, cell proliferation, cell protection, and collagen promotion. Mulberry sericin (B. mori) extracted by WaterSS was the most popular for use in pharmaceuticals, cosmetics, and wound healing applications while non-mulberry sericin extracted by AlkaliSS was mostly developed in tissue engineering.

2.2  ­Modification of Sericin Structure Sericin is a natural polymer produced by silkworms, and primarily contains hydroxyl, carboxyl, and amino groups, and its blend or copolymers show special reactivities and properties (Kunz et  al. 2016). Sericin has been found to exert many biological activities and applications such as antioxidant activity (Dash et al. 2008; Takechi et al. 2014), antibacterial activity (He et al. 2017b; Karahaliloglu et al. 2017), wound healing (Aramwit et al. 2013; Ersel et al. 2016), antitumor effects (Kaewkorn et al. 2012; Kumar and Mandal 2019), medical materials (Lamboni et al. 2015; Wang et al. 2014; Zhang et al. 2015), supports for immobilized enzymes (Zhang et  al. 2004), and cosmetics (Kunz et  al. 2016). Sericin is easily distinguished from other proteins by its nucleophilic groups. Sericin contains a much higher amount of amino acids with nucleophilic groups than fibroin, with levels of up to 50% of all amino acids. The strong polar side groups of sericin can be conjugated covalently with peptides, proteins, and enzyme (Zhang et al. 2006b). These properties attribute high chemical reactivity of sericin. Additionally, another feature of sericin is that it constitutes hydroxyl groups from Ser, Thr, and Tyr residues, accounting for about 90% of the nucleophilic groups. The modification of these hydroxyl groups is important for the efficient alteration of sericin properties (Takasu et al. 2002; Teramoto et al. 2004). The modification of sericin is summarized in Table 2.4.

2.3  ­Chemical Modification In 2004, Teramoto and colleagues reported the chemical modification of silk sericin using LiCl/dimethyl sulfoxide (DMSO) solvent with 4-cyanophenyl isocyanate. Sericin is a highly hydrophilic protein. LiCl/DMSO was found to be a good solvent of sericin and useful for the homogeneous modification of its abundant hydroxyl groups under nonaqueous conditions. The modified sericins presented that 4-cyanophenyl groups were incorporated into sericin molecules mainly through urethane linkages as shown in Figure 2.1. However, the results demonstrate that the chemical modification of sericin using LiCl/DMSO solvent markedly alters its characteristics via the inhibition of intermolecular hydrogen bonds (Teramoto et al. 2004).

2.3  ­Chemical

Modificatio

Table 2.4  Comparative studies of sericin structure modification. Sericin modification

Chemical interactions

References

4-Cyanophenyl isocyanate using the LiCl/DMSO solvent system

Urethane linkages

Teramoto et al. (2004)

2D cross-linked sericin membranes

Cross-linking with glutaraldehyde

Nayak et al. (2012)

Collagen/sericin hybrid scaffolds

Cross-linking with glutaraldehyde

Mitran et al. (2015)

Sericin/collagen membranes Cross-linking with glutaraldehyde

Akturk et al. (2011)

Ser–Col films

Gallo et al. (2020)

Cross-linking with glutaraldehyde Cross-linking with N-(3dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC)

SS–Ins

Cross-linking with glutaraldehyde

Zhang et al. (2006b)

PEI-functionalized silk sericin

Cross-linking with glutaraldehyde

Kwak and Lee (2018)

Silk–collagen scaffold

Cross-linking with DHT

Bi et al. (2015)

Collagen, sericin, and hyaluronic acid

Cross-linking with carbodiimide

Vulpe et al. (2016)

Sericin/poly (vinyl alcohol)

Cross-linking with DMU

Gimenes et al. (2007)

SS–ASNase

Covalent attachment

Zhang et al. (2006a)

SS-TGase

Enzymatic peptide-bridged cross-linking

Guo et al. (2019)

SMH

Photo-cross-linking

Qi et al. (2018)

SMH/GO

Photo-cross-linking

Qi et al. (2020)

DTT

The transition between disulfide and SH groups leading to free sulfhydryl in protein

Sangwong et al. (2016)

β-ME

The transition between disulfide and SH groups leading to free sulfhydryl in protein

Sangwong et al. (2016)

UV light

UV light may cause structural changes

Sangwong et al. (2016)

Protease enzyme

Short-chain peptides

Sangwong et al. (2016)

4-Cyanophenyl isocyanate NH

O C N

O

CN

NH O O

OH Ser residue in sericin

LiCI/DMSO room temperature

O N H

CN

4-Cyanophenyl isocyanatemodified sericin derivatives (CPI-Src)

Figure 2.1  The reaction between sericin and 4-cyanophenyl isocyanate. Source: Based on Guo et al. (2019).

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2  Processing Sericin

2.4  ­Glutaraldehyde Cross-linking Glutaraldehyde is a functional compound mainly used in the chemical modifications of proteins and polymers. This compound binds covalently to the amine groups of lysine or hydroxylysine in the protein molecules, creating a stable structure (Boratynski 2000). Accordingly, the chemical modification of sericin with cross-linking agents can be used for the reinforcement of the protein structures resulting in sericin stabilization. Nayak and colleagues intended to study the behavior of cross-linked sericin membranes, from the cocoon of non-mulberry tropical silkworm, A. mylitta, prepared using glutaraldehyde as the cross-linking agent for improving the mechanical strength and stability. The physical and structural characteristics of the sericin two-dimensional (2D) membranes were analyzed using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction along with swelling and degradation studies. The secondary structure of the sericin 2D membranes shows that cross-linking using glutaraldehyde provides a more integrated structure that significantly improves the stability and mechanical strength of the membranes. Moreover, the biocompatibility of membranes was evaluated by MTT assay and cell cycle analysis of feline fibroblast cells. The adherence, growth, and proliferation patterns of cells on membranes were assessed by confocal microscopy. The results showed cytocompatibility for supporting the growth and proliferation of fibroblasts (Nayak et al. 2012). Because of the high solubility and weak structural properties, sericin was often blended with natural polymers, such as collagen, to prepare membranes, scaffolds, and hydrogels for tissue engineering, wound dressing application, and cosmetics. The interactions between collagen and sericin in solution had been identified by Duan and colleagues. The results indicate that Tyr of collagen and sericin had close proximity (3 Å) and generated excimers, which weaken the hydrogen bonds and changed the conformation of collagen (Duan et al. 2016). Sericin has been shown to be a suitable material for promoting regeneration and improving tissue function. Mitran and colleagues synthesized three-dimensional (3D) hydrogel scaffolds containing collagen (COLL) and variable concentrations of sericin (SS) in order to find the most suitable formula for adipose tissue engineering applications by COLL cross-linking with glutaraldehyde. The collagen/sericin hybrid scaffolds were developed and evaluated for biocompatibility and biological performance. The developed scaffolds showed biocompatibility and cellular colonization. By the addition of different concentrations of sericin into a constant collagen composition, a dose-dependent influence of sericin on the behavior of preadipocytes colonizing the COLL-based scaffolds was revealed. The collagen hydrogel with 40% sericin presented the highest biological property (Mitran et al. 2015). In accordance with Gallo and colleagues, sericin–collagen (Ser–Col) films were produced by air-drying and subsequently cross-linked through two different cross-linking methods for peripheral nervous system (PNS) regeneration. The results showed enhanced cell proliferation, adhesion, and controlled release in an in vitro study (Gallo et al. 2020). For wound healing applications, the wound dressing membranes were prepared by glutaraldehyde cross-linking between sericin and collagen. Akturk and colleagues prepared sericin/ collagen membrane and evaluated the properties of wound dressings. The researchers report the first membrane using sericin and collagen proteins as a wound dressing material.

2.6  ­Carbodiimide Cross-linkin

However, the wound healing property should be further explored via in vivo studies (Akturk et al. 2011). Moreover, the sericin protein has been reported to enhance the therapeutic value after glutaraldehyde cross-linking due to the biocompatibility, degradability, and oxygen permeability of sericin. Zhang and colleagues studied the modification of insulin with silk sericin to improve insulin stability using glutaraldehyde as a cross-linking agent. The silk sericin– insulin (SS-Ins) was synthesized and physicochemical properties were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The biological activities of SS-Ins bioconjugates were investigated using both in vitro and in vivo studies. The results showed that sericin conjugation allows for the preparation of novel insulin constructs with improved pharmacological properties. The pharmacological activity improved long-term control of blood glucose levels (Zhang et al. 2006b). Kwak and colleagues successfully fabricated polyethylenimine (PEI)-functionalized silk sericin beads using cross-linking with glutaraldehyde for the detoxification of aqueous solutions. Chromium (Cr) is a common metal species that is considerably toxic to humans and other species. The silk sericin beads were modified and functionalized using PEI and glutaraldehyde as shown in Figure 2.2. A number of functional surface groups aimed to achieve a high Cr (VI) removal efficiency by appropriate surface modification. The study further clarified that the Cr (VI) removal process involved electrostatic adsorption coupled with a reduction mechanism (Figure 2.2). This study promises desirable properties such as low cost, metal ion removal capacity, and ­ecological friendly properties (Kwak and Lee 2018).

2.5  ­Dehydrothermal (DHT) Cross-linking Bi and colleagues prepared 3D scaffolds to reconstruct an anterior cruciate ligament (ACL) by dehydrothermal (DHT) cross-linking to improve the mechanical properties of the collagen scaffold. The results indicated that the scaffold was appropriate for the ­reconstruction of ACL in a rabbit model and had the potential for clinical application (Bi et al. 2015).

2.6  ­Carbodiimide Cross-linking Several attempts to obtain tridimensional structures based on collagen and sericin have been proposed using glutaraldehyde or genipin cross-linkers. However, the hydrogels obtained by these methods displayed a certain amount of cytotoxicity. Vulpe and colleagues generated a new type of hydrogel based on collagen, sericin, and hyaluronic acid by carbodiimide cross-linking in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) (Figure  2.3). The results indicated that the obtained hydrogel showed improved biocompatibility due to the addition of sericin. Nevertheless, the molecular dynamics and the reaction yields were decreased by sericin. Further studies should be performed to evaluate the therapeutic potential for skin tissue engineering applications (Vulpe et al. 2016).

51

NH2 NH2 NH 2 NH2 NH2

NH2 Sericin bead

NH2 NH 2

NH2

H NH2

Methanol, 24 hour incubation H O

C

C

O

H

C (CH2)3

H

C N

Sericin

H+

u c ti o n –

N

Sericin bead

PEI

H (CH2)3

Cr3+

Red

PEI

NH2

Poly(ethylene imine)

+

rption

HCrO4Adso –

Cr3+ H+

HCrO4

+

–

HCrO4 + NH3 + – HCrO4 NH3

NH3

+

NH3

I

PE

–

Cr3+

HCrO4 +

NH3

–

HCrO4

+

NH3

Sericin

+

NH3

–

HCrO4

Glutaraldehyde

Figure 2.2  The synthesis of polyethylenimine (PEI)-functionalized silk sericin beads using cross-linking with glutaraldehyde and possible mechanism for Cr (VI) adsorption and reduction to Cr (III). Source: Kwak and Lee (2018). © 2018 Elsevier.

2.8  ­Enzymatic Cross-linkin

Collagen

Hyaluronic acid

Cross-linked network of hydrogel Amide bond

Polymers + EDAC NHS

Sericin

SEM image of hydrogel

O C HN

Figure 2.3  Hydrogel preparation based on collagen, sericin, and hyaluronic acid by carbodiimide cross-linking. Source: Vulpe et al. (2016). © 2016 Elsevier.

