VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------***------------- GRADUATION THESIS TITLE: RESEARCH ON THE PROCESS OF SEPARATING COLLAGEN FROM HORSE SKIN HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------***------------- GRADUATION THESIS TITLE: RESEARCH ON THE PROCESS OF SEPARATING COLLAGEN FROM HORSE SKIN Student name : VU THI YEN Student code : 637193 Class : K63CNSHE Faculty : BIOTECHNOLOGY Supervisor : Dr. LE THI BICH THAO : Dr. NGUYEN THANH HAO HANOI – 2022 DECLARATION OF AUTHORSHIP I hereby declare that this is my research work under the guidance of Dr. Le Thi Bich Thao and the co-direction of Dr.
Nguyen Thanh Hao. The data and results presented in the thesis are honest and partly published in the proceedings of the Vietnam National Conference on Biotechnology 2022 with the consent and permission of the co-authors. The rest has not been published by anyone in any other work. Hanoi, day 22 month 2 year 2023 Student Vu Thi Yen i ACKNOWLEDGEMENTS I would like to express my sincere gratitude and boundless gratitude to Dr.
Le Thi Bich Thao, Head of the Protein Biochemistry Department, Institute of Biotechnology, as well as the main instructor in my research. Le Thi Bich Thao not only enthusiastically guided and taught me, but also created conditions to support and encourage me during the past time so that I could complete my thesis. In particular, she also instilled in me a passion for scientific research and helped me have a clear direction for my future research. I have also learned a lot from Dr.
Le Thi Bich Thao's valuable experiences in recent years. Again, I sincerely thank you. I would also like to express my deep gratitude to Dr. Nguyen Thanh Hao (Lecturer of biology, Vietnam National University of Agriculture), who was my co-guide in my research.
In addition to talking with me about experimental results, the doctoral scholar also encouraged me and shared with me the difficulties of scientific research, and I am very grateful for that help. I am extremely grateful and dear to my parents, who gave birth to me and raised me up. My parents have always been a source of encouragement and comfort for me throughout my life, and I would like to share this joy with them as a gift to affirm my growth and maturity. I would like to thank my sister and two cousins, who always bring me joy, help, and sincere encouragement.
I am also very grateful to the aunts and uncles, brothers and sisters, and colleagues at the Department of Protein Biochemistry, Institute of Biotechnology. Bui Thi Huyen and Ms. Pham Thi Hue taught me many practical things, and I learned a lot from the experience when we worked together. Colleagues Ngo Nhat Quang, Nguyen Thuy Dung have shared with me in scientific research and in life.
You always bring me joy, laughter, and comfort so that I can work in a ii friendly and united scientific environment. I always look forward to working with all my uncles and colleagues forever. Finally, I would like to thank the Board of Directors of the institute of Biotechnology, the Leadership of the Key Laboratory of Gene Technology, the aunts and uncles, brothers and sisters, and colleagues working at the Institute of Biotechnology. They collaborated in research as well as created favorable conditions for me to complete the thesis Hanoi, day 22 month 2 year 2023 Student Vu Thi Yen iii CONTENTS DECLARATION OF AUTHORSHIP.
vi LIST OF TABLES. viii LIST OF FIGURES. What is collagen?. How many types of collagen are there?.
Structures of collagen. Application and origin of collagen types. Applications in food technology. Applications in medicine and pharmaceuticals.
Applications in the cosmetic industry. Research situation on collagen extraction in the country and in the world. MATERIALS AND METHODS OF RESEARCH. Chemicals, machinery and equipment.
Methods of research. Preservation of hydrolyzed collagen. Quantitative technique of collagen concentration dissolved in extract solution. 29 iv PART III.
RESULTS AND DISCUSSION. Effect of enzymes on the concentration of soluble collagen in pepsin. Effect of ultrasonic on the concentration of soluble collagen in pepsin. Effect of temperature on the concentration of soluble collagen in pepsin.
