Research on the process of separating collagen from horse skin (khóa luận tốt nghiệp)

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 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 Dr 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 Ms 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 year 2023 Student Vu Thi Yen iii CONTENTS DECLARATION OF AUTHORSHIP i ACKNOWLEDGEMENTS ii CONTENTS iv ABBREVIATIONS vi LIST OF TABLES viii LIST OF FIGURES ix PREAMBLE PART I: INTRODUCTION 1.1 What is collagen? 1.2 How many types of collagen are there? 1.3 Structures of collagen 1.4 Application and origin of collagen types 1.4.1 Applications in food technology 1.4.2 Applications in medicine and pharmaceuticals 1.4.3 Applications in the cosmetic industry 11 1.5 Research situation on collagen extraction in the country and in the world 13 PART II MATERIALS AND METHODS OF RESEARCH 18 2.1 Materials 18 2.2 Chemicals, machinery and equipment 18 2.3 Methods of research 19 2.3.1 Sample treatment 21 2.3.2 Collagen extraction 22 2.3.3 Preservation of hydrolyzed collagen 26 2.3.4 Quantitative technique of collagen concentration dissolved in extract solution 27 2.3.5 Data processing 29 iv PART III RESULTS AND DISCUSSION 30 3.1 Effect of enzymes on the concentration of soluble collagen in pepsin 32 3.2 Effect of ultrasonic on the concentration of soluble collagen in pepsin 36 3.3 Effect of temperature on the concentration of soluble collagen in pepsin 38 3.4 Determination of the efficiency of collagen extraction 39 3.5 Surface morphology of PSC from horse skin 42 CONCLUSIONS 43 REFERENCE 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 α2 Alpha β Beta o Celsius µl Microliter C vii LIST OF TABLES Table 1.1 Collagen classification table (Pope et al., 1983) Table 2.1 Chemicals 18 Table 2.2 Machinery and equipment 19 Table 3.1 Results of collagen mass obtained when extracted at room temperature (25-26oC) using 2% pepsin ratio (w/w) with ultrasonic 39 Table 3.2 Results of collagen mass obtained when extracted at room temperature (25-26oC) using 1% pepsin ratio (w/w) 41 viii telopeptide region at both the N-terminus and the C-terminus, which is a crosslinked structure This cross-linking structure limits its solubility in acids without the presence of enzymes Therefore, enzymatic hydrolysis has been developed to address some of the shortcomings of traditional methods and can be used in combination with traditional chemical methods Enzymatic hydrolysis offers better reaction selectivity and less damage to collagen Therefore, it has the potential to maximize collagen production and the purity of the extracted product These enzymes can be of animal origin (trypsin, pepsin, etc.), plant origin (bromelain, papain, ficin, etc.), or derived from single or mixed enzymes produced by microorganisms (proteinase K, collagenase, etc.) Pepsin, trypsin, and papain are enzymes commonly used in collagen extraction because they act only on the untwisted part of the collagen peptide chain (the heads) and leave the structurally important helix intact However, there are some other proteins in collagen that are digested by papain, so pepsin was selected for collagen extraction in this study (Yu et al., 2018) In addition, the extracted pepsin-soluble collagen is generally of higher purity because the non-colloidal proteins are efficiently hydrolyzed by enzymes Pepsin treatment also increases the acid solubility of collagen, increasing the extraction yield if used in combination with acid extracts (Matinong et al., 2022) Therefore, pepsin was selected for collagen extraction in this study 33 kDa M 245 18 β 13 α1 α2 100 63 48 35 Figure 3.2 SDS-PAGE of PSC extracted from horse skin using different ratios of pepsin M: Marker; 1: Pepsin 1%; 2: Pepsin 2%; 3: Pepsin 4% (w/w) During the preparation of collagen for SDS-PAGE, boiling denatured the collagen, and the triple helix structure of the collagen was lost Therefore, the individual polypeptide sequences assume a random coil configuration Covalent bonds occur between polypeptide chains in collagen, and they can link two or all three chains together SDS-PAGE results of PSC samples (Figure 3.2) extracted from horse skin using different ratios of pepsin enzyme (1%, 2%, and 4% (w/w)), there is three bands in each line with size 250, 140 and 120 kDa, for