MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION GRADUATION THESIS MAJOR: FOOD TECHNOLOGY EFFECT OF ACID SOLVENT ON PROPERTIES OF SERICIN PROTEIN EXTRACTED FROM SILK COCOON INSTRUCTOR: PHAM KHANH DUNG STUDENT: BUI NGOC HOANG VI Ho Chi Minh city, June 2024 HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY OF INTERNATIONAL EDUCATION GRADUATION PROJECT Thesis code: 2024 - 19116036 EFFECT OF ACID SOLVENT ON PROPERTIES OF SERICIN PROTEIN EXTRACTED FROM SILK COCOON BUI NGOC HOANG VI Student ID: 19116036 Major: FOOD TECHNOLOGY Supervisor: Ms. PHAM KHANH DUNG, PhD. Ho Chi Minh City, June 2024 HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY OF INTERNATIONAL EDUCATION GRADUATION PROJECT Thesis code: 2024 -19116036 EFFECT OF ACID SOLVENT ON PROPERTIES OF SERICIN PROTEIN EXTRACTED FROM SILK COCOON BUI NGOC HOANG VI Student ID: 19116036 Major: FOOD TECHNOLOGY Supervisor: Ms. PHAM KHANH DUNG, PhD.
Ho Chi Minh City, June 2024 i DISCLARATION We affirm that the work presented in this graduation thesis is entirely our original creation. We also verify that all sources and references used have been properly and thoroughly cited in accordance with the appropriate guidelines. Ho Chi Minh city, 20th June, 2024 Signature ii ACKNOWLEDGEMENTS First and foremost, we would like to express our heartfelt gratitude to all Department Food Technology Programe - Faculty Of International education - Ho Chi Minh City University of Technology and Education for their dedicated teaching and imparting knowledge during the past years and created all conditions for facilities and equipment to help us complete the thesis in the best way. We extend our sincere thanks to the faculty and staff of Ho Chi Minh City University of Technology and Education for providing the necessary facilities and support throughout the course of our thesis work.
Most importantly, we wish to convey our deepest appreciation to Ms. Pham Khanh Dung Ph.D, who guided and mentored us with great dedication, imparting invaluable knowledge and creating the most favorable conditions for us throughout the development of our thesis. Her insightful comments, guidance, and helpful suggestions have greatly broadened our understanding and helped us identify and address numerous important issues during the completion of this thesis. Inevitably, due to time constraints and our limited knowledge, shortcomings have arisen during the process of studying and writing this thesis.
We welcome valuable feedback from our teachers to help improve our group's thesis. Finally, we wish all the teachers and faculty members good health and continued success in their noble careers. We sincerely thank iii iv v vi vii viii ix TABLE CONTENT CHAPTER 1. Object and scope of the research.
Scientific and practical significance. Layout of the research. Overview of Silkworm Cocoon. Introduction of Silkworm Coocon.
Chemical compostion silkworm Cocoon. Biological characteristics of silkworm cocoons. Overview of Sericin. Introduction of Sericin.
The chemical composition Sericin. Methods for sericin extracting. Appication 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 .1 In the world. MATERIALS AND METHODS. Silkworm cocoon shell. Chemical used in experiment.
Research process diagram. Extraction process of sericin from silkworm cocoon. Surveying raw materials. Determination of moisture content.
Determination of ash content. Determine the lipid contet by Soxhlet method of method of cocoon. Determine the carbohydrate content of cocoon. Determine the total nitrogen content by Kjeldahl method of cocoon.
Survey other types of acids when used in the process of extracting Sericin from silkworm cocoons and compare. Survey the concentration of citric acid at different concentrations when used in the process of extracting Sericin from silkworm cocoons and compare. Investigating the optimal temperature during the extraction of Sericin from silkworm cocoons. Investigating the optimal time in the process of extracting Sericin from silkworm cocoons.
The Lowry technique is used to determine the sericin content in the extract. Characterization of sericin solution from Acid extraction method. SDS-Page Analysis. Isoelectric point of sericin (pI).