Determination of the efficiency of collagen extraction. Surface morphology of PSC from horse skin. 44 v ABBREVIATIONS Abbreviations AEE Associated equine encephalitis BSE Bovine spongiform encephalopathy DNA Deoxyribonucleic acid FMD Foot and mouth disease FE-SEM Field Emision Scanning Electron Microscopy SEM Scanning Electron Microscopy HCl Hydrogen Chloride (24)h (24) hours kDa Kilo Dalton kHz Kilohertz kV Kilovolt M Molarity Min Minute nm Nanometer NaCl Sodium Chloride NaOH Sodium Hydroxide Pa Pascal pH Potential of hydrogen PSC Pepsin-soluble collagen Pt Platium rpm Revolutions per minute SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis SDS Sodium Dodecyl Sulphate-Polyacrylamide vi UV-Vis Ultraviolet and visible U/g Unit/gram W Watt w/w Weight/weight w/v Weight/volume α1 Alpha 1 α2 Alpha 2 β Beta o C Celsius µl Microliter vii LIST OF TABLES Table 1. Collagen classification table (Pope et al.
Machinery and equipment. Results of collagen mass obtained when extracted at room temperature (25-26oC) using 2% pepsin ratio (w/w) with ultrasonic. Results of collagen mass obtained when extracted at room temperature (25-26oC) using 1% pepsin ratio (w/w). 41 viii LIST OF FIGURES Figure 1.
What is collagen? (Pham Anh, 2021). Structure of collagen (Lin et al. Collagen sausage casings (Amazon. Some collagen drinks (AliExpress).
Some collagen peptide products (Amazon. Collagen used in dentistry (Hawryluk, 2021). Collagen used in “beauty mask” (Amazon. Schematic diagram of the method of extracting collagen from horse skin.
Error! Bookmark not defined. Skin samples before and after pretreatment. Pepsin 2000 U/g of Novaco(Vietnam) is used in the collagen extraction process. SDS-PAGE procedure.
Scanning electron microscope (SEM). UV-vis spectrophotometer. Pepsin-soluble collagen (PSC) is extracted from horse skin. SDS-PAGE of PSC extracted from horse skin using different ratios of pepsin.
Spectrum investigating the effect of enzyme on collagen extraction efficiency. Spectrum investigating the effect of ultrasonic on collagen extraction efficiency. SDS-PAGE of PSC extracted from horse skin by ultrasonic M: Marker; 1: Ultrasonic; 2: No ultrasonic. SDS-PAGE of PSC extracted from horse skin with different temperature.
Quantitative spectrum of collagen by UV-vis method. SEM image of lyophilization PSC from horse skin (left, magnification × 5. 42 x PREAMBLE Surely we have come across an advertisement or heard someone talk about a certain cosmetic or functional food containing collagen ingredients and the wonderful effects they bring. In fact, as age increases, the body's endogenous collagen production level decreases, which affects the firmness of the skin.
Therefore, exogenous collagen is a very sought-after product today. They are used for medical and cosmetic purposes, with the general purpose of providing repair and restoration materials for body tissues. Contributes to maintaining endogenous collagen production, thereby slowing down the aging process of the body. Typically, exogenous collagen products contain collagen types I, II, III, or a mixture of all three.
Collagen is supplied to the body mainly in three forms: hydrolyzed collagen, gelatin, and collagen in raw form. In particular, the best form of collagen absorbed by the body is hydrolyzed collagen. That is, exogenous collagen in any form introduced into the body is hydrolyzed into amino acids, which can be easily absorbed by the body to build collagen and other necessary protein forms. Most cosmetics and collagen supplements on the market are derived from animals, especially cows, pigs, and fish.