beta chains (dimer), α1 and α2 chains of collagen, respectively Furthermore, the results of UV-visible spectroscopic analysis investigating the effect of enzymes on PSC concentration in Figure 3.3 showed the presence of helix polypeptide chains in the triple helix collagen structure and it also indicated that collagen extracted from horse skin is type I collagen having two α1 chains and one α2 chain This type of collagen is found in the skin, tendons, bones, ligaments, and interstitial tissue of animals It has been isolated from the skin of fish such as PSC from Aluterus monocerous, PSC from Scomberomorous niphonius, Istiophorus platypterus, 34 from the body wall of sea cucumbers Holothuria cinerascens, Bohadschia bivitatta, or from human placenta, bovine, pig, etc Effect of enzyme on the efficiency of collagen extraction Absorbance 1.5 0.5 -0.5 190 210 -1 230 250 270 290 310 330 350 Wavelengh (nm) Pepsin 1% Pepsin 2% Figure 3.3 Spectrum investigating the effect of enzyme on collagen extraction efficiency Besides, the protein bands β, α1, α2 in track are darker than those in tracks and at the same position, and the absorbance of PSC at 2% pepsin ratio is 0.67 times higher than that of PSC at 1% pepsin ratio This proves that the concentration of PSC pepsin in the extract solution with a 2% pepsin ratio is 0.67 times higher than the PSC concentration in the extract solution using a 1% pepsin ratio Additionally, based on the SDS-PAGE results, we can see that the 2% and 4% pepsin ratios give the bands with the same density, demonstrating that the extraction efficiency at two ratios of 2% and 4% is the same Furthermore, the aim of this study is to optimize the process as well as to optimize the production cost, so using pepsin with a concentration ratio of 2% (w/w) obtained the efficient collagen quality at the better cost This is completely true with the research results of Weiwei Feng et al (Feng et al., 2013) in the study on optimizing collagen extraction with the support of enzymes from the skin of Chinese sturgeon (Acipenser sturio Linnaeus) 35 3.2 Effect of ultrasonic on the concentration of soluble collagen in pepsin Ultrasonic is a process that uses the energy of sound waves generated at frequencies higher than human hearing (higher than 16 kHz) Ultrasonic waves primarily affect fluid systems due to a phenomenon called cavitation Ultrasonic causes cavitation bubbles to grow quickly, violently collapse, and generate extremely high heat and pressure The cavitation zone becomes turbulent and severe as a result Effect of ultrasonic waves on the efficiency of collagen extraction Absorbance 1.5 0.5 190 210 230 250 270 290 310 330 350 Wavelengh (nm) Ultrasonic No ultrasonic Figure 3.4 Spectrum investigating the effect of ultrasonic on collagen extraction efficiency In this study, ultrasonic was used in conjunction with pepsin hydrolysis to extract collagen from horse skin According to the results of SDS-PAGE (Figure 3.5), protein bands at about 250, 140, and 120 kDa positions in line are darker than those in line 2, indicating that using ultrasonic has a better effect on collagen extraction from horse skin However, line also consists of more protein bands with smaller sizes than line number two, which can be explained by the fact that ultrasonic was used for a long time, which can increase the temperature and shear strength, as well as the high pressure in the environment due to cavitation It can also break hydrogen bonds and van der Waals forces in polypeptide chains and can lead to protein denaturation In addition, the results of UV-visible spectrum analysis investigating the influence of ultrasound on PSC concentration in Figure 3.4 showed that the absorbance during ultrasonic collagen extraction was higher 0.4 times that of collagen extract without using ultrasound This proves that PSC 36 concentration in ultrasonic extraction solution is 0.4 times higher than in nonultrasonic extraction solution kDa M β 245 180 135 α1 α2 100 75 63 48 35 25 Figure 3.5 SDS-PAGE of PSC extracted from horse skin by ultrasonic M: Marker; 1: Ultrasonic; 2: No ultrasonic According to a study by Kim et al (Kim et al., 2012) ultrasonic treatment at 20 kHz in 0.5 M acetic acid boosted extraction efficiency and lowered extraction time when used for the extraction of acid-soluble collagen