Evaluation of amino acid composition. Evaluation of the antioxidant activity of sericin. Raw silk cocoon composition. Identifying the optimal types of acids for sericin extraction from silkworm cocoons.
Identifying the optimal the concentration of acids for sericin extraction from silkworm cocoons. Identifying the optimal temperature for sericin extraction from silkworm cocoons. Identifying the optimal time for sericin extraction from silkworm cocoons. Evaluation of the characterization of sericin solution from Acid extraction method 44 4.
UV Spectroscopy of sericin solution. SDS-Page Analysis. Isoelectric point of sericin (pI). Evaluation of amino acid composition.
Evaluation of the antioxidant activity of sericin. Evaluation of antibacterial activity of sericin. CONCLUSION AND RECOMMENDATIONS. 68 xiii LIST OF FIGURES Figure 2.1 Bombyx mori L’s cocoon .2 Diagram of three sericin layers of silkworm cocoons.3 Depicting the chemical structure of silk cocoon .1 Diagram of experimental research .2 Process diagram for extracting sericin from silkworm cocoons.3 Chopped silk cocoon shells .1 Sericin content extracted by acid under different acid conditions.2 Sericin content extracted with acid under conditions of varying acid concentration.3 Sericin content extracted by acid under changing temperature conditions.4 Sericin content extracted by acid under changing time conditions.5 UV spectrum of sericin from extraction method using Citric acid solvent (orange) and distilled water (blue).6 Results of protein electrophoresis of sericin from extraction method with citric acid solvent.7 FTIR spectrum of sericin extracted using citric solvent (blue) and distilled water (orange) .8 The experimental results for measuring the isoelectric point (pI) of sericin.9 Results of ABTS free radical scavenging ability of sericin solution extracted from B.
mori using citric acid solvent.10 Antibacterial activity of sericin against S. aureus after 24 hours of incubation for (a) Control plate, (b) Sericin supplemented plate. aureus bacterial cell density (%) in different rations of sericin extract.12 Antibacterial activity of sericin against E. coli after 24 hours of incubation for (a) Control plate, (b) Sericin supplemented plate.
coli bacterial cell density (%) in different rations of sericin extract. 55 xv LIST OF TABLES Table 2.1 The amino acid makeup of Fibroin and Sericin .2 The secondary structure ratio of B. mori sericin protein obtained using extraction techniques .3 The amino acid makeup of sericin extracted using different techniques (in mole%) .1 Chemicals used in the research .2 Equipment used in the research .3 Arrange experiments to investigate the extraction process of sericin in different types of acids.4 Arrange the experiment to investigate the extraction process of sericin with citric acid solution at different concentrations.5 Arrange the experiment to investigate the sericin extraction process with citric acid solution at different temperatures.6 Arrange the experiment to investigate the extraction process of sericin with citric acid solution at different times.9 Experimental setup for isoelectric method of sericin extract .10 Plate Count Agar (PCA) .1 Some criteria for raw materials of B. mori silkworm cocoon shells.2 The amino acid content in sericin is extracted with citric acid solvent.
49 xvi 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 S. aureus: Staphylococcus aureus E. coli: Escherichia coli BSA: Bovine Serum Albumin CFU: Colony Forming Unit DPPH: Diphenylpicrylhydrazyl pI: Isoelectric point PCA: Plate Count Agar SDS-PAGE: Sodium Dodecyl Sulfate–PolyacrylAmide Gel Electrophoresis APS: Ammonium persulfate TEMED: N, N, N, N-tetramethyl ethylenediamine UV-Vis: Ultraviolet–Visible spectrophotometry LOD: Limit of detection. NO : Not detected xvii ABSTACT This study explores Effects of acid solvents on properties of sericin protein extracted from silk cocoon in the laboratory.