However, the use of pigs and cows as raw materials for collagen extraction is limited because of immune responses, the risks of bovine or porcine infectious diseases, and religious constraints. Therefore, a new source of raw materials is required to replace and improve on these limitations. Recently, collagen extracted from horses has been of interest as an attractive, safer alternative because they are virtually disease-free (Silverstein et al., 2014) and no immune response has been noted. Therefore, we carried out a research project on horse skin in order to develop a method to extract and collect collagen from horse skin with high efficiency.
In this study, we extracted collagen from horse skin by an improved enzymatic hydrolysis method with the 1 expectation that the hydrolyzed collagen extract had high purity (>90%), while maintaining the intact properties of collagen. From there, find an alternative source of raw materials for industrial applications to serve the needs of the current market. What is collagen? Figure 1. What is collagen? (Pham Anh, 2021) Collagen is a long fibrous protein that functions quite differently from globular proteins as an enzyme; it is the most abundant structural protein in animals (Shoulders and Raines, 2009).
Tough bundles of collagen, known as collagen fibers, are major components of the extracellular matrix that form a scaffold that provides the strength to support most tissues and internal structures in the body. It is an essential component of connective tissue and plays an important role in linking the body's cells together. Together with collagen and elastin, soft collagen is responsible for the strength and elasticity of the skin, and its degradation leads to the appearance of wrinkles that accompany aging. In addition, collagen contributes to the strengthening of blood vessels and plays an extremely important role in tissue growth.
Collagen is distributed mainly in the extracellular matrix and is found in certain cells. It has very high tensile strength and is a major component of fascia, cartilage, ligaments, tendons, bones, and skin. How many types of collagen are there? At least 29 types of collagen have been identified and reported. Collagens are of different types: such as fibril – forming, network forming, fibril – associated collagens with interrupted triple helices (FACIT), membrane – associated collagens with interrupted triple helices (MACIT), and multiple triple – helix domains and interruptions (MULTIPLEXINs).
Collagen is classified based on its variety of structure, function, complexity, and combinations of α chains. The distribution and classifications of collagen types are summarized in the table below (Pope, Nicholls, Dorling, and Webb, 1983). Collagen classification table (Pope et al., 1983) Class Type Distribution I Bone, skin, tendon, ligaments, cornea II Cartilage, vitreous humor in the eyes III Skin, blood vessels V Bone, dermis, co-distribution with type I Fibril-forming (Fibrillar) XI Cartilage, inverterbral discs, co- distribution with type II XXIV Bone, cornea XXVII Cartilage VII Bladder, dermis, IX Cartilage, cornea, XII Tendon, dermis Fibril-associated XIV Bone, dermis, cartilage collagens with XVI Kidney, dermis interrupted tripped XIX Basement membrane helices (FACIT) XX Cornea of chick XXI Kidney, stomach XXII Tissue junctions XXVI Ovary, testis IV Basement membrane VI Muscle, dermis, cornea, cartilage Network-forming VIII Brain, kidney, skin, heart X Cartilage XXVIII Dermis, sciatic nerve 4 Membrane associated XIII Dermis, eyes, endothelial cells collagens with XVII Hemi desmosomes in epithelia interrupted triple helices XXIII Heart, retina (MACIT) XXV Heart, testis, brain Multiple triple-helix XV Capillaries, testis, kidney, heart domains and XVIII Liver, basement membrane interruptions (MULTIPLEXINs) Collagen types I, II, III, X, and XI are fibrous structural collagens found in large quantities in vertebrates, and among fibrous collagens, type I collagen is predominant. Collagen type I: Type I collagen makes up 95–99% of tendons and 90% of collagen in ligaments.
The type I collagen molecule is 300 nm long, 1.5 nm in diameter, and has three subunits: two α1 chains and one α2 chain, each of which has 1050 amino acids and is wrapped around each other to form a triple helix structure toward the right. It is this twisted structure that gives strength and elasticity to the collagen fiber bundles as well as tendons and ligaments. It helps tendons and ligaments function as a spring cushion connecting muscle - bone and bone - bone, ensuring the operation of the body and minimizing injuries.