from the skin of Japanese sea bass (Lateolabrax japonicus) The 1, 2, and chains of collagen, specifically, were unaffected by ultrasonic extraction According to study of Ran and Wang's (Ran & Wang, 2014) (20 kHz pulse, 20 seconds), the use of ultrasonic in conjunction with pepsin enhances the extraction of collagen from beef tendons Boost natural collagen extraction productivity without sacrificing the integrity of the resulting collagen The pepsin enzyme was employed by Li and colleagues (Li, Mu, Cai, & Lin, 2009) to extract collagen from beef tendons using ultrasonic waves (40 kHz, 120 W) Ultrasonic thereby speeds up the extraction process and can improve extraction by up to 124% These findings were explained as a rise in substrate activity and solubility due to ultrasound-induced dispersion of pepsin and collagen fiber opening, which promoted enzyme activity 37 3.3 Effect of temperature on the concentration of soluble collagen in pepsin kDa M 200 150 120 100 85 70 60 50 40 Figure 3.6 SDS-PAGE of PSC extracted from horse skin with different temperature M: Marker; 1: Sample pretreatment and dialysis were performed in a refrigerator o at C, and hydrolysis with pepsin was performed at room temperature (25– o o 26 C).; 2: hydrolysis with pepsin at 35 C The collagen extraction process was carried out under two conditions: in a constant-temperature bath of 35oC and at room temperature (25-26oC) The results of SDS-PAGE examining the effect of temperature on collagen extraction efficiency in figure 3.6 show that the extraction process at room temperature gives a clearer band; while the SDS-PAGE results of the extraction process at 35oC did not show the band line Higher temperature extraction produces low molecular weight peptide fragments, which reduces the yield of type I collagen obtained (Aukkanit & Garnjanagoonchorn, 2010) This proves that the extraction temperature greatly affects the yield and properties of the obtained collagen PSC extraction at 25°C produced the higher yield of type I collagen with consistent molecular properties The results obtained in this study are different from the study on collagen extraction from silver-line grunt skin (4–10oC) of (Aukkanit & 38 Garnjanagoonchorn, 2010) and from Olatanji's study on collagen extraction from croaker scales (100oC) (Olatunji & Denloye, 2017) Differences in studies show that collagen extraction temperatures will be right for different materials 3.4 Determination of the efficiency of collagen extraction Quantification of collagen by UV-Vis 1.8 Absorbance 1.6 1.4 1.2 0.8 0.6 0.4 0.2 190 210 230 250 270 290 310 330 350 Wavelengh (nm) Figure 3.7 Quantitative spectrum of collagen by UV-vis method The obtained collagen was quantified by the UV-Visible spectroscopy and the extraction and purification efficiency was determined Typically, amino acids have an absorption peak at 280 nm However, in our study we found that the maximum absorption peak of collagen isolated from horse skin is generally at 236 nm based on the results of spectroscopic analysis obtained through iterative collagen extraction experiments repeat This is due to the absence of aromatic amino acids, such as tyrosine and phenylalanine, which are UV-sensitive color carriers at 283 nm and 251 nm Therefore, collagen soluble by extracted pepsin is not sensitive to UV at 283 nm and 251 nm Besides, due to the presence of -COOR or -COOH structures, collagen extracted from horse skin has the lowest absorbance at 222 nm The results of this study are similar to those of He et al (He, Lan, Wang, Ahmed, & Liu, 2019), on extracting collagen from grass carp 39 skin, grass carp scales, and carp skin with maximum absorption at 235 nm, 235 nm, and 234 nm Table 3.1 Results of collagen mass obtained when extracted at room temperature (25-26oC) using 2% pepsin ratio (w/w) with ultrasonic Experimental Initial weight of hourse skin (g) Mass of pepsin-soluble collagen obtained after acid hydrolysis (g) 1st 20.00 2.79 2nd 20.22 3.34 3rd 19.39 2.74 Average value 19.87 2.96 2.96 𝑥100 = 14.88% 19.87 The efficiency of PSC extraction from horse skin at room temperature (25𝐶𝑜𝑙𝑙𝑎𝑔𝑒𝑛 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 26oC) with the influence of ultrasonic waves and using the enzyme pepsin 2000 U/g at a rate of 2% (w/w) was 14.88% (table 3.1) This yield