effect of high pressure and high temperature on sericin protein properties from silkworm

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, January 2024 EFFECT OF HIGH PRESSURE AND H

MINISTRY OF EDUCATION AND TRAINING

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING

Ho Chi Minh City, January 2024

EFFECT OF HIGH PRESSURE AND HIGH TEMPERATURE ON SERICIN PROTEIN PROPERTIES FROM SILKWORM

LECTURER: PHAM KHANH DUNG, PH.D STUDENT: NGUYEN THAO HIEN NGUYEN NGOC LAM VY

S K L 0 1 2 5 2 9

GRADUATION PROJECT FOOD TECHNOLOGY

Ho Chi Minh City, January 2024

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY

AND EDUCATION FACULTY OF INTERNATIONAL EDUCATION

GRADUATION PROJECT

Thesis code [2023-19116040]

EFFECT OF HIGH PRESSURE AND HIGH TEMPERATURE ON SERICIN PROTEIN PROPERTIES FROM SILKWORM

Supervisor: PHAM KHANH DUNG, PH.D

Student: NGUYEN THAO HIEN 19116040

Student: NGUYEN NGOC LAM VY 19116039

Independence – Freedom– Happiness

GRADUATION THESIS ASSIGNMENT

Student name: Nguyen Thao Hien Student ID: 19116040 Student name: Nguyen Ngoc Lam Vy Student ID: 19116039 Major: Food Technology Class: 19116CLA Supervisor: Pham Khanh Dung, Ph D

Email: dungpk@hcmute.edu.vn

Date of assignment: 10/08/2023 Date of submission: 19/1/2024

1 Thesis title: Extraction and characterization of sericin cocoon silk by high temperature and high pressure

2 Thesis assignment:

- Evaluate chemical composition of silk cocoon: total protein, lipid, carbohydrate, ash

and moisture content

- Effect of temperature on the yield of sericin extraction from silk cocoon

- Effect of extraction time on the yield of sericin extraction from silk cocoon

- Study the structure of silk sericin through SDS-Page, FTIR spectroscopy

- Study the characteristics of silk sericin through antioxidant, antibacterial

- Study the amino acid compound in silk sericin

The content and requirements of the graduation thesis have been approved by the Chair of the Food Technology program

CHAIR OF THE PROGRAM Ho Chi Minh, 19th January 2024

(Sign with full name)

ii

DECLARATION

We hereby declare that all content presented in the graduation thesis is our own work We hereby certify that the contents referenced in the graduation thesis have been cited accurately and completely in accordance with regulations

January,2024

Signature

ACKNOWLEDGEMENT

Firstly, we would like to express our sincere and deep gratitude to the teachers in the Food Technology Industry - High Quality Training Faculty - Ho Chi Minh City University of Technology and Education for their dedicated teaching and imparting knowledge during the past four years and created all conditions for facilities and equipment to help us complete the thesis in the best way

We would especially want to thank PhD Pham Khanh Dung, the teacher who guided this graduation project Throughout the course of the project, she carefully guided us and imparted to us the knowledge and abilities required to use the instruments and operate the lab equipment Finally, we would like to thank Ms Ho Thi Thu Trang for supporting measurement tools and equipment throughout the project implementation process, helping us complete it in the best possible way

During the process of studying and writing the thesis, due to time constraints and limited knowledge, shortcomings are inevitable We hope to receive valuable feedback from teachers to improve the group's thesis

Finally, we would like to wish all teachers good health and the opportunity to contribute a lot to their noble careers

We sincerely thank

1.5 Scientific and practical significance 4

CHAPTER 2: LITERATURE REVIEW 5

2.1 Overview of Silkworm Cocoon 5

2.1.1 Introduction of Silkworm Cocoon 5

2.1.2 The chemical composition silkworm cocoon 6

2.4 Applications of Sericin in the Food Industry 13

2.4.1 In the food packaging and food coating 13

2.4.2 In the food industry 13

2.5 The current research status of sericin 13

2.5.2 Vietnam 15

CHAPTER 3: MATERIALS AND METHODS 17

3.1 Materials 17

3.1.1 Silkworm cocoon shell 17

3.2 Equipment for study 17

3.2.1 Chemicals used in experiments 17

3.2.2 Equipment 18

3.3 Methods 19

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3.3.1 Research process diagram 19