2.7  ­Dimethylolurea (DMU) Cross-linking Gimenes and colleagues developed sericin/poly (vinyl alcohol) blend membranes for ethanol dehydration by pervaporation using an appropriate cross-linking agent including dimethylolurea (DMU). This cross-linking agent appears to be appropriate as the crosslinking will occur primarily through the hydroxyl functionality of the polymers. The residue amino groups in sericin are free to interact with water, thereby improving the dehydration performance of the membrane. The permeation and sorption of the permeant in the membranes occurred via a strong coupling effect (Gimenes et al. 2007).

2.8  ­Enzymatic Cross-linking The polar amino acids with hydroxyl, carboxyl, and amino groups of sericin were modified with l-asparaginase (ASNase) to produce silk sericin peptide–l-asparaginase (SS–ASNase) by covalent attachment. Zhang and colleagues investigated the bioconjugation of ASNase with reducing the immunological response and enhancing the drug’s effects in blood. The SS–ASNase is active, stable, has a lower immune response, and has extended half-lives in vitro in human serum due to its resistance to trypsin digestion. The result presents that the affinity of the enzyme to its substrate l-asparagine greatly increases when bioconjugated with silk sericin. The in vivo experiments also show that the silk sericin peptides have no immunogenicity, and the antigenicity of the enzyme is obviously decreased when coupled covalently with the silk sericin peptides (Zhang et al. 2006a). Quo and colleagues fabricated the enzymatic cross-linking of sericin proteins. Transglutaminase (TGase) was combined with sericin using two custom peptides of GQGEGQG (p-Q) and KKKK (p-K), containing exogenous glutamine and lysine residues, respectively, to increase the number of reactive sites and promote the cross-linking of sericin proteins. The structure, water solubility, and mechanical properties were investigated. The results present the potential applications of p-Q for the preparation of sericin-based biomaterials (Guo et al. 2019).

53

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2  Processing Sericin

2.9  ­Physical Modification 2.9.1  Photo-Cross-linking Cartilage injury and age/disease-related cartilage degeneration require the development of minimally invasive strategies for effective cartilage repair. This sericin hydrogel is a promising tissue engineering scaffold for the generation of artificial cartilage. Qi and colleagues proposed preparing an injectable sericin hydrogel by photo-cross-linking to allow a minimally invasive delivery for cartilage repair. The methacryloxy groups were added to the reactive side groups (amine and hydroxyl) of sericin to obtain photo-­cross-linkable sericin methacryloyl (SerMA). When exposed to ultraviolet (UV) light, the SerMA ­solution was rapidly cross-linked in situ, forming a pure sericin hydrogel (SMH) through the photo-polymerization of methacryloyl groups. The SerMA hydrogels exhibited excellent biocompatibility, cell adhesiveness, proliferation-promoting effects, and photoluminescence. Additionally, SMH effectively supports the attachment and growth of chondrocytes and promotes the generation of cartilage-specific extracellular matrix components in in vitro study. It effectively formed an in situ artificial cartilage with a morphological, cellular, molecular, and mechanical resemblance to native cartilage. This is the first study to develop a minimally invasive cartilage repair strategy using sericin hydrogel as a 3D biomimetic extracellular matrix via photo-cross-linking (Qi et  al. 2018). To improve its mechanical property and enhance its osteogenic induction ability, SerMA solution was mixed with different concentrations of graphene oxide (GO) to form SMH/ GO composite hydrogels (SMH/GO) as a biomimetic scaffold. The SMH/GO hydrogels are capable of effectively increasing the number of osteoblasts both at the early and at late stages of bone regeneration. This modification may provide a new type of medical material for effective bone regeneration due to its flexibility, low cost, and practicality (Qi et al. 2020). There are many methods that contribute to the structural and biological effects of sericin. The conformational and functional changes have been explored by the relationship between modification methods and sericin properties. Sangwong and colleagues investigated the relationship between sericin protein treated with chemical and physical modifications. Sericin from the cocoons of polyvoltine silkworm strain, Nangnoi, was extracted and fractionated by salting out and modified with dithiothreitol (DTT), β-mercaptoethanol (β-ME), UV light exposure, and hydrolysis by a protease from Streptomyces griseus. The relationship between modified sericin protein and its antioxidant activity was determined by ABTS and DPPH assays. The antioxidant activity of crude sericin extract (CSE) treated with protease enzyme exhibited an excellent antioxidant activity compared to that of untreated CSE, while DTT, β-ME, and UV light treatments were found to significantly decrease the antioxidant activity of CSE. Later, CSE was fractioned by salting out. The colorless supernatant (SNT) fraction showed the highest antioxidant activity. However, the antioxidant property of SNT presented results similar to those of treated CSE. This study suggests that sericin modification with protease enzymes could be used to enhance the antioxidant activity of sericin in biological applications (Sangwong et al. 2016). The development of functional substitutes through chemical and physical modification is considered a possibility for the alteration of protein functions. Our information highlights

2.10  ­Forms of Sericin Processin

the modification of sericin, which affects the structural stability of sericin and provides the modified sericin protein with numerous valuable applications.

2.10  ­Forms of Sericin Processing After processing, sericin can be formed as 2D or 3D structure for various applications. All forms of processed sericin are mainly used in medical applications such as wound healing, tissue engineering, or cell culture applications while some of them are used in food and engineering industries.

2.10.1  Two-dimensional Structure of Sericin 2.10.1.1  Hydrogel

Hydrogel is the most common form of 2D structure of biomaterials due to the ease of processing and wide range of applications. Sericin can be easily formed into a hydrogel using various techniques such as alcohol precipitation (Teramoto et al. 2005) or using chemical cross-linking agents genipin (Siritientong et al. 2013) or glutaraldehyde (Wang et al. 2014). Sericin hydrogel also can be formed without using any chemicals but using mechanical techniques such as varying temperatures to cause cross-linking process by freeze–thaw (Aramwit et al. 2018) or ultrasonication (Zhang et al. 2019). As mentioned earlier, sericin hydrogel recently has been used for wound treatment. However, cell culture and drug delivery purposes are also applicable (Figure 2.4).

Figure 2.4  Sericin hydrogel fabricated using the freeze–thaw technique.

55

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2  Processing Sericin

2.10.1.2  Film

Sericin itself can be formed as a film but may have weak mechanical properties. In order to solve this problem, several polymers e.g. polyvinyl alcohol, polyoxyethylene– polyoxypropylene block copolymer (Maikrang and Aramwit 2009) have been used to blend with sericin. Moreover, sugar alcohols such as glycerin are widely used to improve the mechanical properties of sericin film (Yun et al. 2016). Cross-linking agents may also be used to stabilize the film. Sericin film is simply formed using the casting technique (Figure 2.5).

2.10.2  Three-dimensional Structure of Sericin Apart from medical applications, three-dimensional forms are very useful in various purposes, including bioengineering and cell culture. The different techniques and comaterials used result in variety of morphologies with different mechanical properties. There are several techniques for fabricating a scaffold, sponge, or 3D hydrogel from sericin. The most common are electrospinning, freeze-drying, salt leaching, and gas forming. 2.10.2.1  Electrospinning Silk Sericin and Its Blends

Sericin is a unique protein that is available in large quantities, but utilized to a limited extent. Unlike the silk protein fibroin, sericin is water-soluble, which is both an advantage and a disadvantage depending on the application. Considerable efforts have been made to develop sericin-based materials. Since electrospinning produces fibers similar to that of extracellular matrix and with properties that are desirable for tissue engineering and other medical applications, attempts have also been made to develop electrospun sericin in pure and blended forms (Khan and Tsukada 2014). Although most studies use sericin with synthetic polymers such as PVA and polycaprolactone (PCL), blends with gelatin and other natural polymers have also been produced. In addition, researchers have developed electrospun blends of fibroin and sericin. Although most studies use sericin from B. mori cocoons, electrospinning of sericin from wild silks has also been reported.

Figure 2.5  Sericin with genipin (cross-linking agent) film.

2.10  ­Forms of Sericin Processin

2.10.3  Pure Sericin Electrospun Fibers In one of the earliest studies on developing pure sericin electrospun fibers, sericin was dissolved in 1.3–22.9 wt% solutions in trifluoroacetic acid (TFA) at 25 °C. Dissolved sericin was electrospun into fibers using a 21 gauge needle, voltage of 25 V, and extrusion rate of 3.2 ml/h (Khan et al. 2013). The ability to form fibers and the morphology of the fibers were heavily dependent on the concentration of sericin used. For instance, only beads were formed at concentrations between 1.3% and 3.8%, and spindle-like beads and fibers with beads were obtained from 9.6% to 16.5%. Fibers with smooth surfaces (Figure  2.6) were obtained at concentrations between 20.9% and 22.9%, with the optimum concentration suggested to be 20.9%. The structure of sericin transforms from β-sheet to random coil in solution but reverts to β-sheet after heating at 160 °C for 60 minutes. The fibers had good thermal stability and TFA could be removed by heating, making the fibers suitable for various medical and other applications. Sericin could also be made into electrospun fibers by dissolving 50–60% in water at 85 °C for 30 minutes. The average diameter of the fibers obtained was between 330 and 395 nm at a spinning voltage between 13 and 25 kV (Zhang et al. 2011c). The properties of sericin affect not only electrospinability but also the properties of the fibers and scaffolds developed. In a study by Yang et al., the effects of different molecular weight sericin on electrospinning and the properties of the fibers obtained were studied.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Figure 2.6  SEM pictures depict the formation of beads and fibers with increasing concentration of sericin from 8.5% to 22.9%. Various concentrations used were 8.5% (a), 9.6% (b), 11.7% (c), 14.2% (d), 16.5% (e), 20.9% (f), and 22.9% (g). Source: Khan et al. (2013). Reproduced with permission from Springer.

57

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2  Processing Sericin

Table 2.5  Comparison of the amino acids in high- and low-molecular-weight sericins (Mol%). Amino acid

High molecular weight

Low molecular weight

Ala

4.0

5.0

Gly

14.5

15.2

Try

3.4

3.0

Ser

35.8

34.8

Asp

15.7

15.0

Arg

3.1

3.1

His

1.5

1.6

Glu

4.7

4.5

Lys

2.7

2.7

Val

3.2

3.7

Leu

1.0

0.6

Ile

0.8

0.7

Phe

0.7

0.6

Pro

0.6

1.2

Thr

8.1

8.0

Met

0.2

0.1

Cys

0.2

0.2

Source: Based on Yang et al. (2014).

Sericin with low molecular weight (LMW, 12 kDa) and with high molecular weight (HMW, 66 kDa) was extracted from B. mori cocoons. Extracted sericin was dissolved in hexafluoro acetic acid (HFA) at room temperature for 24 hours in 5–10% concentrations. Electrospinning was done at a voltage of 15 kV and flow rate of 1.2 ml/h; the fibers obtained were immersed in 90% ethanol for 12 hours and dried. Considerable differences in the composition (Table 2.5) and structure of the LMW and HMW sericins were observed, which affected the fiber morphology and properties. LMW sericin formed cylinder-like particles of 200–300 nm, whereas HMW sericin formed agglomerated particles of about 1 μm. Lower concentrations (four weeks). The fabricated sericin-based nanofibrous matrices indicated biocompatibility and potential wound healing efficacy (Sapru et al. 2018). Researchers have successfully fabricated scaffolds with sericin, PVA, glycerin (as a plasticizer), and genipin (as a crosslinking agent). The physical and wound healing properties of genipin-cross-linked sericin–PVA scaffolds were evaluated in the dorsal skin of Sprague– Dawley rats. These genipin-cross-linked sericin–PVA scaffolds represent a promising candidate biomaterial for the accelerated healing of full-thickness wounds, as confirmed by the wound size reduction, the level of inflammatory reactions, collagen formation, and epithelialization (Aramwit et al. 2013). Moreover, antibacterial scaffolds were synthesized to minimize the bacterial infection of burns and other wounds using chitosan–silk sericin 3D porous scaffolds. These scaffolds were synthesized and characterized to ensure a suitable material. The physicochemical properties, and biocompatibility, with excellent antibacterial activity, provide promising scaffolds as wound dressing composite materials (Karahaliloglu et al. 2017) (Figure 3.6). Figure 3.6  Soft and flexible sericin–PVA– glycerin hydrogel for medical and food applications.