was lower than that of the studies that isolated PSCs from the skin of Magalaspis cordyla tuna (22.5%), yellow croaker Otolithes ruber (25.7%), Japanese sea bass (51.4%), chub mackerel (49.8%), bullhead shark (50.1%); ocellate puffer (44.7%), catfish (38.4%), deep sea redfish (92.2%), and the Asian bullfrog Rana tigerina (22.59%– 28.30%) (Sampath Kumar & Nazeer, 2013) Compared with these reports, the collagen yield from the present study was lower due to differences in the source materials 40 Table 3.2 Results of collagen mass obtained when extracted at room temperature (25-26oC) using 1% pepsin ratio (w/w) Experimental Initial weigh of horse skin (g) Mass of pepsin-soluble collagen obtained after acid hydrolysis (g) 1st 20.13 1.59 2nd 24.28 0.089 3rd 20.00 1.18 Average value 21.47 2.86 𝐶𝑜𝑙𝑙𝑎𝑔𝑒𝑛 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 2.86 𝑥100 21.47 = 13.32% In addition, the efficiency of PSC extraction from horse skin at room temperature without ultrasonic waves and using 1% (w/w) pepsin enzyme was 13.32% (table 3.2) The extraction efficiency at this condition was 1.56 times lower than that for PSC extraction from horse skin in the ultrasonic-assisted condition with a pepsin ratio of 2% (w/w) at room temperature A comparison of the extraction efficiency of collagen-extracting PSCs under this condition confirmed that the extraction efficiency was better than that of the horse skin extract at room temperature with ultrasonic support and using 2% (w/w) of the enzyme pepsin However, our study on the efficiency of PSC extraction by 2% pepsin in room temperature, with the aid of ultrasonic waves showed a higher efficiency than that of the bullfrog skin collagen extraction study by Li et al (2004) (12.6%) and PSC from marine eels (4.7%) This result proves that our method improvement process is quite effective 41 3.5 Surface morphology of PSC from horse skin At least 29 different types of collagen have been reported, classified according to their structure into: striated (fibrous), non-fibrous (network forming), microfilamentous, and those types related to fibril A B 2 1 Figure 3.8 SEM image of lyophilization PSC from horse skin (left, magnification × 5.00k; right magnification × 2.00k α1 or α2 twisted fibers β twisted fibers SEM images of pepsin-soluble collagen scafford were performed to evaluate scafford collagen features such as structural features and alignment of collagen surface features When observing PSC extracted from horse skin under the Hitachi s-4800 high-resolution scanning electron microscope (FE-SEM, Hitachi, Japan), PSCs were observed mainly as short, thin monofilaments These two fibers are approximately twice the length of each other (Figure 3.5.1.B) Short fibers (1) can be alpha or alpha twisted fibers (about 120-140 kDa), and long fibers (2) can be beta twisted fibers (size about 250 kDa) These structures also matched and agreed with previous research This confirms that we have successfully isolated collagen from horse skin 42 CONCLUSIONS After months of conducting optimal research and investigating the influence of factors (temperature, enzyme concentration, ultrasonic wave), we obtained the above results and came to the conclusion In this study, pepsinsoluble collagen was successfully extracted from horse skin with an efficiency of 14.88% at room temperature (25-26oC) under the influence of ultrasonic waves and using pepsin enzyme at a rate of 2% (w/w) The efficiency of this study is higher than some previous studies by Li et al (2004), which proves that the study on extracting horse skin has better yield and has given some extraction conditions should be used to reduce costs The study results expressed by SDS-PAGE clearly showed α1 (120 kDa), α2 (140 kDa), and β (250 kDa) bands The extracted collagen has two forms: single strands are polypeptide chains, corresponding to α and β filaments was confirmed by SEM technique The results of our research show that horse skin can be an alternative raw material, a potential source of collagen for applications in food, nutrition, cosmetics, and medicine, etc., and improve the disadvantages of collagen derived from bovine and porcine 43 REFERENCE Reference Vietnamese Anh, P (Producer) (2021, 5) COLLAGEN - Lớp đệm vững cho da Retrieved from 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