3.3.2 Extraction process of sericin from silkworm cocoon 20

3.3.3 Experimental procedure 20

3.4 Research Methods 23

3.4.1 Raw materials survey 23

3.4.1.1 Method for moiture content determination 23

3.4.1.2 Method for determining total ash content of cocoon 23

3.4.1.3 Determination of total nitrogen content by Kjeldahl method of cocoon 24

3.4.1.3 Determine the lipid content by Soxhlet method of cocoon 25

3.4.1.4 Determine the carbohydrate content of cocoon 26

The carbohydrate content in the water sample is determined from the components of protein, lipid, moisture, and ash, and is calculated using the following formula: 26

3.4.2 Determination of the optimal temperature during the sericin extraction process 26 3.4.3 Determine the optimal time during the sericin extraction process 27

3.6.4 Isoelectric point of sericin 32

3.6.5 Evaluation of amino acid composition 33

3.7 Evaluation of the antioxidant activity of sericin 33

3.8 Evaluation of antibacterial activity of sericin 34

CHAPTER 4: RESULTS AND DISCUSSIONS 36

4.1 Raw silk cocoon composition 36

4.2 Determination of the optimal temperature during the sericin extraction process 37

4.3 Determine the optimal time during the sericin extraction process 39

4.4 Evaluation of the properties of sericin solution from various extraction methods 41

4.4.1 UV spectroscopy of sericin solution 41

4.4.2 SDS – Page Analysis 42

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4.4.3 FTIR Analysis 44

4.4.4 Isoelectric Point of Sericin 45

4.4.5 Evaluation of amino acid composition 46

4.4.6 Evaluation of the antioxidant activity of sericin 48

4.4.7 Evaluation of antibacterial activity of sericin 49

CHAPTER 5: CONCLUSION 55

REFERENCES 57

APPENDICES 64

LIST OF FIGURES

Figure 2.1 Bombyx mori L’s cocoon 6

Figure 2.2 Chemical structure of silk sericin ( Saha., et al 2019) 8

Figure 2.3 : Three layers of sericin in a silk cocoon (Saha., et al 2019) 9

Figure 3.1: Diagram of experimental research 20

Figure 3.2: Flow chart of sericin extraction process from silkworm cocoons 20

Figure 3.3 Chopped silk cocoon shells 21

Figure 3.4 Sericin filtration 22

Figure 4.1: Sericin content in various temperature extraction 38

Figure 4.2: Sericin content in various time extraction 40

Figure 4.3: UV spectroscopy of silk sericin 41

Figure 4.4: SDS-PAGE analysis of sericin samples 43

Figure 4.5: FTIR spectrum of high temperature and high pressure (HT-HP) and NaOH degummed sericin 44

Figure 4.6 Determination of the isoelectric point (pI) of sericin through experimental results 45

Figure 4.7: Standard curve of ABTS free radical scavenging ability of sericin concentration 48

Figure 4.8 Antibacterial activity S aureus of sericin after 24 hours 50

Figure 4.9: S aureus bacterial cell density (log CFU/mL) in different rations of sericin extract 51

Figure 4.10 Antibacterial activity E coli of sericin after 24 hours 52

Figure 4.11 E.coli bacterial cell density (log CFU/mL) in different concentrations of sericin extract 53

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LIST OF TABLES

Table 2.1 The amino acid composition of sericin extracted using various methods (in

mole%) (Aramwit et al 2009) 9

Table 2.2 Pharmaceutical and Healthcare Applications 15

Table 3.1 Chemicals used in the study 17

Table 3.2 Equipment used in the study 18

Table 3.4 Arrange the experiment to investigate the sericin extraction process at different temperatures 26

