MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION PROJECT FOOD TECHNOLOGY 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 SKL012529 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 Ho Chi Minh City, January 2024 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.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. 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) SUPERVISOR (Sign with full name) 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 ii 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. iii TABLE OF CONTENTS CHAPTER 1: INTRODUCTION.3 Object and scope of the research .5 Scientific and practical significance. 4 CHAPTER 2: LITERATURE REVIEW. Overview of Silkworm Cocoon.
Introduction of Silkworm Cocoon .2 The chemical composition silkworm cocoon. Overview of Sericin. Introduction of Sericin. Methods for sericin extracting.
Applications of Sericin. Antioxidant potential of sericin. Anti-aging capability. Applications of Sericin in the Food Industry.
In the food packaging and food coating. In the food industry. The current research status of sericin. 15 CHAPTER 3: MATERIALS AND METHODS.1 Silkworm cocoon shell .2 Equipment for study.
Chemicals used in experiments .1 Research process diagram .2 Extraction process of sericin from silkworm cocoon .1 Raw materials survey.1 Method for moiture content determination .2 Method for determining total ash content of cocoon .3 Determination of total nitrogen content by Kjeldahl method of cocoon. Determine the lipid content by Soxhlet method of cocoon. 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: .2Determination of the optimal temperature during the sericin extraction process 26 3.3 Determine the optimal time during the sericin extraction process .1 Estimation of sericin quality and quantity the protein content by Lowry's method .6 Characterization of sericin solutions from HTHP extraction method .2 SDS – Page Analysis .4 Isoelectric point of sericin .5 Evaluation of amino acid composition .7 Evaluation of the antioxidant activity of sericin .8 Evaluation of antibacterial activity of sericin.
34 CHAPTER 4: RESULTS AND DISCUSSIONS. Raw silk cocoon composition .2 Determination of the optimal temperature during the sericin extraction process .3 Determine the optimal time during the sericin extraction process .4 Evaluation of the properties of sericin solution from various extraction methods. UV spectroscopy of sericin solution .2 SDS – Page Analysis. Isoelectric Point of Sericin.
Evaluation of amino acid composition. Evaluation of the antioxidant activity of sericin. Evaluation of antibacterial activity of sericin. 64 vi LIST OF FIGURES Figure 2.
Bombyx mori L’s cocoon. Chemical structure of silk sericin ( Saha.3 : Three layers of sericin in a silk cocoon (Saha.1: Diagram of experimental research.2: Flow chart of sericin extraction process from silkworm cocoons. Chopped silk cocoon shells .1: Sericin content in various temperature extraction .2: Sericin content in various time extraction .3: UV spectroscopy of silk sericin .4: SDS-PAGE analysis of sericin samples .5: FTIR spectrum of high temperature and high pressure (HT-HP) and NaOH degummed sericin. Determination of the isoelectric point (pI) of sericin through experimental results .7: Standard curve of ABTS free radical scavenging ability of sericin concentration.
aureus of sericin after 24 hours. aureus bacterial cell density (log CFU/mL) in different rations of sericin extract. coli of sericin after 24 hours .coli bacterial cell density (log CFU/mL) in different concentrations of sericin extract. 53 vii LIST OF TABLES Table 2.1 The amino acid composition of sericin extracted using various methods (in mole%) (Aramwit et al 2009).
Pharmaceutical and Healthcare Applications. Chemicals used in the study. Equipment used in the study. Arrange the experiment to investigate the sericin extraction process at different temperatures.
Arrange the experiment to investigate the sericin extraction process at different times. Separation gel composition. Stacking gel composition. Experimental arrangement for the isoelectric method of sericin extract.
Some Parameters of Bombyx Mori Silk Cocoon Shell Raw Material. FTIR Peaks of Sericin Powder Obtained by the Salting-Out Proces (M. Senthil Kumar, Roli Purwa 2008) .3: Results of amino acid composition table of sericin extract. 46 LIST OF ABBREVIATION viii 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 ix 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. 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.1 Problem The silkworm is the larva (the active immature form of an insect) or caterpillar of the Bombyx mori moth.
Silk has been made for at least 5000 years in China (Dhanushree et al, 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. 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 2 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).2 Research objective The study was conducted to investigate the effects of high pressure and high temperature on the extraction of sericin from silkworm cocoons. -Determine the optimal temperature of sericin extract by investigating different temperatures. -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.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.4 Research content Investigate and evaluate the effects of temperature and time on sericin content obtained from the high temperature and high pressure (HTHP) extraction methods: - Evaluate chemical composition of silk cocoon: total protein, lipid, carbohydrate, ash and moisture content.