3.3  ­Biotechnology Application

The fabrication of injectable in situ sericin hydrogels has also been reported. This work presented the formulation of non-mulberry tropical Tasar cocoon sericin hydrogels of Antheraea mylitta through the physical entrapment of sericin within the 3D hydrophilic network of polyacrylamide aimed at dermal reconstruction. This formulation enables the migration of fibroblasts during the healing processes, as confirmed by confocal and scanning electron microscopy. Cell adhesion along with biocompatibility suggests its potential as an in situ tissue sealant (Kundu and Kundu 2012). In an in vivo study, the regeneration of skin using non-mulberry silk protein sericin blend hydrogels promoted the reconstruction of the skin tissue after being implanted subcutaneously in Wistar rats. The infiltration of skin tissue cells into the hydrogels mark their biocompatibility and nontoxicity (Sapru et al. 2019). Furthermore, fibroblast growth factor-1 (FGF1) has a potential effect on wound healing. The fabricated FGF1 sericin hydrogels presented injectability, a porous microstructure, biocompatibility, and no immunogenicity, which contributed to cell adhesion and survival in a mouse embryonic fibroblast cell line. Moreover, FGF1 protected against degradation during sericin processing and gelation and achieved long-term stability in the sericin hydrogels (Wang et al. 2018). Many fabrications of sericin-based biomaterials for tissue repair indicated sericin as an excellent prospect to be used for skin regeneration. 3.3.1.2  Sericin-Based Materials for Bone and Cartilage Tissue Engineering

Researchers have successfully formed sericin-based materials for bone tissue engineering. Normally, the healing processes in bone or cartilage regeneration are limited by the source of tissue, infection, and poor biocompatibility of tissue (Amini et al. 2012; Ikada 2006). The delivery of bioactive agents (i.e. growth factors, genes, and anti-inflammatory drugs) promotes beneficial effects in bone tissue engineering. Antibiotics have been used to prevent infection after bone implantation. Gentamicin sulfate (GS) is the antibiotic of choice for the addition of poly(l-lactic acid)–sericin hybrid scaffolds because of the broad antibacterial spectrum of action, the low rate of primarily resistant pathogens, and the low allergy rate, making this suitable for bone regeneration (Klemm 2001). The GS-loaded poly (l-lactic acid)–sericin hybrid scaffolds were successfully synthesized by the particulate leaching method. The scaffold properties were observed to show inhibitory activity against bacteria. The inhibitory effect of scaffolds for the incorporation of GS into the scaffolds presented bacterial growth inhibition of E. coli TISTR 780 and S. aureus TISTR 1466 (Pankongadisak et al. 2018). Osteogenic differentiation is an important process in the generation of multiple cell types, including adipocytes, chondrocytes, and osteocytes (Birmingham et al. 2012). The regulation of osteogenic differentiation plays a possible role in bone engineering applications. To provide sericin-based materials as bio-functions for osteogenic differentiation, the human platelet-derived growth factor (PDGF-BB)–sericin hydrogel was evaluated for cell proliferation and osteogenic differentiation. PDGF-BB is a potent mitogen, which regulates bone formation. The fabrication of PDGF-BB-functionalized sericin hydrogel materials in this study showed the efficacy of PDGF-BB–sericin hydrogel-promoted proliferation and enhanced BMP-9-induced osteoblastic differentiation of mesenchymal stem cells (MSCs) in both in vitro and in vivo studies (Wang et al. 2020). Sericin has the potential to be used as a membrane for bone tissue engineering. In one such study, cellulose acetate membrane was functionalized with sericin; the functionalized

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membrane was characterized by different techniques to confirm the successful synthesis of sericin covalent immobilization onto the cellulose acetate membrane. The functional properties of the membrane were observed in MC3T3-E1 pre-osteoblasts while showing noncytotoxicity, good cell adhesion, and a typical polygonal morphology (Voicu et al. 2016). The nucleation of hydroxyapatite (HAP) is involved in bone biomineralization. Previous reports of solid films or films on the surface of raw silk fibers have been used as a template to form HAP crystals (Takeuchi et al. 2005; Yang et al. 2014). Sericin has the potential to induce HAP nucleation because of its high molecular weight and beta-sheet structure (Takeuchi et al. 2005). Moreover, Ca2+ in the HAP precursor solution induces the conformational change of sericin from random coils into beta-sheets resulting in the proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs) (Yang et al. 2015). There are many patients who suffer from cartilage injury and age-/disease-related cartilage degeneration. Cartilage regeneration is limited by poor self-healing potential. The development of an alternative method for effective cartilage repair using biomaterial is desired. Scaffolds that promote cartilage regeneration were developed using collagen and sericin-based materials, supplemented with prochondrogenic factors such as chondroitin sulfate or hyaluronic acid for cartilage tissue engineering. The scaffold properties showed a porous structure with pore sizes between 20 and 150 μm, high capacity for water absorption, an undenatured triple helical structure of collagen, and biocompatibility. Furthermore, another report fabricated photo-cross-linkable sericin hydrogels to provide a favorable microenvironment for cartilage repair by chondrocyte attachment, proliferation, and the accumulation of cartilage-specific ECM components (Qi et  al. 2018). However, tissue regeneration using sericin-based materials could be further investigated to confirm the efficacy in cartilage tissue in a clinical study (Dinescu et al. 2013). These studies demonstrate that sericin-based materials can provide useful biomaterials to support cell and tissue outcomes in bone and cartilage tissue engineering. 3.3.1.3  Sericin-Based Biomaterials for Other Tissues

The bacterial cellulose scaffolds incorporating sericin were developed for gut repair. The physical characterization of bacterial cellulose–sericin scaffolds was observed in the release of sericin, its stability, and the swelling of scaffolds in water. The incorporation of sericin stabilized the network and enabled the slow release of free sericin under physiological conditions with increased material stiffness and decreased water swelling ability. Moreover, the incorporation of sericin into cellulose provides a smooth surface that becomes advantageous for gastrointestinal applications (Lamboni et al. 2019). Chronic nerve compression (CNC) impairs the structure and microvasculature of peripheral nerves and is often the result of nerve damage or a malfunctioning nervous system (Rayan 1992). The restoration of nerve function and the microstructure of compressed nerves was developed using a tissue-engineered scaffold to deliver an appropriate cytokine to injured sites. A chitosan–sericin composite scaffold was fabricated to deliver a nerve growth factor. The advantages presented were high porosity, adjustable mechanical properties, swelling ratios, the support of Schwann cells growth, and improved nerve

3.5  ­Conclusio

regeneration. In the CNC model, the scaffold showed the ability to deliver a nerve growth factor for the treatment of peripheral nerve compression (Zhang et al. 2017). Sericin was identified as a biomaterial for minimally invasive myocardial infarction repair. Sericin was prepared in the form of an injectable hydrogel and its therapeutic outcomes were evaluated in a mouse myocardial infarction model. Sericin hydrogel was injected into areas of myocardial infarction. The scar formation and infarct size were drastically decreased in association with the downregulation of pro-inflammatory cytokines (TNF-α and IL-18) and chemokines. Additionally, sericin enhanced angiogenic activity by promoting migration and the tubular formation of human umbilical vessel endothelial cells (HUVECs) (Song et al. 2016). Sericin is a suitable material for tissue engineering, such as adipose tissue engineering, due to its various biological properties. The scaffold was synthesized as a hybrid scaffold with collagen and sericin. The addition of variable concentrations of sericin showed that sericin improved the cell adhesion and subsequent cell proliferation in the scaffold. This study concluded that the highest biological performance in the scaffold containing 40% sericin provides a useful material for soft tissue engineering (Mitran et al. 2015) (Table 3.5).

3.4 ­Miscellaneous Application 3.4.1  Sericin-Coated Material as an Air filter Sericin coatings have been commonly found in the textile industry, with several purposes such as easy ironing, and nonfading fabric dye. In addition, sericin is also coated on the surface of polyester fabrics for use as an air filter (Verma et al. 2019). Using a simple dipcoating method followed by thermal fixation, changes in surface functionality and the morphology of polyester fibers were studied. Sericin-coated filters were able to remove PM2.5 and PM10 from the 1000 μg/m3 level to 5 μg/m3 in a 6.28 m3 chamber within 27 and 23 minutes of operation, respectively. Additionally, the sericin-coated air filter also proved very effective for the removal of volatile organic compounds (benzene, toluene, ethylbenzene, and xylene) from an indoor chamber at a varying initial concentration of 100–1000 μg/ m3. The use of sericin in this aspect is quite attractive, since particulate matter and volatile organic compounds have emerged as prime environmental concerns with increasing air pollution now seen worldwide.

3.5 ­Conclusion The application of sericin has shown benefits in several areas. The main concern, especially for medical purposes, is still the purity and characterization of sericin from various strains and via different extraction methods. Even though this gumming silk protein has been widely investigated, the commercial products from this biomaterial are still limited. The availability and quality control of this protein needs to be standardized to promote its use commercially.

121

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3  Applications of Sericin

Table 3.5  Sericin biomaterials for tissue engineering. Type of material

Supported cells/organisms

Advantage properties

References

Skin tissue repair with sericin-based materials Sericin/collagen membrane

Fibroblasts and keratinocytes

Sericin-based nanofibrous matrices

Human keratinocytes (HaCaT), Wistar rats

Genipin-crosslinked sericin– PVA scaffolds

Sprague–Dawley rats

Chitosan–silk sericin 3D porous scaffolds

Human keratinocytes (HaCaT)

●● ●●

●●

●●

●●

●●

●●

Sericin– polyacrylamide hydrogel

The feline fibroblast (AH 927)

Sericin–chitosan hydrogel

Human keratinocytes, dermal fibroblasts, and Wistar rats

●●

●●

●●

Mouse embryonic fibroblast FGF1cell line (NIH/3T3) functionalized sericin hydrogels

●●

Support cell adhesion Present a stable property four weeks in water

Akturk et al. (2011)

Improve mechanical strength and stability Provide biocompatibility and wound healing efficacy

Sapru et al. (2018)

Accelerate healing of full-thickness wounds

Aramwit et al. (2013)

Improve proliferation and extended viability for HaCaT cells Exhibit antibacterial activity

Karahaliloglu et al. (2017)

Provide cell adhesion and enable migration of fibroblasts

Kundu and Kundu (2012)

Enhance adhesion, proliferation, and migration of skin cells Promote the reconstruction of skin after implantation in rats

Sapru et al. (2019)

Show no cell toxicity or inflammatory response

Wang et al. (2018)

Sericin-based materials for bone and cartilage tissue engineering GS-loaded poly (l-lactic acid)–sericin hybrid scaffolds

Mouse osteoblast cell line (MC3T3-E1), E. coli TISTR 780, and S. aureus TISTR 1466

●●

●●

PDGF-BB– sericin hydrogel

Mouse embryonic fibroblast cell line (NIH/3T3), mouse macrophage cell line (RAW 264.7), mouse mesenchymal stem cell line (C3H10T1/2), and athymic nude mice

Sericin–cellulose Preosteoblasts (MC3T3-E1) acetate membrane

●●

●●

●●

Improve the attachment and proliferation of cells on the surface of the scaffolds Present inhibitory activity against bacteria

Pankongadisak et al. (2018)

Wang et al. Contribute the cell (2020) adhesion and growth. Provide synergy to support the osteoblastic differentiation of mesenchymal stem cells Contribute cell adhesion, viability, and proliferation

Voicu et al. (2016)

  ­Reference

Table 3.5  (Continued) Type of material

Supported cells/organisms

Sericin nanofibrous

Human bone marrow-derived mesenchymal stem cells (BMSCs)

Injectable sericin Mouse myoblast cells hydrogel (C2C12), human umbilical vein endothelial cells (HUVEC), and primary chondrocytes from human fresh knee cartilage tissue.