Table 3.5 Arrange the experiment to investigate the sericin extraction process at different times 27

Table 3.6 Separation gel composition 30

Table 3.7 Stacking gel composition 30

Table 3.8 Experimental arrangement for the isoelectric method of sericin extract 32

Table 4.1 Some Parameters of Bombyx Mori Silk Cocoon Shell Raw Material 36

Table 4.2 FTIR Peaks of Sericin Powder Obtained by the Salting-Out Proces (M L Gulrajani, K P Brahma, P Senthil Kumar, Roli Purwa 2008) 45

Table 4.3: Results of amino acid composition table of sericin extract 46

LIST OF ABBREVIATION

HTHP: High Temperature and High Pressure FTIR: Fourier Transform Infrared Spectroscopy

ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

B.mori: Bombyx mori

CFU: Colony Forming Unit

S aureus: Staphylococcus aureus E coli: Escherichia coli

pI: Isoelectric point

SDS-PAGE: Sodium Dodecyl Sulfate–PolyacrylAmide Gel Electrophoresis APS: Ammonium persulfate

TEMED: N, N, N, N-tetramethyl ethylenediamine

BSA: Bovine Serum Albumin

UV-Vis: Ultraviolet–Visible spectrophotometry LOD: Limit of detection

NO : Not detected

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ABSTACT

This study determined the effects of sericin extraction using high-temperature and high-pressure methods From there, the optimal temperature and time for sericin in this study were determined to be 120 oC and 30 minutes Furthermore, it has been studied using several methods for the determination of amino acid composition The main amino acids present in sericin are glycine, serine, and aspartic acid The characterization of secondary structure using Fourier transform infrared spectroscopy (FTIR), the presence of water and a high concentration of hydroxyl groups contributes to a broad absorption peak around 3273.57 cm-

1 in sericin Antibacterial properties were evaluated by agar dilution method with gram-

positive bacteria Staphylococcus aureus and gram-negative Escherichia coli as test microorganisms Antibacterial properties against S aureus and E coli depend on sericin

concentration, which affects the ability to prevent microorganisms from growing and reduce bacterial density Antioxidant properties were determined by 2,2′-azinobis-(3- ethylbenzothiazoline-6-sulfonic) (ABTS+) radical scavenging showed that the IC50 result of this study sample was 2.77 mg/g, strong antioxidant activity but lower than that of the standard antioxidant (ascorbic acid) SDS-PAGE analysis results in two bands of protein in 13 kDa and 24 kDa

CHAPTER 1: INTRODUCTION 1.1 Problem

The silkworm is the larva (the active immature form of an insect) or caterpillar of the

2022) The main components of silk are fibroin and sericin, which play an important role in increasing strength and hardness and maintaining the structural integrity of the cocoon The amount of protein in both main components accounts for about 75% and 25% of the total silk weight, respectively (Biswal et al, 2022) Sericin silk is a natural polymer produced by silkworms that is frequently removed by the textile industry but may be recovered and reused Nearly 50,000 tons of sericin are generated globally as a by-product of the degumming process, causing economic as well as environmental concerns (Vaishnav & Singh, 2023) Wastewater from industry is discharged, leading to an increase in the level of chemical oxygen demand (COD) and biological oxygen demand (BOD) Consequently, wastewater from the silk industry leads to water and environmental pollution (Fabiani et al, 1996) There have been ongoing efforts to recover and reuse it as a natural biopolymer in a variety of applications Sericin has recently been researched for its biological, physicochemical, and structural properties, indicating that it has considerable potential in the cosmetic, food, and textile industries In biomedicine and pharmaceuticals, sericin is considered a functional food due to its antioxidant properties, and various studies have reported that dietary intake of sericin significantly reduces cholesterol levels, serum, and free fatty acids (Barajas-Gamboa et al, 2016) In foods, sericin has the potential to improve color and texture while inhibiting polyphenol oxidase enzyme activity (Thongsook et al, 2011)