The study investigates a variety of variables, including organic acid kinds, acid concentrations, temperature, and extraction time. The optimum conditions were calculated as follows: Using a 20% citric acid solution, the maximal sericin level was 124.24 mg/g after 30 minutes at 100°C. Furthermore, the extracted sericin solution was analyzed for amino acid composition, which revealed that glycine, serine, and aspartic acid were the main components. The secondary structural properties of sericin were investigated using Fourier transform infrared spectroscopy (FTIR), which revealed strong absorption peaks around 3292 𝑐𝑚−1 due to the presence of O-H stretching, the N-H stretching vibration peak at 3066 𝑐𝑚−1.
Protein bands at between 26 and 37 kDa were detected using SDS-PAGE analysis. The antioxidant activities of the sericin extract were assessed using ABTS free radical scavenging tests, which yielded an IC50 of 3. Furthermore, it was demonstrated that sericin has antibacterial activity to against Staphylococcus aureus and Escherichia coli, with effectiveness proportional to sericin concentration, indicating its ability to limit bacterial development and lower bacterial density. Based on these findings, sericin appears to be a potential substance for use in the food business due to its functional qualities.
Further study might look at its use as an antioxidant and antibacterial agent in food compositions. Keywords: Sericin, Bombyx mori, acid solvent extraction, Fourier transform infrared spectroscopy, antioxidant activity, antibacterial activity, food industry applications 1 CHAPTER 1. Problem Silk is a natural material made up of proteins secreted by silkworms while they construct cocoons to build nests. To mitigate these environmental impacts and utilize the full potential of silk production, there is ongoing research and development focused on finding sustainable solutions.
One approach involves exploring innovative methods to recover and utilize sericin from silk processing wastewater. By doing so, we can reduce environmental pollution, minimize waste of valuable biological resources, and promote more sustainable practices in the silk industry. These efforts align with advancements in food technology, aiming to enhance efficiency and sustainability across the entire silk production (Wu, Yue, & Zhang, 2014). The removal and use of sericins can have significant economic, social, and environmental consequences, particularly in mulberry-growing nations such as China, India, and Brazil.
Wastewater from industry enters wastewater streams, resulting in increased Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) (Fabiani, Pizzichini, Spadoni, & Zeddita, 1996). As a result, effluent from the silk industry pollutes both water and the environment. Reclaiming and reusing sericin, often discarded by the textile industry, not only benefits the environment but also carries significant scientific and commercial value (Kunz, Brancalhão, Ribeiro, & Natali, 2016). With a growing demand for biocompatible and biodegradable materials, there is increasing interest in exploring alternative applications of sericin proteins.
Recent studies have highlighted its potential in polymers, biomaterials, cosmetics, and the food industry (Zhao & Zhang, 2020). This underscores ongoing efforts in food technology to explore diverse applications of sericin, aiming to capitalize on its versatile properties and contribute to sustainable practices in various industries. Sericin is regarded a useful food in biomedicine and pharmaceuticals due to its antioxidant capabilities, and several studies have found that consuming sericin decreases cholesterol levels considerably. Furthermore, silk protein membrane is a safe biological substance that does not influence serum biochemical parameters when administered via the skin and is unlikely to induce skin sensitivity and irritation.
It is a novel wound coagulation material due to its outstanding biocompatibility and anti-infective qualities (Padol et al. 2 Our group researched this topic with the aim of minimizing the pollution of industrial wastewater and using sericin source in silkworm cocoons to research and apply in biology and application of Food Technology. Therefore, we have the idea of doing analytical research to extract sericin from silkworm cocoons using acid as a solvent to optimize the efficiency of obtaining the amount of sericin in silkworm cocoons. Research objective The purpose of the study was to see how acid affected the features and qualities of Sericin from silkworm cocoons.
Determine the acid type and concentration of the solvent used to extract sericin from silkworm cocoon shells. Surveying several times and temperatures can help you determine the best time and temperature combination. Furthermore, assess sericin's physicochemical qualities and biological features (antioxidant, antibacterial) in order to determine whether it is a promising raw material for the food sector.