Advantage properties ●●

●●

●●

References

Promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs)

Yang et al. (2015)

Contribute the cell adhesion and growth. Provide favorable microenvironment for cartilage repair

Qi et al. (2018)

Improve cell growth and differentiation on the structured bacterial cellulose

Lamboni et al. (2019)

Deliver a nerve growth factor Support Schwann cells growth Improve nerve regeneration

Zhang et al. (2017)

Sericin-based biomaterials for other tissues Sericin–bacterial Primary smooth muscle and enteric nervous system cells cellulose from the gut of postnatal scaffolds BALB/c mice Chitosan–sericin Schwann cells (RSC96), mouse macrophage cell line composite (RAW264.7), scaffold pheochromocytoma cells (PC12), and rats Sericin hydrogel

Human umbilical vein endothelial cells (HUVECs), human iPSC-derived cardiomyocytes (iPSC-CMs), mouse fibroblast cells (NIH3T3), mouse macrophages (RAW264.7), and rat cardiomyocytes (H9c2), and C57BL/6 mice

Collagen–sericin Preadipocytes (3T3-L1) scaffold

●●

●●

●●

●●

●●

●●

●●

Song et al. Influence the intrinsic capability of inducing cells (2016) to produce VEGF, antagonizing endothelial cells and cardiomyocytes apoptosis, and inhibiting inflammatory responses in in vitro Promotes cardiac functional recovery in in vivo Promote cell survival, proliferation, and cellular colonization

Mitran et al. (2015)

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Kaewkon, W., Aonsri, C., Tiyaboonchai, W. et al. (2012). Sericin consumption suppresses development and progression of colon tumorigenesis in 1,2-dimethylhydrazine-treated rats. Biologia 67 (5): 1007–1012. Kaewkorn, W., Limpeanchob, N., Tiyaboonchai, W. et al. (2012). Effects of silk sericin on the proliferation and apoptosis of colon cancer cells. Biological Research 45 (1): 45–50. Kanoujia, J., Singh, M., Singh, P., and Saraf, S.A. (2016). Novel genipin crosslinked atorvastatin loaded sericin nanoparticles for their enhanced antihyperlipidemic activity. Materials Science and Engineering C: Materials for Biological Applications 69: 967–976. Karahaliloglu, Z., Kilicay, E., and Denkbas, E.B. (2017). Antibacterial chitosan/silk sericin 3D porous scaffolds as a wound dressing material. Artificial Cells, Nanomedicine and Biotechnology 45 (6): 1–14. Katiyar, S.K., Korman, N.J., Mukhtar, H., and Agarwal, R. (1997). Protective effects of silymarin against photocarcinogenesis in a mouse skin model. Journal of the National Cancer Institute 89 (8): 556–566. Kato, N., Sato, S., Yamanaka, A. et al. (1998). Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Bioscience, Biotechnology, and Biochemistry 62 (1): 145–147. Kim, Y.H., Kim, D., Hong, A.R. et al. (2019). Therapeutic potential of rottlerin for skin hyperpigmentary disorders by inhibiting the transcriptional activity of CREB-regulated transcription coactivators. Journal of Investigative Dermatology 139 (11): 2359–2367. e2352. Kitisin, T., Maneekan, P., and Luplertlop, N. (2013). In-vitro characterization of silk sericin as an anti-aging agent. Journal of Agricultural Science 5 (3): 54. Klemm, K. (2001). The use of antibiotic-containing bead chains in the treatment of chronic bone infections. Clinical Microbiology and Infection 7 (1): 28–31. Kumar, J.P. and Mandal, B.B. (2017). Antioxidant potential of mulberry and non-mulberry silk sericin and its implications in biomedicine. Free Radical Biology and Medicine 108: 803–818. Kumar, J.P. and Mandal, B.B. (2019). Silk sericin induced pro-oxidative stress leads to apoptosis in human cancer cells. Food and Chemical Toxicology 123: 275–287. Kumar, P., Kumar, D., Sikka, P., and Singh, P. (2015). Sericin supplementation improves semen freezability of buffalo bulls by minimizing oxidative stress during cryopreservation. Animal Reproduction Science 152: 26–31. Kumar, J.P., Alam, S., Jain, A.K. et al. (2018). Protective activity of silk sericin against UV radiation-induced skin damage by downregulating oxidative stress. ACS Applied Bio Materials 1 (6): 2120–2132. Kundu, B. and Kundu, S.C. (2012). Silk sericin/polyacrylamide in situ forming hydrogels for dermal reconstruction. Biomaterials 33 (30): 7456–7467. Lakatos, P.L. and Lakatos, L. (2008). Risk for colorectal cancer in ulcerative colitis: changes, causes and management strategies. World Journal of Gastroenterology 14 (25): 3937–3947. Lamboni, L., Li, Y., Liu, J., and Yang, G. (2016). Silk sericin-functionalized bacterial cellulose as a potential wound-healing biomaterial. Biomacromolecules 17 (9): 3076–3084. Lamboni, L., Xu, C., Clasohm, J. et al. (2019). Silk sericin-enhanced microstructured bacterial cellulose as tissue engineering scaffold towards prospective gut repair. Materials Science and Engineering C: Materials for Biological Applications 102: 502–510. Lapphanichayakool, P., Sutheerawattananonda, M., and Limpeanchob, N. (2017). Hypocholesterolemic effect of sericin-derived oligopeptides in high-cholesterol fed rats. Journal of Natural Medicines 71 (1): 208–215.

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Okazaki, Y., Kakehi, S., Xu, Y. et al. (2010). Consumption of sericin reduces serum lipids, ameliorates glucose tolerance and elevates serum adiponectin in rats fed a high-fat diet. Bioscience, Biotechnology, and Biochemistry 74 (8): 1534–1538. Padamwar, M.N., Pawar, A.P., Daithankar, A.V., and Mahadik, K.R. (2005). Silk sericin as a moisturizer: an in vivo study. Journal of Cosmetic Dermatology 4 (4): 250–257. Panilaitis, B., Altman, G.H., Chen, J. et al. (2003). Macrophage responses to silk. Biomaterials 24 (18): 3079–3085. Pankongadisak, P., Jaikaew, N., Kiti, K. et al. (2018). The potential use of gentamicin sulfateloaded poly(l-lactic acid)-sericin hybrid scaffolds for bone tissue engineering. Polymer Bulletin 76 (6): 2867–2885. Patwardhan, J. and Bhatt, P. (2015). Ultraviolet-B protective effect of flavonoids from Eugenia caryophylata on human dermal fibroblast cells. Pharmacogn Magazine 11 (Suppl 3): S397–S406. Qi, C., Liu, J., Jin, Y. et al. (2018). Photo-crosslinkable, injectable sericin hydrogel as 3D biomimetic extracellular matrix for minimally invasive repairing cartilage. Biomaterials 163: 89–104. Rattana, S., Katisart, T., Butiman, C., and Sungthong, B. (2017). Antihyperglycemic effect of silkworm powder, fibroin and sericin from these three Thai silkworm (Bombyx mori Linn.) in streptozotocin-induced diabetic rats. Pharmacognosy Journal 9 (4): 559–564. Rayan, G.M. (1992). Proximal ulnar nerve compression. Cubital tunnel syndrome. Hand Clinics 8 (2): 325–336. Rikans, L.E. and Hornbrook, K.R. (1997). Lipid peroxidation, antioxidant protection and aging. Biochimica et Biophysica Acta 1362 (2–3): 116–127. Sahu, N., Pal, S., Sapru, S. et al. (2016). Non-mulberry and mulberry silk protein sericins as potential media supplement for animal cell culture. BioMed Research International 2016: 7461041. Sapru, S., Das, S., Mandal, M. et al. (2018). Prospects of nonmulberry silk protein sericin-based nanofibrous matrices for wound healing – In vitro and in vivo investigations. Acta Biomaterialia 78: 137–150. Sapru, S., Das, S., Mandal, M. et al. (2019). Nonmulberry silk protein sericin blend hydrogels for skin tissue regeneration – in vitro and in vivo. International Journal of Biological Macromolecules 137: 545–553. Sasaki, M., Kato, N., Watanabe, H., and Yamada, H. (2000a). Silk protein, sericin, suppresses colon carcinogenesis induced by 1,2-dimethylhydrazine in mice. Oncology Reports 7 (5): 1049–1052. Sasaki, M., Yamada, H., and Kato, N. (2000b). A resistant protein, sericin improves atropineinduced constipation in rats. Food Science and Technology Research 6 (4): 280–283. Sasaki, M., Kato, Y., Yamada, H., and Terada, S. (2005). Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin. Biotechnology and Applied Biochemistry 42 (Pt 2): 183–188. Scrivano, L., Iacopetta, D., Sinicropi, M.S. et al. (2017). Synthesis of sericin-based conjugates by click chemistry: enhancement of sunitinib bioavailability and cell membrane permeation. Drug Delivery 24 (1): 482–490. Senakoon, W., Nuchadomrong, S., Sirimungkararat, S. et al. (2009). Antibacterial action of eri (samia ricini) sericin against escherichia. Asian Journal of Food and Agro-Industry 2 (Special Issue): S222–S228.

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Yalcin, E., Kara, G., Celik, E. et al. (2019). Preparation and characterization of novel albuminsericin nanoparticles as siRNA delivery vehicle for laryngeal cancer treatment. Preparative Biochemistry and Biotechnology 49 (7): 659–670. Yang, M., Shuai, Y., Zhou, G. et al. (2014). Nucleation of hydroxyapatite on Antheraea pernyi (A. pernyi) silk fibroin film. Bio-Medical Materials and Engineering 24 (1): 731–740. Yang, M., Zhou, G., Shuai, Y. et al. (2015). Ca(2+)-induced self-assembly of Bombyx mori silk sericin into a nanofibrous network-like protein matrix for directing controlled nucleation of hydroxylapatite nano-needles. Journal of Materials Chemistry B 3 (12): 2455–2462. Zhang, Y.Q., Tao, M.L., Shen, W.D. et al. (2004). Immobilization of l-asparaginase on the microparticles of the natural silk sericin protein and its characters. Biomaterials 25 (17): 3751–3759. Zhang, Y.Q., Ma, Y., Xia, Y.Y. et al. (2006). Silk sericin-insulin bioconjugates: synthesis, characterization and biological activity. Journal of Controlled Release 115 (3): 307–315. Zhang, Y., Liu, J., Huang, L. et al. (2015). Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery. Scientific Reports 5: 12374. Zhang, L., Yang, W., Tao, K. et al. (2017). Sustained local release of NGF from a chitosansericin composite scaffold for treating chronic nerve compression. ACS Applied Materials and Interfaces 9 (4): 3432–3444. Zhao, R., Li, X., Sun, B. et al. (2014). Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings. International Journal of Biological Macromolecules 68: 92–97. Zhaorigetu, S., Sasaki, M., Watanabe, H., and Kato, N. (2001). Supplemental silk protein, sericin, suppresses colon tumorigenesis in 1,2-dimethylhydrazine-treated mice by reducing oxidative stress and cell proliferation. Bioscience, Biotechnology, and Biochemistry 65 (10): 2181–2186. Zhaorigetu, S., Yanaka, N., Sasaki, M. et al. (2003a). Inhibitory effects of silk protein, sericin on UVB-induced acute damage and tumor promotion by reducing oxidative stress in the skin of hairless mouse. Journal of Photochemistry and Photobiology B: Biology 71 (1–3): 11–17. Zhaorigetu, S., Yanaka, N., Sasaki, M. et al. (2003b). Silk protein, sericin, suppresses DMBATPA-induced mouse skin tumorigenesis by reducing oxidative stress, inflammatory responses and endogenous tumor promoter TNF-alpha. Oncology Reports 10 (3): 537–543. Zhaorigetu, S., Sasaki, M., and Kato, N. (2007). Consumption of sericin suppresses colon oxidative stress and aberrant crypt foci in 1,2-dimethylhydrazine-treated rats by colon undigested sericin. Journal of Nutritional Science and Vitaminology (Tokyo) 53 (3): 297–300.