The presence of highly hydrophobic amino acids and its antioxidant capacity make sericin applicable in the food and cosmetic industries In order to completely exploit the supply of sericin found in silkworm cocoons for biomedical and food applications, environmental pollution is to be minimized (Kunz et al, 2016) In this report, the objective is to consider the physicochemical, biological, and structural properties of sericin by the method of protein extraction from silkworm cocoons At the same time, determine the influence of the properties and dynamics of sericin by the methods of high temperature and

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high pressure, as well as the antimicrobial capacity of sericin The fractionation of sericin into various components is carried out by dissolving it in hot water for different time periods, during which the sericin undergoes hydrolytic cleavage (Gulrajani et al, 1992) Sericin is likewise degraded by this approach, however the decomposition preserves sericin's key characteristics (Rangi et al, 2015)

-Determine the optimal time for sericin extraction by investigating different times

-Simultaneously, evaluate the physicochemical properties and biological properties (antioxidant, antibacterial) of sericin with the aim of evaluating sericin as a potential raw material for the food and pharmaceutical industries

1.3 Object and scope of the research

-Research Object: Sericin extract from silkworm cocoons, Bombyx mori

-Scope of the study: This study was carried out on a laboratory scale

-Research topic on the influence of high-temperature and high-pressure extraction methods on the sericin content extracted from silkworm cocoon shells At the same time, evaluate some properties of the sericin solution

- Study the amino acid compound in silk sericin

1.5 Scientific and practical significance

Preliminary research on the content and bioactivity of sericin obtained from silkworm cocoon shells From there, assess the potential application of sericin in food A number of factors affecting the variability of the basic characteristics of sericin extract have been identified The application of sericin not only helps create innovation in the food industry but also contributes to environmental protection by limiting industrial waste from the garment industry

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CHAPTER 2: LITERATURE REVIEW 2.1 Overview of Silkworm Cocoon

2.1.1 Introduction of Silkworm Cocoon

Silk is a natural fiber produced by silk worms, including those belonging to the

Bombycidae, Saturnidae, and Lasiocampidae families, as well as spiders (Humenik et al.,

2011) [9] Mulberry silk, derived from Bombyx mori, a member of the Bombycidae family,

is specifically obtained from worms fed with mulberry leaves The cultivation of these silk worms, which requires human care due to historical domestication, is known as sericulture

Sericulture is a demanding sector where the cultivation of mulberry trees (Moraceae family, Morus genus) and the reproduction of silk worms are key components The primary

objective is to acquire both yarn and textile goods This process involves preserving eggs, breeding silk worms, disease prevention, feeding with mulberry leaves, collecting mature larvae, and transferring them to the area for cocoon formation (Takeda, 2009) Globally, approximately 100 thousand tons of silk are produced each year Among these, China contributes 70 %, followed by Brazil, Japan, India, Thailand, and Vietnam (Pescio.,et al 2008)

Silkworms feed on leaves, with these leaves containing 81.72 % water, 0.57 % fat, 1.55 % protein, 1.47 % fiber, and 14.21 % carbohydrates Leaf proteins are synthesized by the silk gland cells of the silkworm, and immediately after, they are stored in the lumen, where they transform into silk fibers During the spinning process, these silk fibers pass through the anterior gland and are then ejected through the die opening The result is a delicate double fibroin filament, coated by a gum called sericin Sericin aids in the formation of the silk cocoon by acting as a binder, maintaining its structural integrity The obtained oval-shaped structure serves as a safe haven during the larval metamorphosis process, transitioning from a larva to a pupa (Patel & Modasiya, 2011; Takeda, 2009)

Figure 2.1 Bombyx mori L’s cocoon

2.1.2 The chemical composition silkworm cocoon

The structural composition of the silk cocoon layer involves two main proteins, fibroin and sericin Fibroin, serving as the central component, is a fibrous protein, while sericin, acting as the adhesive element, is a globular protein that wraps around the fibers and binds them together Additionally, certain impurities such as carbohydrates, salts, and waxes, referred to as "non-sericin" constituents, contribute to the water repellent properties of the silk cocoon

Concerning various silkworm types and methods for extracting components based on nutrition sources, the cocoon primarily consists of fibroin, sericin, and additional impurities such as pigments, waxes, carbohydrates, and phytochemicals These components make up approximately 75–83 %, 17–25 %, and around 1–4 % of the cocoon respectively (Hossein Biganeh., et al 2022) The amino acid residues present in silk proteins can be categorized into three classes, encompassing charged residues like aspartic acid, polar residues like serine, and hydrophobic residues like glycine

2.1.3 Biological characteristics of silk cocoon

In recent years, there has been a growing interest in the biological properties of silkworm, especially its main proteins such as fibroin and sericin Scientific studies have focused on exploring the pharmacological effects of silkworm and its specific components These applications include cardiovascular protection, antioxidant capabilities, cancer cell inhibition, diabetes treatment, lipid reduction, gastric mucosa protection, and improvement of skin health (Biganeh.,et al 2022) Silkworm has also been extensively researched and

2.2 Overview of Sericin 2.2.1 Introduction of Sericin

Sericin can be extracted from silk fibers by removing it from the fibroin component Since only the fibroin part is essential in the silk industry, the removal of sericin is necessary and is typically carried out through the degumming process, after which sericin is discarded Recycling sericin from degumming water not only helps reduce the load in wastewater but also produces a biopolymer with various important properties There are several methods for extracting sericin, including boiling [69], alkaline treatment [70], organic solvents lactic acid and citric acid [35] Another method involves using high temperature and high pressure to eliminate sericin from silk fibers

2.2.2 The chemical composition Sericin

Silk secirin an innate luminous globular protein, is obtained from the silk cocoon of

Bombyx mori (Ki CS, 2007; Poza P, 2002; Wu JH, 2007) Its chemical structure is depicted

in Figure 2.2

Where R = -CH3 or -CH2C6H4OH

Figure 2.2 Chemical structure of silk sericin ( Saha., et al 2019)

Fibroin serves as the core structure of the silk fiber, while sericin acts as the adhesive substance enveloping it Additionally, the composite structure of fibroin consists of the amino acids Gly-Ser-Gly-Ala-Gly-Ala, forming beta-pleated sheets referred to as "β- keratin." Simultaneously, hydrogen bonds are established between chains and side chains, both above and below the plane of the hydrogen bond network In this context, R represents H (glycine), R represents CH3 (alanine), and R represents CH2OH (serine).( Saha., et al 2019)

Sericin is a natural polymer found in the cocoon of the B mori silkworm, connecting

with fibroin molecules through hydro bonds, constituting 25 – 30 % of the total cocoon weight Sericin is a hydrophilic protein with the ability to dissolve in hot water To separate sericin from fibroin or the silkworm cocoon, degumming techniques can be employed and implemented through various methods (Saha., et al 2019) Sericin typically exists in an amorphous state, exhibiting adhesive properties, enveloping fibroin protein fibers, forming the cocoon's structure, and aiding in maintaining the integrity of the cocoon's structure

Sericin is a macromolecule of hydrophilic amino containing hydrophilic amino acids, comprising a total of 18 amino acids It possesses potent polar groups, such as amino, carboxyl, and hydroxyl groups, which can engage in copolymerization, crosslink formation, and amalgamation with other polymers (Lamboni.,et al 2010) The elemental composition analysis of sericin reveals it consists of 6 % hydrogen, 46.5 % carbon, 16.5 % nitrogen, 31 % oxygen, and 0.9 % sulfur (Kunz., et al, 2016; Jena,K., et al, 2018) The CN ratio shows a correlation with hot water solubility; thus, a lower C/N ratio results in higher solubility in hot water (Jena,K., et al, 2018)