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4 Non-silk Applications of Mulberry Plants 4.1 ­Introduction Mulberry is synonyms with silkworms and sericulture and is the only feed for Bombyx mori silkworms. Although there are innumerable species and different varieties of mulberry, Morus spp., a genus belonging to the Moraceae family of the Urticales subclass, is most commonly used for rearing B. mori silkworms. In addition to the leaves that are used for feeding the silkworms, mulberry plants also produce fruits, branches, and stems. The fruits are collected as a source of food, whereas the stems are discarded as waste and typically burnt or buried. In addition to using mulberry leaves as feed for B. mori, researchers have shown that the leaves and stems contain several valuable compounds that have high medicinal properties (Figure 4.1). However, the composition and yield of the compounds depend on the age of the plant and part of the plant being considered and the extraction methods used. For example, variations in the composition between leaves, edible branches, and stems obtained from plants after 6, 8, and 10 months after plantation have been reported. Extensive studies have been done to develop methods to extract the components in mulberry plants and understand their composition, biochemical functions, and applications. Although considerably fewer studies have been done in comparison to leaves, mulberry stems have also reported to contain antioxidant and antimicrobial chemicals. The mulberry leaves and stems have also been used as a source for paper, fibers, pulp, nanomaterials, composites, sorption of various pollutants, etc. In this chapter, we present the nonsilk uses of mulberry leaves and stems. Although the focus of the book is on the utilization of silk and mulberry by-products and leaves of mulberry may not be considered a by-­ product, application of the leaves, branches, and stems for non-silkworm applications has been extensively reported and hence included to provide comprehensive information on the potential of mulberry plants. Similarly, although not from the same species as Morus alba, the potential of using paper mulberry (Morus papyrifera L) tree as a source of fibers and pulp has also been reported.

Sustainable Uses of Byproducts from Silk Processing, First Edition. Narendra Reddy and Pornanong Aramwit. © 2021 WILEY-VCH GmbH. Published 2021 by WILEY-VCH GmbH.

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4  Non-silk Applications of Mulberry Plants Mulberry sources

Representative functional ingredients

Polysaccharides

Mulberry fruits

Mulberry leaves

Mulberry stems, branches and roots,

Phenols: resveratrol, oxyresveratrol, chlorogenic acid, mulberroside, maclurin, moracins

Functionality -Anti-diabetic -Antioxidant -Anti-inflammatory -Anti-apoptotic -Promoting dendritic cell maturation -Antibacterial -Anti-obesity -Immunostimulatory effect -Antioxidant -Anti-inflammatory -Anti-apoptotic -Antibacterial -Antihyperlipidemic -Neuroprotective -Antibrowning -Inhibitory of α-glucosidase

Anthocyanins*: cyanidin-3glucoside, cyanidin-3-rutinoside, geranium-3-glucoside

-Antioxidant -Anti-inflammatory -Anticancer -Neuroprotective activity -Amelioration of insulin resistance

Non-anthocyanins Flavonoids: rutin, quercertin, kaempferol-3rutinoside

-Anti-inflammatory -Antibacterial -Anti-immobility -Anti-apoptotic -Hepatoprotective activity -Inhibitory effect on NO production -Inhibitory of tyrosinase -Inhibitory of tyrosine phosphatse 1B

Alkaloids: DNJ, fagomine

-Inhibitory of α-glucosidase -Anti-diabetic -Amelioration of insulin resistance -Anti-obesity -Antiviral -Anticarcinogenic

Figure 4.1  Major components in mulberry plants and their phytochemical significance. Source: Wen et al. (2019). © 2019 Elsevier.

4.2 ­Medicinal Applications of Mulberry Plant Extracts 4.2.1  Polysaccharides Polysaccharides from mulberry plants are reported to have high antioxidant, anti-tumor, anti-diabetic, and several other medicinal properties (Yuan et al. 2015; Zhang et al. 2014). A study was conducted to understand the possibility of extracting polysaccharides from mulberry leaves (Thirugnanasambandham et al. 2015) using a microwave-assisted process. To obtain the polysaccharides, mulberry leaves were made into a fine powder and heated

4.2  ­Medicinal Applications of Mulberry Plant Extract

with 80% ethanol. Treated material was dispersed in water and placed in a microwave extractor and heated at different microwave power and durations. Extractants were precipitated using ethanol, washed with acetone, and analyzed for their yield and composition. A yield of 9.4% polysaccharides was obtained under the optimum treatment conditions of 170 W microwave power and extraction time of 10 minutes using 20 g sample. No major changes to the structure of the polysaccharides were observed. Ethanolic extracts (antioxidants) from mulberry leaves were also able to protect rats from injuries caused due to immobilization stresses. Considerable increase in nitrite in plasma and adrenal glands and elevated levels of thiobarbituric acid in plasma, kidney, and spleen were observed. It was reported that even low doses of mulberry antioxidants were more effective than rutin (4 mg/day), a common inflammation reducing drug (Lee et al. 2007). Interestingly, methanolic extracts from M. alba (mulberry) at a dose of 200 mg/g were able to induce sedative effects and could possess anxiolytic properties (Yadav et al. 2008). Several methods have been used to extract polysaccharides from mulberry leaves (He et al. 2018) (Table 4.1). In a comparative study on the extraction of polysaccharides from mulberry leaves using ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and conventional solvent extraction (CSE), it was found that crude polysaccharides obtained through UAE had the highest yield of 10.79% compared to 9.5 and 4.7 for the MAE and CSE processes, respectively. The UAE polysaccharides could be further purified into two components having molecular weights of 25 and 61 kDa. Major components in one fraction were Sor, Ara, Xyl, and Glc, whereas in the second component were Rha, Ara, Xyl, Glc, Gal, and Man (Ying et  al. 2011). In a similar study, polysaccharides were extracted from mulberry leaves using ultrasonic treatments (Zhang et al. 2016). Mulberry leaves immersed in distilled water were treated using an ultrasonic bath at 100 W, heating power of 600 W, and temperature between 20 and 80 °C. Extracts were filtered using Table 4.1  Comparison of the methods, conditions used for extraction of polysaccharides using water as a solvent from mulberry leaves, and yield obtained. Extraction time, min

Solid–liquid ratio

Temperature, °C

Yield, %

90

1:24

70

2.64

60

1:10

70

2.91

85-twice

1:17

80

4.67

300-thrice

1:18

85

12.00

210-twice

1:34

92

10.00

Ultrasound 20, 60 W

1:15

80

10.79

80, 100 W

1:53

57

6.92

11, 436 W

1:25

70

9.53

10, 170 W

—

—

9.41

Microwave

Source: He et al. (2018). Reproduced with permission from Elsevier.

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4  Non-silk Applications of Mulberry Plants

membranes and precipitated using ethanol. Crude polysaccharides obtained were further purified by deproteinizing and dialyzing. Flavonoids and phenols were removed by treating with the resin (ADS-17 resin). After the three-step purification, mulberry leaf polysaccharides with carbohydrate content varying between 37 and 83% were obtained. Crude polysaccharides obtained had considerable antioxidant activity which decreased with increasing purity but adding quercetin further enhanced the activity. Ethanol-treated mulberry leaves were reheated with deionized water at various temperatures and times to extract polysaccharides. Further purification of the polysaccharides was done using chromatography, and various fractions of monosaccharides and phenols were collected (Yuan et al. 2015). A crude polysaccharide yield of 10% was obtained using a temperature of 92 °C, extraction time of 3.5 hours, and two extraction cycles. Polysaccharides with two molecular weights (80.9 and 3.6 kDa) were obtained along with several other monosaccharides and phenols (Table 4.2). The crude polysaccharides had high scavenging and chelating properties suitable for medicinal and functional food applications. Further, it has also been demonstrated that crude polysaccharides from mulberry leaves when ingested into mice were able to increase insulin secretion and consequently improve glucose and lipid metabolism and hence useful for treating diabetes mellitus (Zhang et  al. 2014). A within-subject clinical study by Aramwit et al. in 2011 indicated that patients who met the National Cholesterol Education Panel-Adult Treatment Plan (NCEP-ATP III) criteria guideline for dyslipidemia and failed a four-week diet therapy who took 280 mg mulberry leaf tablet three times a day before meals for a period of 12 weeks showed significant decrease of total cholesterol, triglyceride, and low-density lipoprotein (LDL) by 4.9, 14.1, and 5.6%, respectively, from baseline, whereas high-density lipoprotein (HDL) was significantly Table 4.2  Properties of extractants obtained from mulberry leaves. Component/property

Carbohydrate, %

Crude

52.1 ± 1.1

Treatment I

Treatment II

89.7 ± 0.3

37.2 ± 0.59

Protein, %

2.2 ± 0.02

0.83 ± 0.03

0.22 ± 0.02

Uronic acid, %

32.5 ± 0.9

6.53 ± 0.13

65.3 ± 1.52

Sulfuric radical, % Total polyphenols (mg GAE/100 mg) Molecular weight, kDa

1.7 ± 0.1

1.40 ± 0.02

1.22 ± 0.02

1.93 ± 0.02

0.17 ± 0.01

0.16 ± 0.01

—

80.99

3.64

Monosaccharides, mol -Man

0.51

0.77

—

-Rha

5.13

4.53

1.57

-GluA

2.23

0.81

0.20

-Glu

3.02

1.21

6.10

-Gln

2.13

3.47

—

-Gal

2.95

12.55

1.27

-Ara

2.55

11.14

0.89

Source: Yuan et al. (2015). © 2015 Elsevier.