Sericin exhibits a molecular weight ranging from 10 to 300 kDa, depending on the solvent used (acidic or alkaline solvents) for sericin dissolution The content and molecular size of sericin are influenced by factors such as temperature, pH, processing time, and solvent concentration (Zhang YQ, 2002) ).The silk cocoon is divided into three layers of sericin-outer, middle, and inner-each containing 15 %, 10.5 %, and 4.5 % sericin respectively

(Cao, T.-T., & Zhang, Y.-Q 2016) , as illustrated in Figure 2.3 Sericin remains insoluble in

cold water but becomes soluble in hot water, as the extended protein molecules break down into smaller fractions under high-temperature conditions, making them easily hydrolyzed and dispersed (Cao, T.-T., & Zhang, Y.-Q., 2016; Zhang YQ, 2002)

Table 2.1 The amino acid composition of sericin extracted using various methods (in mole%) (Aramwit et al 2009)

Amino acid Extraction method of sericin

Asp 15.64 18.31 15.93 19.88 Ser 33.63 31.27 31.86 30.01 Glu 4.61 5.27 5.75 5.93 Gly 15.03 11.23 10.49 11.01

His 1.06 3.26 2.47 1.72 Arg 2.87 5.41 4.92 4.92 Thr 8.16 8.36 8.51 6.49 Ala 4.10 4.33 3.72 4.21 Pro 0.54 1.46 0.78 1.24 Cys 0.54 0.39 0.53 0.23 Tyr 3.45 0.36 5.56 5.24 Val 2.88 2.96 2.95 2.94 Met 3.39 0.12 0.06 0.15 Lys 2.35 3.14 3.48 2.89 Ile 0.56 0.96 0.87 0.75 Leu 1.00 1.58 1.43 1.56 Phe 0.28 0.60 0.71 0.81

The working conditions of the enzyme restrict the use of this method for extraction purposes (Sothornvit, R., et al, 2010) The hot water extraction method is the most common due to the mentioned constraints for sericin extraction In this method, silk is heated in hot distilled water without adding any chemicals Both time and temperature play important roles in the amount of extracted sericin Although this method causes a reduction in the quality of sericin, the extent of the reduction is not to the point where sericin loses its essential properties It has been reported that temperature can be applied to the system through boiling at atmospheric pressure (Aramwit, Pornanon; et al 2009), HTHP (high temperature, high pressure) (Gupta, D., et al, 2014; Haggag, K.,2007) However, the HTHP method for sericin extraction does not use chemicals but produces smoke, is messy, time- consuming, and may damage fibroin (Aramwit, Pornanong; 2009) In this report, sericin was extracted from silk cocoon using the high-temperature and high-pressure method Below is the basic procedure for this method

-Prepare Silk Cocoons:

+ Obtain silk cocoons from silkworms

+ Sort and clean the cocoons to remove any impurities -Autoclave Setup:

+ Place the cleaned silk cocoons inside the autoclave machine Add the calculated amount of distilled water

-Adjust Parameters: Set the autoclave machine or pressure cooker to high temperature and high pressure conditions The specific temperature and pressure values may vary, but commonly, temperatures around 121 °C in 15 and 30 mininutes

-Cooling: After the extraction process is complete, allow the autoclave to cool down before opening it

-Separate Sericin: Extract the silk cocoons from the autoclave and separate the sericin from the fibroin This can be done by washing the cocoons and gently separating the sericin layer -Characterization: Optionally, you can characterize the extracted sericin for its properties, such as molecular weight, composition, and functional groups

+ This general procedure provides an overview, and the actual process may be adapted based on specific research or production requirements

2.3 Applications of Sericin 2.3.1 Antibacterial Agent

2021) Extracted through a degumming process involving sodium carbonate, sericin has

Waraluk, et al (2009) Sericin obtained through water degumming has exhibited the ability to inhibit S aureus, another foodborne pathogenic bacterium.

2.3.2 Antioxidant potential of sericin

Due to the presence of polyphenols and flavonoids, sericin exhibits antioxidant

of amino acids, hydroxyl groups, high-molecular-weight sericin, polyphenols, and

JIN‐BO, et al 2009) Therefore, sericin is a valuable multifunctional substance that can be explored for applications in the cosmetics industry and food preservation, as a natural and safe component against oxidative processes affecting food quality and shelf life (Miguel, Gabriela Andrea, et al)

2.3.3 Anti-aging capability

The powder made from sericin (5–30 %) and silk fibers (70–95 %) exhibits anti- static and moisture-absorbing properties (Kundu, Subhas C., et al 2008) Silk sericin, with its high water solubility, becomes a crucial element in the cosmetics industry (Yazicioglu, Alkin, et al, 2017) Sericin not only acts as an adhesive but is also widely recognized in skincare, haircare, and nail products, contributing to improved skin elasticity and anti- wrinkle effects (Gillis G., Bojanowski, et al, 2009) Particularly noteworthy is sericin's ability to inhibit apoptosis and stimulate the synthesis of collagen type I Additionally, it surpasses vitamin C in its anti-aging capabilities by effectively limiting oxidative stress (Kitisin, T., et al 2013)

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2.4.1 In the food packaging and food coating

The current primary materials for food packaging are synthetic polymers Synthetic polymers are non-biodegradable and non-renewable, contributing to environmental pollution This issue can be mitigated by utilizing biodegradable polymers as alternatives to

exhibit excellent reactivity and possesses numerous high biological functions, such as biodegradability, biocompatibility, antibacterial properties, and antioxidant capabilities (Zhang YQ, 2002).

Another article suggests that the use of a coating material containing sericin, chitosan, aloe vera, and glycerol has the potential to extend the shelf life of tomatoes when stored at 25°C and 70% relative moisture This material helps maintain the quantity of fruits and prevents the aging process, creating conditions similar to those after harvest ATR–FTIR

T., & Takamura, H, 2014).

2.4.2 In the food industry

Sericin is used in the production of bread When combined with 2–4 g sericin/1 kg of flour to make bread, there is a tendency to reduce the height and bulk density of the bread, as well as alter its color However, sericin maintains the uniform internal surface structure

H, 2014) The author asserts that sericin, lacking immune-stimulating properties (Takechi, T., & Takamura, H, 2014), can be employed as an emulsifying agent instead of natural food emulsifiers like egg yolk and casein, which occasionally pose the risk of potential allergic reactions Furthermore, the emulsifying activity of sericin can be enhanced through acylation

2.5 The current research status of sericin 2.5.1 The world

Currently, research on sericin in the field of food technology is attracting attention from the scientific community and the food industry worldwide Sericin, a protein derived

from silk, is being explored and applied in various applications within the food industry Some studies focus on using sericin as a thickening agent, preservative, or antioxidant in food Its biological properties, including its ability to form gels and adhesion, offer potential for enhancing the quality and stability of food products Moreover, sericin is being investigated to create protective coatings to shield food from oxidation, contamination, and to maintain freshness and longevity

While still in the research and development phase, the application of sericin in food technology holds significant potential for improving the quality and attractiveness of food products, thereby creating opportunities for innovation in the food industry

Sericin is still being researched in various fields:

- Medical applications: There's particular interest in the medical potential of sericin Research focuses on medical material applications, including tissue regeneration, self- dissolving properties, and even the production of smart medical products

- Textiles and Fashion: Sericin is considered a potential raw material in the textile and fashion industries Its flexibility can lend unique characteristics to textile products

- Processing and Extraction: Research into sericin processing and extraction methods revolves around optimizing processes to ensure high quality and efficiency