4.2  ­Medicinal Applications of Mulberry Plant Extract

increased by 19.7% (Aramwit et al. 2011). Regarding the effect on blood sugar level, clinical study has also found that consuming 4.6 g of dry mulberry leaves could reduce fasting plasma glucose by 3.86 ± 5.99 mg/dl and glycated hemoglobin (HbA1c) by 0.11 ± 0.22% when compared with the baseline levels (Thaipitakwong et al. 2020). Moreover, mulberry leaves tended to ameliorate insulin resistance without any serious side effects. The ability to decrease glucose levels was observed in leaves of different varieties and also mulberry leaves from different locations. Ethanol insoluble but hot water soluble extracts from the leaves showed decrease in blood glucose levels of up to 81% (Chen et al. 1995). Mulberry leaf polysaccharides were also found to be effective for delivering genes to mesenchymal stem cells. Polysaccharides were extracted from mulberry leaves by treating in distilled water at 85 °C for three hours. The extractants were separated by centrifuging, and the precipitate obtained was washed, dialyzed and lyophilized. The crude polysaccharides obtained were further separated using thin-layer chromatography to form pure polysaccharides. These polysaccharides were further modified to form an ethylenediamine–polysaccharide conjugate. Average molecular weight of the polysaccharide was 53 kDa before and 13 kDa after modification. Treating with ethylenediamine (EDA) resulted in grafting of amino groups (up to 2.43 μmol/g). Modified polysaccharides were made into nanoparticles and used to load plasmid transforming growth factor β1. The drug-loaded nanoparticles were able to transfect into bone marrow mesenchymal stem cells without causing any cytotoxicity and at higher rates than common drugs. It was suggested that modified mulberry polysaccharides could be used to deliver therapeutic genes into cells (Deng et al. 2012).

4.2.2  Phenols and Flavanoids In addition to polysaccharides, a multitude of micro- and macronutrients, anticancer, cardiovascular, hypolipidemic, and anti-inflammatory compounds have been extracted from mulberry leaves and stems. However, part of the plant and extraction processes used plays a major role in determining the composition, activity, and applications of the extracts depending on total phenol contents, flavonoid content, and minimum inhibitory concentrations (Gryn-Rynko et  al. 2016). A comparative study was done to determine the phytochemical composition and antimicrobial properties of stems and leaves of different varieties of Morus species (Thabti et al. 2014). The phenolic and flavonoid contents in the bark and leaves of three varieties of mulberry plants are given in Table 4.3. Both the methanol and aqueous extracts show high flavonoid and phenolic content, but the amount obtained differs considerably depending on the species and part of the plant. In general, aqueous extracts were found to provide higher yields than the leaves. However, aqueous extracts had considerably low antimicrobial activity compared to the methanolic extracts from both bark and leaves. The extracts had very low inhibition for Escherichia coli but substantial reduction for Salmonella typhimurium, Staphylococcus epidermis, and Enterococcus feacalis. Up to 13 different phenolic compounds could be extracted using methanol from mulberry stems of Morus nigra L. (Abbas et  al. 2014). A new compound stilbene (2′,3,4′,5,5′-pentahydroxy-cis-stilbene) was identified and found to have high (IC50 of 4.69 μM) antioxidant activity. Similarly, the compound oxyresveratrol in mulberry stems when fed to diabetic mice (0.6 g/kg body weight) led to considerably high reduction in plasma glucose levels, increase in hepatic glucose transporter 2 transcription and glycogen

135

136

4  Non-silk Applications of Mulberry Plants

Table 4.3  Comparison of the phenolic and flavonoid content in three varieties of mulberry leaves and stems extracted using methanol and water. Total phenols Mulberry

Leaves

Stems (bark)

Variety

Methanol

Water

Total flavonoids Methanol

Water

Morus alba var. alba

560 ± 97

759 ± 74

283 ± 4

717 ± 45

Morus alba var. rosa

345 ± 0.6

998 ± 34

194 ± 4

504 ± 15

Morus rubra

631 ± 21

1129 ± 21

398 ± 8

816 ± 46

Morus alba var. alba

304 ± 10

807 ± 48

173 ± 0.7

450 ± 234

Morus alba var. rosa

295 ± 12

442 ± 45

128 ± 5

379 ± 45

Morus rubra

254 ± 9

285 ± 29

83 ± 4

247 ± 43

Source: Thabti et al. (2014). Reproduced from Taylor and Francis by permission through open access publishing.

content. It was suggested that the mulberry extract was able to stimulate hepatic glucose and glycogen storage and hence reduce glucose generation (Ahn et al. 2017). Substantial differences in phenolic type and content were noticed in white and black mulberry leaves (Table  4.4). Leaves were found to have high caffeoylquinic acids (6.8–8.5 mg/g) and flavanols (3.7–9.8 mg/g) and could be used in food and pharmaceutical industry (Sánchez-Salcedo et al. 2015). In terms of flavanols, it was reported that 60% ethanol was able to extract highest amounts with major components being rutin (573 mg/100 g), isoquercitrin (194 mg/100 g), and quercetin 3-(6-malonylglucoside) (900 mg/100 g) (Katsube et al. 2006). However, the concentration of flavonoids in the mulberry leaves was found to vary between the different varieties and seasons. Spring leaves had flavonoid contents between 1.7 and 26.6 mg/g, whereas autumn leaves had 9.8 to 29.6 mg/g of flavonoids. These flavonoids were found to have up to 46.5% scavenging potential for superoxide ions (Zhishen et  al. 1999). In addition to common flavonoids and phenols, 13 different compounds (Table  4.5) were isolated from three varieties of mulberry leaves. These extracts were found to be flavonol glycosides and phenolic acids, and three novel components kaempferol-7-O-glucoside, quercetin-3-O-β-glucoside-7-O-α-rhamnoside, and quercetin-3-O-rhamnoside-7-O-glucoside were extracted for the first time (Figure  4.2) (Thabti et al. 2012). In another study using M. alba and M. nigra, 31 phytochemicals were extracted using Ultra High-Performance Liquid Chromatography–Mass Spectrometry and it was reported that seven unique compounds could be identified (Sánchez-Salcedo et al. 2016). In what could be a path-breaking study, it has been found that extracts from the leaves and stems of mulberry could have antiviral activity even against new pathogens such as human coronavirus (HVoC 229E) and different members of Picornaviridae family. Extracts (12) obtained using water and ethanol from three different species of mulberry plants showed ability to reduce viral titers and also have cytopathogenic effects (Table 4.6) (Thabti et al. 2020), suggesting that mulberry extracts could be used to treat respiratory infections caused due to viral and other pathogens. In another study, it has been demonstrated that mulberry leaf extracts (ethanol) could eliminate neuroblastoma cells when used between

138

4  Non-silk Applications of Mulberry Plants

Table 4.5  Antiviral activities of water and methanol extracts obtained from the stems and barks of three different varieties of mulberry. HCoV 229E

PV1

Source

Variety

Viral Extractant titer (log10)

Viral Inhibition, % titer (log10)

Inhibition, %

Stems (50 μg/ ml)

M. alba

MeOH

2.16 ± 0.52

41

5.44 ± 0.10

7

Water

Leaves (200 μg/ml)

2.40 ± 0.67

35

5.67 ± 0.29

3

M. alba rosea

MeOH

1.98 ± 0.29

45

5.10 ± 0.36

12

Water

2.50 ± 0.87

36

5.61 ± 0.19

4

M. rubra

MeOH

2.30 ± 0.17

37

4.96 ± 0.48

15

Water

2.42 ± 0.54

34

5.62 ± 0.33

3

MeOH

0

100

5.40 ± 0.17

7

Water

M. alba

1.88 ± 0.67

48

5.61 ± 0.67

4

M. alba rosea

MeOH

1.05 ± 0.59

71

5.44 ± 0.10

6

Water

2.24 ± 0.65

39

5.46 ± 0.24

6

M. rubra

MeOH

1.19 ± 0.60

67

5.12 ± 0.67

12

Water

2 ± 0.50

45

5.43 ± 0.23

7

Source: Thabti et al. (2020). Reproduced from MDPI with permission through open access publishing. O

HO

CO2H O

HO

OH HO

O OH

HO Cafeic acid

OH

OH

HO2C

5-caffeoylquinnic acid

O O

HO

OH

OH O

R2 O

OH

1-caffeoylquinnic acid

OH

O O OH

O

OH

OH

OH

R2 O

OH

R1

R1 = rhamnopyranosyl(1,6)glucopyranoside, R2 = H: Kaempferol-3-O-(6 malonyl)glucoside R1 = R2 = H: kaempferol-3-O-glucopyranoside R1 = H, R2 = glucose : kaempferol-7-O-glucoside R1 = rhamnopyranosyl(1,6)glucopyranoside, R2 = H : Kaempferol-3-O-rhamnopyranosyl-(1-6)glucopyranoside

O OH

R1

O

R1 = malonyl-glucoside, R2 = H: quercetin-3-O(6-malonyl)glucopyranoside R1 = glucose, R2 = rhamnose: quercertin-3-Oglucoside-7-O- rhamnoside R1 = rhamnose, R2 = glucose : quercertin-3-Orhamnoside - 7-O- glucoside R1 = R2 = glucose : quercertin-3,7-D-O-β-Dglucopyranoside

Figure 4.2  Chemical structure of the three unique compounds extracted from mulberry leaves. Source: Thabti et al. (2012). Reproduced from Elsevier with permission through open access publishing.

4  Non-silk Applications of Mulberry Plants 4

a

OD 560

3

(a)

0

a

b

c

b

d c

a a b c 48 h

96 h

144 h

1.00

a b

0.75 0.50

ctrl 10 μg/mL 20 μg/mL 40 μg/mL

b

2 1

OD 560

140

a b b c

a

b b

ctrl 10 μg/mL 20 μg/mL 40 μg/mL

b c

c

0.25

(b)

0.00

48 h

96 h

144 h

Figure 4.3  Decrease in the viability of BE(2)C cell (a) and FI cells (b) seeded with three different concentrations of the mulberry leaf methanol extracts and incubate for up to six days. Source: Park et al. (2012). Reproduced from Taylor and Francis with permission through open access publishing.

10 and 40 μg/ml in cells. The extracts were able to decrease the expression of stem cell markers and increase the expression of differentiation markers. Similarly, phosphorylation of the extracellular signal regulated kinase was increased which showed chemopreventive effects of the extracts on neuroblastoma cells and hence potential for treating cancer (Park et  al. 2012) (Figure  4.3). Similarly, the anti-inflammatory ability of ethanol extract from mulberry was studied using lipopolysaccharide-stimulated RAW 264.7 macrophage cell lines. It was found that mulberry extract in concentrations between 10 and 40 μg/ml had cell viability higher than 80% and at concentrations between 50 and 40 μg/ml inhibited nitric oxide production and reduced cyclooxygenase-2 mRNA (messenger ribonucleuc acid) expression leading to anti-inflammation (Soonthornsit et al. 2017).

4.2.3  Pectins and Lignins In addition to carbohydrates, phenols, flavonoids, and proteins, the possibility of extraction and properties of other components in mulberry stems such as pectins and lignins have also been studied (Liu et al. 2010). In an attempt to extract pectins, mulberry barks with and without the epidermis were treated with hydrochloric acid (0.05 to 0.25 M) at 90 °C for 40 to 120 minutes. The treated material was centrifuged, and the supernatant was collected and concentrated and later dispersed in isopropanol and pH adjusted to 3.5 for the pectins to precipitate. Some of the properties of the pectins obtained are given in Table 4.7. It was

142

4  Non-silk Applications of Mulberry Plants

suggested that the mulberry stems could be used to obtain pectins with different levels of esterification. Removal of the epidermis assists in better conversion and obtaining pectin with high viscosity and hence gelling ability desirable for food applications. Microorganisms (endophytes) extracted from mulberry plants were able to generate indole acetic acid (IAA). Amount of IAA produced varied between 45 and 60 μg/ml and depending on the part of the plant and microorganisms used. IAA generated by mulberry plants was higher than that in most plants, and it was suggested that IAA could promote the growth of mulberry plants (Bhuvaneshwari et al. 2019).