- Pharmaceutical and Healthcare applications: Several studies emphasize sericin's potential in antioxidant, antibacterial properties, and other features in pharmaceuticals and healthcare products

Kumar J.P., et al(1981) Anti-inflammatory activity Kaewkorn W., et al (2012)

Anti-aging activity Wu J.-H., et al ( 2007) Anticancer activity Khampieng T., et al ( 2015)

Deenonpoe R.,et al (2019) Anti-tyrosinase activity Kundu S.C.,et al (2008)

- Medical Applications: There's particular interest in the medical potential of sericin Research focuses on medical material applications, including tissue regeneration, self- dissolving properties, and even the production of smart medical products ( Fakharany, et al 2020)

- Textiles and Fashion: Sericin is considered a potential raw material in the textile and fashion industries Its flexibility can lend unique characteristics to textile products (Kumar, et al 2022)

- Processing and Extraction: Research into sericin processing and extraction methods revolves around optimizing processes to ensure high quality and efficiency (Lamboni et al 2015)

2.5.2 Vietnam

The research on sericin in Vietnam is drawing attention from both the research community and related industries Sericin, a protein derived from silk, is being explored and

applied across various fields in Vietnam, including healthcare, food industry, pharmaceuticals, and textile industry

Studies on sericin in Vietnam are focused on optimizing the extraction process from silk and analyzing its properties and applications in different sectors (Le; et al., 2022)

In the healthcare sector, sericin is being researched for applications in tissue regeneration, the production of smart medical materials, and other advanced medical products

Within the food industry, sericin is being considered to enhance the quality and preservation characteristics of food products

In pharmaceuticals and healthcare, research is concentrating on sericin's antioxidant, antibacterial properties, and other beneficial characteristics for health-oriented product development

While still in progress, research on sericin in Vietnam holds significant potential for extensive development and application across various domains, especially when combining fundamental research with practical applications

The research findings on the extraction of silk proteins from silkworm cocoons, conducted by Le Hong Van and colleagues, were investigated using three methods: Na2CO3 salt utilization, neutral soap application, and the high-temperature, high-pressure method It was found that the extraction of sericin and fibroin by boiling silkworm cocoons in pure water at a temperature of 126°C and a pressure of 0.14 Mpa for 5 hours was determined to be the optimal method The sericin and fibroin powders were assessed to have high purity and were easily soluble in water Consequently, the powders of sericin and fibroin are regarded as potential raw materials for application in the cosmetic or food industry (Le; et al., 2022)

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CHAPTER 3: MATERIALS AND METHODS 3.1 Materials

3.1.1 Silkworm cocoon shell

The B mori silkworm cocoons used in the study are from silkworm farms in the Nam

Dinh area After a small portion of the shell is sliced apart to reveal the silkworm pupae, the cocoons will be collected and transported to facilities that need silkworm cocoon shell materials The cocoon shell of the silkworm must be structurally intact, unstained, and purified

The silkworm harvest is in the fall from September 5 to 10 and ends in November every year, and the group purchases cocoon shells near the end of September

3.2 Equipment for study

3.2.1 Chemicals used in experiments

Table 3.1 Chemicals used in the study

1 Sodium hydroxide (NaOH) China 2 Sodium carbonate (Na2CO3) China

11 Peptone China 12 Sodium chloride (NaCl) China 13 Yeast extract powder India 14 Folin Ciocalteu phenol reagent Germany 15 Potassium Persulfate China

16 Potassium sodium tartrate tetrahydrate

(KNaC4H4O6) China

3.2.2 Equipment

Table 3.2 Equipment used in the study

1 2 and 4-digit analytical

balance Sartorius, Germany 2 Kjeldahl machine Germany 3 pH meter Italy 4 Autoclave machine Gernamy 5 Thermostat tank Germany 6 FTIR – 4700 meters (Jasco, Japan)

7 Ultraviolet–Visible (UV-VIS)

spectrophotometry Japan

8 Biological Safety Cabinet

Class II (BSC) Singapore