4.2.4  Production of Paper Common mulberry plant (M. alba) and another species of mulberry M. papyrifera L. commonly called as paper mulberry have been used for the production of paper. The paper made from mulberry and commonly called “mulberry paper” has distinct properties and several unique applications. Tensile strength of paper made from mulberry leaves was 16.8 kN/m, and water absorption was 462% at a density of 383 kg/m3. To improve mechanical properties and resistance to water, mulberry paper was coated with latex leading to increased strength from 37 to 79 MPa. Lamination also provided a glossy appearance and increased the suitability for printing and cleaning (Memon et al. 2011). It has been reported that unlike commercial paper, fibers used in making mulberry fiber are 2–2.5 longer and form a considerably strong mechanical network that is resistant to wetting. Presence of high amounts of holocellulose makes the fiber hydrophilic and suitable for dip coating various active materials, for example developing flexible storage systems. Also, mulberry paper is typically made using weak alkaline solutions which makes the paper have good resistance to oxidation and acidolysis and hence good chemical toughness. In a unique application, paper-based flexible supercapacitors were developed (Yun et al. 2018) from mulberry paper. A simple coating was used to add carbon black and poly(3,4ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) in aqueous solution to mulberry paper. Some of the properties of the mulberry paper are given in Figure  4.4 in comparison to commercial paper. As seen from the figure, paper made from mulberry had higher tensile strength (12.7 MPa) and elongation (2.8%) compared to commercial paper but lower modulus (430 MPa) along with good resistance to sulfuric acid (pH 1) after immersion for 48 hours with 95.5% modulus retention. Electrodes developed from the coated paper had specific capacitance of 374 F/g at a current density of 1/Ag, power density between 2.8 and 13.9 Wh/kg, and energy density of 29.8–39.8 Wh/kg. The electrodes were able to retain 90.7% of initial capacity after 15 000 charge discharge cycles. Energy capacity of the electrode could be increased linearly with increasing area, and a capacitance of 18.7 F was obtained when the surface area was 100 cm2. Mulberry paper-based supercapacitor was suggested to have properties suitable for developing wearable energy storage devices (Yun et al. 2018). Incorporating silver nanowire/silver nanoparticles onto mulberry paper led to substantially higher mechanical stability and specific capacitance of 100 F/g and ability to withstand bending for up to 10 000 cycles without decrease in performance (Seo and Hwang 2019).

1.0

Cellulose HO

O

OH

HO O

OH

O

O

OH

OH

OH

1.2

O

Lignin O

OH3

O

OH OH

OH3 O

O

O OH

O

O HO 3 OH3

OH

0.8

0.4

OH

O

O H3O

Commercial paper

O OH3

O

Degradation of mechanical and chemical resistance

0.0

(b) 15

CP

MP

CP MP Holocellulose

Lignin

O

HO O

OH

O

O HO

O OH

HO

O

OH

HO O

O

OH HO

O

HO

10 O

HO

O

O OH O

Stress (MPa)

OH OH

Enchancement of (a) mechanical and chemical resistance

(d)

5

0 0.0

0.5

1.0

8s

Mulberry paper HIgh

2.5

3.0

3.5

Mulberry paper O-C-O O-C=O C=O

C-O-C C-OH

C-C C-H

C-C C-H

291 290 289 288 287 286 285 284 283 Binding energy (eV)

0.8 0.6

0.2 0.0

(e)

Mulberry paper Commercial paper

0.4

0

10

20 30 40 Exposure time (h)

50

17 s 85.3°

50.4°

(f)

1.5 2.0 Strain (%)

C-O-C C-OH

O-C=O

1.0 Normalized modulus

O

HO HO

HO HO

(c)

Mulberry paper Commercial paper

Holocellulose OH

O-C-O C=O

OH

HO O

Mass ratio

OH

OH O HO

Intensity (arb.units)

OH HO

Cellulose filter Absorption rate

110.5°

115.5°

Commercial paper Low

Figure 4.4  Properties of the mulberry paper used to develop supercapacitors. Chemical structure of constituents (a), comparison of lignin and holocellulose with commercial paper (b), XPS analysis (c), tensile properties (d), modulus retention after immersion in pH 1 solution for 48 hours (e), and wettability and contact angle (f). Source: Yun et al. (2018). Reproduced with permission from John Wiley and Sons.

144

4  Non-silk Applications of Mulberry Plants

4.2.5  Fermentation and Biogas Production Ability of mulberry leaves to support the production of fermented products including bacteria and enzymes has been studied. Two varieties of mulberry leaves were harvested and ensiled with either 106 CFU of Lactobacillus casei (LC) or 2% sucrose (S) or a mixture of both (LC + S). The mixture was placed (180 g) in plastic films and stored in ambient conditions for 1 to 60 days, and amount of bacteria was determined. The presence of LC and S considerably increased the fermentation quality of mulberry silage and increased availability of Lactobacillus and Pseudomonas but inhibited the growth of Enterobacter (Wang et al. 2019). In a similar study, the effects of cellulase and LC ability on the nutritive value and antioxidant properties of two varieties of mulberry leaves were studied. Microbial populations, carbohydrate and protein content, and antioxidant activities changed considerably due to the presence of cellulase and LC (He et al. 2019). As seen from Table 4.8, the carbohydrate and protein fractions vary considerably between the inoculated and non-inoculated samples and also between cultivars. Similar variations were also observed in the antioxidant activities and gas production. Hence, mulberry silage was considered as a high value feed. When used as a source for production of gas, the methane levels achieved varied between 2.5 and 12.5/200 g of dry matter after 24 hours of incubation and between 1.75 and 18.75 ml/200 mg of dry matter after 72 hours of incubation (Tables 4.9 and 4.10) (Eshetu et al. 2018). It was suggested that using branches and stems after 10 months of cultivation could provide better yield of gas compared to using the leaves.

4.2.6  Synthesis of Nanomaterials Synthesis of gold nanoparticles with anti-bacterial activity was possible using mulberry leaf extracts. Leaves were heated in de-ionized water at 60 °C for 15 minutes, and the extracts obtained were filtered and used for producing the nanoparticles. To prepare the gold nanoparticles, the leaf extract (0.2 to 1 ml) was added into 10 ml of 2 × 10−4 M HAuCl4 3H2O and maintained at room temperature for about 36 hours. Average size of the particles formed was 50 nm, and zeta potential was −16 mV indicating good stability. The nanoparticles were crystalline and had higher inhibition for gram-negative Vibrio cholera compared to the gram-positive Staphylococcus aureus (Adavallan and Krishnakumar 2014). Since mulberry leaves and stems are lignocellulosic and contain large amounts of cellulose, attempts have been made to convert them into nanocellulose in the form of nanoparticles, whiskers, and fibers. Cellulose nanowhiskers were produced from mulberry stems using an alkali conversion process (Li et  al. 2009). Stems (barks) were treated in 1% NaOH at 80 °C for two hours, and the epidermis was removed. Barks were again immersed in 1% NaOH and 1% sodium sulfide mixture and treated at 80 °C or 130 °C for 1.5 hours to obtain the fibers. The fibers obtained were also bleached using sodium chlorite at 80 °C for 1.5 hours to delignify and obtain white fibers. To extract whiskers, the bleached fibers were hydrolyzed in sulfuric acid solution at 60 °C for 30 minutes and later dialyzed in distilled water for three days. About 1% of the bark was converted into cellulose whiskers with diameters between 25 and 30 nm, length between 400 and 500 nm, and crystallinity of 73%. The whiskers obtained were suggested to be suitable for developing composites, pharmaceuticals, and in optical applications. Mulberry leaves have also been used as substrate to

4.2  ­Medicinal Applications of Mulberry Plant Extract

Table 4.8  Changes in the carbohydrate and protein fractions of mulberry leaf silage depending on the presence of cellulase and LC and between two cultivars. Non-inoculated Item

DM %

Cultivar

E0

E50

Inoculated E100

E0

E50

E100

V1

29.44

32.30

32.24

31.04

31.27

29.53

V2

33.80

36.17

35.67

36.55

37.25

37.12

-Crude protein

V1

20.94

21.37

21.77

21.08

21.55

21.94

V2

21.04

21.92

21.58

20.06

21.16

21.61

- Total protein

V1

11.80

13.87

13.85

13.16

15.62

14.83

V2

10.45

12.60

12.59

13.14

13.83

14.24

- NPN

V1

9.14

7.37

7.91

7.92

5.98

7.11

V2

10.59

9.32

7.81

6.92

7.87

7.36

Protein fraction, %

Carbohydrates, % NDF ADF ADL Hemicellulose Cellulose WSC

V1

21.09

17.81

16.47

18.64

16.98

16.73

V2

21.87

19.42

19.80

20.06

18.32

17.64

V1

10.33

9.19

8.61

9.00

8.80

8.97

V2

12.43

11.31

11.53

10.65

9.91

9.69

V1

2.56

2.50

2.11

1.74

2.14

2.37

V2

3.85

3.40

3.21

2.51

2.83

2.71

V1

10.76

8.61

7.86

9.64

8.18

7.77

V2

9.44

8.12

8.26

9.41

8.41

7.95

V1

7.76

6.80

6.50

7.25

6.67

6.60

V2

8.58

7.90

8.32

8.15

7.08

6.98

V1

1.69

2.35

2.01

1.10

1.76

1.83

V2

2.75

2.40

2.20

2.12

2.06

2.16

Source: He et al. (2019). © 2019 American Dairy Science Association.

generate bacterial celluloses. Proteins were extracted from mulberry leaves using alkali and hydrolyzed using hydrochloric acid. Hydrolysate obtained was detoxicated and concentrated. The hydrolysate was steam sterilized at 121 °C for 20 minutes and injected into the mulberry leaf hydrolysate to form the fermentation media. The inoculum (Acetobacter xylinum NUST 4.2) was added into the media and maintained at 30 °C for seven days. Bacterial cellulose films formed after fermentation were collected and freeze-dried to form the powder (Chen et al. 2019). Yield of bacterial cellulose obtained using the mulberry hydrolysate was 2.04 g/l compared to 4.05 g/l for lactic acid. The cellulose obtained was in the form of nanofibers having 3D network with a diameter of about 200 nm. Mulberry cellulose had lower mechanical properties than lactic acid cellulose but showed excellent antimicrobial activity against both gram-positive and gram-negative bacteria and also supported the

145

148

4  Non-silk Applications of Mulberry Plants

attachment and growth of human mesenchymal stem cells suggesting suitability for medical applications (Chen et al. 2019).

4.2.7  Environmental Remediation In a unique study, the ability of mulberry plants (specifically leaves) to capture particulate matter in air and remove heavy metals was investigated. M. alba when planted road side as avenue trees in polluted regions were able to accumulate heavy metals on their leaves and later transport these metals into the roots and stems and into the soil. It was found that the amount of particulate accumulation and heavy metal removal was dependent on the type of foliage, morphology of the foliage, the particulate size, and particulate load (Sharma et al. 2020). Accumulation of Zn in various parts of the mulberry plant and after different periods of exposure is shown in Figure 4.5. It was suggested that planting trees such as mulberry would be an effective approach to reduce particulate pollution, especially heavy metals. Mulberry plants grown in heavily cadmium-contaminated soil show ability for remediation (Lei et  al. 2019) but amount sorbed varied between different locations and planting. Roots, stems, branches, and leaves accumulated cadmium to different levels with highest amount found in the roots and lowest (16%) in leaves (Figure 4.6). Interestingly, silkworms fed with cadmium-containing leaves produced cocoons with permissible levels of cadmium (120

120 max

0.5 max —

Source: Sarma and Ganguly 2016. © 2016 Academy for Environment and Life Sciences.

fat, and defatting increases the protein content to about 75%. Pupae proteins contain almost all essential amino acids similar to whole egg protein, except for tryptophan (Rao 1994). However, the amount and type of amino acids extracted from silkworm pupae are dependent on the condition of the pupae and also extraction methods used (Table  5.9). Also, amount of amino acids in pupae was found to vary between male and female insects and their stage of growth. Protein content increased continuously with increasing growth and reached about 50% after 216 hours with female pupae having 45 g of proteins compared to 39 g in male pupae (Priyadarshini and Revanasiddaiah 2013). In addition to alkali extraction, using enzymes not only increases yield but also improves water solubility and antioxidant activity making the amino acids suitable for human and animal food uses (Anootthato et al. 2019). Similarly, heat, alkali treatment, enzymes, freeze drying, or spray drying also caused substantial changes to the amino acids obtained from silkworm pupae (Shi et al. 2017). Alkali treatment followed by enzymatic hydrolysis with trypsin and flavourzymes increased protein recovery. The protein hydrolysate obtained was stable in pH 4 to 8, but the antioxidant activity was affected by alkaline pH but unaffected by pasteurization or autoclaving. Peptides obtained from silkworm pupae have been studied for food and non-food applications. Since pupae proteins contain all the 18 amino acids, effect of including about 0.5% pupae peptides on the functional and nutritional properties of fermented milk products

166

5  Pupae and Its Applications

Table 5.10  Changes in the textural properties of yogurt containing different levels of peptides isolated from silkworm pupae. Peptide concentration (%)

Firmness (g)

Consistency (gs)

Cohesiveness (g)

Viscosity index (gs)

0

225 ± 6

763 ± 10

120 ± 5

100 ± 2

0.1

403 ± 7

1407 ± 10

271 ± 5

142 ± 3

0.3

722 ± 8

2118 ± 11

343 ± 3

193 ± 5

0.5

856 ± 12

2948 ± 12

293 ± 5

86 ± 3

0.7

550 ± 7

1876 ± 8

219 ± 4

84 ± 2

0.9

600 ± 6

1812 ± 7

213 ± 3

81 ± 5

Source: Wang et al. (2017). Reproduced with permission from John Wiley and Sons.

(yogurt) was studied (Wang et al. 2017). Considerable changes in the acidification potential, water holding capacity, texture, amino acid content, and angiotensin-converting enzyme (ACE) inhibition were observed depending on the peptide content (0–0.9%). Rate of fermentation increased depending on the concentration of the peptides with acid production between 13.2 and 23.4 T/h, whereas water holding capacity had decreased. Textural parameters of the yogurt such as firmness, consistency, and cohesiveness increased substantially with increasing peptide content (Table 5.10). Similarly, ACE inhibitory activity was higher (50%) at 0.9% peptide content compared to only 4% for the yogurt without the peptides (Wang et al. 2017). Although amino acid content also showed a favorable increase, the yogurt was reported to have an undesirable flavor above peptide content of 0.3%. B. mori pupae were defatted using hexane and later acid hydrolyzed by treating with 1N HCl for two hours at 1 : 10 ratio of acid to pupae. Dissolved hydrolysates were centrifuged, washed, and dried for use as insect flour to prepare emulsion meat sausages (Kim et al. 2016). Acid hydrolyzed flour had a protein content of 74%, comparable to commercially available soyproteins. Defatted pupae and insect flour had emulsion capacities of 54% and 53% and solubilities of 124 and 119 mg/g, respectively. Major changes were observed in the appearance, physiochemical, and textural properties of the emulsion sausages prepared using defatted pupae and acid hydrolyzed meal flour (Table 5.11). It was suggested that silkworm meal flour could be added as an ingredient for the preparation of sausages without any adverse impact. Addition of 5–15% of silkworm pupae (without any extraction) or transglutaminase did not adversely affect the properties of meat batter (Park et  al. 2017). Overall improvement in nutrition values (Table 5.12), reduced cooking loss, pH, and viscosity was possible due to the addition of the pupae. Proteins extracted from pupae (protein hydrolysate) have been used for developing novel surfactants. Defatted pupae proteins were treated with 1.5 M HCl for 30 minutes at 125 °C to obtain amino acids. The amino acids obtained were added into acetone and fatty acyl chloride to which sodium hydroxide was added and the mixture maintained at pH 9 for four hours. The pH of the solution was decreased to 1–2, and the N-fatty acyl amino acid formed was filtered and collected. The product obtained was dissolved in ethyl alcohol, and the pH was adjusted between 7 and 8 and stirred for 30 minutes at room temperature to obtain the reddish product called sodium N-fatty acyl amino acid surfactant (SFAAA).

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5  Pupae and Its Applications

Table 5.12  Changes in the composition and nutritional values of meat batter prepared with three different levels of silkworm pupae with and without transglutaminase. Moisture (%) Treatment

With

Without

Protein (%) With

Without

Fat (%) With

Ash (%)

Without

With

Without

Control

73 ± 0.08

72 ± 0.07

18 ± 1.7

20 ± 0.6

5.6 ± 0.4

52 ± 0.3

2.4 ± 0.05

2.5 ± 0.03

5% Pupae

69 ± 0.24

68 ± 0.17

19 ± 0.2

22 ± 0.2

6.2 ± 0.4

6.5 ± 1.1

2.5 ± 0.01

2.5 ± 0.03

10% Pupae

66 ± 0.33

64 ± 0.01

20 ± 1.1

24 ± 1.7

7.1 ± 0.6

7.4 ± 1.1

2.6 ± 0.04

2.7 ± 0.03

15% Pupae

63 ± 0.35

62 ± 0.10

27 ± 1.5

30 ± 2.1

7.1 ± 0.5

7.7 ± 1.6

2.6 ± 0.04

3.1 ± 0.02

Source: Park et al. (2017). © 2017 Korean Society for Food Science of Animal Resources.

About 65% of the protein hydrolysate was converted into the surfactant. Considerable changes were observed in the amino acid content of the surfactant compared to the protein hydrolysate (Table  5.13). Decrease in threonine, valine, tyrosine, and proline whereas increase in glycine was observed. Surface tension of SFAAA decreased as the concentration Table 5.13  Amino acid composition of pupae protein hydrolysate and sodium N-fatty acyl amino acid surfactant (SFAAA). Proportion (mol%) Amino acid

Molecular weight (KDa)

Protein hydrolysate

SFAAA

Asparagine

133

16.87

16.31

Threonine

119

2.51

1.32

Serine

105

6.58

6.34

Glutamine

147

17.55

15.25

Glycine

75

8.08

20.23

Alanine

89

8.02

7.53

Cysteine

121

0.72

0

Valine

117

4.95

2.36

Methionine

149

3.57

2.66

Isoleucine

131

2.96

2.26

Leucine

131

6.85

5.49

Tyrosine

181

5.19

3.26

Phenylalanine

165

1.67

1.23

Lysine

146

6.01

5.56

Histidine

155

1.44

1.13

Arginine

174

1.08

0.98

Proline

115

4.51

2.13

Source: Reproduced with permission through open access publication.

5.2  ­Proteins from Silkworm Pupa

increased, whereas emulsifying power was higher (40–65 ml compared to 15–20 ml) than that of hydrolysate, suitable for use as detergent for commercial applications (Wu et  al. 2014). Further, it was shown that the surfactants did not show any toxicity when included as part of the diet for mice for up to eight weeks. Blood indices, blood lipid, antioxidant capacity, and lipid metabolism in kidney and liver did not show any changes, and hence, the surfactants were considered to be safe to environment and humans (Ding et al. 2017).

5.2.1  Medical Application of Pupae Peptides Pupae consists of four different types of proteins, and a novel peptide in the albumin portion has angiotensin I-converting enzyme inhibitory activity that can be used to control hypertension. Defatted pupae were subject to various treatments to extract different proteins depending on their solubility in water, 5% NaCl, 0.1M NaOH, and 70% ethanol. Albumin fraction was obtained after treating with water, the globulin after treating with NaCl, glutenin by treating with 0.1M NaOH and finally the prolamin fraction after treating with ethanol with properties shown in Table  5.14. The four components obtained were further treated with acid protease at 35 °C for about five hours to obtain protein hydrolysates. The peptides in the hydrolysates were separated using gel filtration chromatography, and their ACE inhibitory activity was determined. The proteins obtained had varying molecular weights between 15 and 200 kDa with glutenin having higher molecular weight peptides. Total protein inhibition was between 81% and 9% depending on the extent of hydrolysis. Further separation of the peaks from albumin showed that an inhibitory rate of 73% with IC50 of 0.047 mg/ml could be achieved. The specific peptide sequence responsible for inhibition was found to be “APPPKK”, and the peptide could readily bind to ASP415, ASP453, Thr282, His353, and Glu162 pockets in ACE causing the inhibitory effect. Based on the level of inhibition possible, it was suggested that peptides in silkworm pupae could be considered as food supplement for treating hypertension (Wang et al. 2011). Peptides in silkworm were also able to inhibit α-glucosidase to varying extents. Among 600 peptides extracted, four peptides having sequence of Gln-Pro-GLy-Arg, Ser-Gln-Ser-Pro-Ala, and Gln-Pro-Pro-Thr having minimum inhibitory concentrations of 65.8, 20, 560, and 205 μmol/l, respectively, were obtained. Hence, the peptides from silkworm could be used as an antidiabetic compound (Zhang et al. 2016). Studies have also shown that hydrolyzed peptides from silkworm pupae inhibit Gastric Cancer SGC-7901 cells. Defatted pupae Table 5.14  Properties of proteins separated from silkworm pupae.

Fraction

Yield (g/100 g)

Protein content (%)

Albumin

27.2 ± 2.4

93.8 ± 1.8

Pure protein (g/100g) Inhibition (%)

Degree of hydrolysis (%)

Isoelectric point (pI)

25.6

81

17

2.5

Globulin

4.2 ± 4.6

71.1 ± 2.7

2.9

61

12

2.7

Glutelin

23.7 ± 3.6

90.2 ± 2.7

21.4

9

5

4.0

Prolamine

11.8 ± 3.1

62.1 ± 0.8

7.3

10

15

4.5

Source: Wang et al. (2011). © 2011 Springer Nature.

169

170

5  Pupae and Its Applications

(a)

(b)

(c)

(d)

Figure 5.2  Changes in the morphology and number of SGC-7901 cells incubated for 36 hours with control (a) 80 μg/ml (b), 160 μg/ml (c), and 320 μg/ml (d) of silkworm hydrolysate (Li et al. 2018). Source: Reproduced with permission through open access publishing.

proteins were hydrolyzed using Alcalase at 50.8 °C at pH 9 for four hours using an enzyme concentration of 3500 U/g to obtain the peptides. Seven different cancer cell lines were chosen, and various concentrations of the peptides were added into the cell culture solution and incubated for 24–48 hours. MTT assay was done to determine the cell viability and cytotoxicity. Figure 5.2 shows that the morphology and number of cells decreased substantially with increasing concentrations of the peptides indicating induced apoptosis (Li et al. 2018). It was found that the protein hydrolysates were able to increase the number of cells in S phase (40–50%), whereas the cells in the G1 phase decreased (53–47%) and G2 phase also decreased from 8% to 3% demonstrating that the hydrolysate was able to arrest cell cycles. Similarly, reactive oxygen species in the cells increased in a dose-dependent manner. Changes in caspase signal pathways (Figure 5.3) were also responsible for the apoptosis. Ability of the hydrolysate to induce these changes showed potential to develop anticancer agents, specifically for treating gastric tumors (Li et al. 2018). Silk pupae protein hydrolysate with molecular weight of about 5000 Da was extracted and later hydrolyzed using acid proteases at 35 °C for five hours at pH 2.0. In the hydrolysate, two types of peptides (one with MW between 307 and 5000 Da (88%) and the other