Pepsin 2000 U/g of NovacoVietnam is used in the collagen extraction process .... The type I collagen molecule is 300 nm long, 1.5 nm in diameter, and has three subunits: two α1 chains an
INTRODUCTION
What is collagen?
Collagen is the most abundant structural protein in animals, functioning differently from globular proteins like enzymes (Shoulders and Raines, 2009) It forms tough bundles known as collagen fibers, which are crucial components of the extracellular matrix, providing strength and support to tissues and internal structures As an essential part of connective tissue, collagen links the body's cells and, along with elastin, contributes to the skin's strength and elasticity; its degradation results in wrinkles associated with aging Additionally, collagen strengthens blood vessels and plays a vital role in tissue growth, being predominantly found in the extracellular matrix and certain cells With its high tensile strength, collagen is a major component of fascia, cartilage, ligaments, tendons, bones, and skin.
Figure 1.1 What is collagen? (Pham Anh, 2021)
How many types of collagen are there?
There are at least 29 identified types of collagen, which can be categorized into several groups, including fibril-forming, network-forming, fibril-associated collagens with interrupted triple helices (FACIT), membrane-associated collagens with interrupted triple helices (MACIT), and MULTIPLEXINs, which feature multiple triple-helix domains and interruptions Collagen classification is based on structural variety, functional roles, complexity, and combinations of α chains A summary of the distribution and classifications of collagen types is provided in the accompanying table (Pope, Nicholls, Dorling, and Webb, 1983).
Table 1.1 Collagen classification table (Pope et al., 1983)
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
XI Cartilage, inverterbral discs, co- distribution with type II XXIV Bone, cornea
Fibril-associated collagens with interrupted tripped helices (FACIT)
IX Cartilage, cornea, XII Tendon, dermis XIV Bone, dermis, cartilage XVI Kidney, dermis
XX Cornea of chick XXI Kidney, stomach XXII Tissue junctions XXVI Ovary, testis
VI Muscle, dermis, cornea, cartilage VIII Brain, kidney, skin, heart
X Cartilage XXVIII Dermis, sciatic nerve
Membrane associated collagens with interrupted triple helices
XIII Dermis, eyes, endothelial cells XVII Hemi desmosomes in epithelia XXIII Heart, retina
XXV Heart, testis, brain Multiple triple-helix domains and interruptions
XV Capillaries, testis, kidney, heart XVIII Liver, basement membrane
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
Type I collagen constitutes 95–99% of tendons and 90% of ligaments, playing a crucial role in their structure and function Each type I collagen molecule measures 300 nm in length and 1.5 nm in diameter, consisting of three subunits: two α1 chains and one α2 chain, each containing 1050 amino acids These subunits are intertwined to form a right-handed triple helix, which imparts strength and elasticity to collagen fiber bundles This unique structure enables tendons and ligaments to act as spring cushions, connecting muscles to bones and bones to each other, thereby facilitating body movement and reducing the risk of injuries.
Collagen type II consists of three identical α1 polypeptide chains, each containing 1060 amino acid residues It features a large uninterrupted triple-helical region and shorter nonhelical telopeptides, with 19 amino acids in the N-telopeptide and 27 in the C-telopeptide Unlike the triple-helical regions, these telopeptides lack the Gly-Xaa-Yaa repeating structure Although the lengths of the α-chains are identical, they are offset by one residue in the triple helix, facilitating proper supercoiling.
Collagen type III is a homotrimer made up of three α1 chains, similar to other fibrillar collagens A distinctive feature of type III collagen is the presence of a disulfide or cystine knot, located between the triple helical region and the C-terminal telopeptide This knot, formed by three interchain disulfide bonds, plays a crucial role in stabilizing the triple helical structure (Kuivaniemi and Tromp, 2019).
Collagen type V: Type V collagen is a regulatory fibril – forming collagen
It has at least three different molecular isoforms: α1 (V) 2 α2 (V), α1 (V) 3 and α1
(V) α2 (V) α3 (V) – formed by combinations of three different polypeptide α chains: - α1 (V), α2 (V), α3 (V) Collagen type V is a relatively minor collagen of the extracellular matrix (ECM) (Mak, Png, and Lee, 2016)
Collagen type XI features a significant triple-helical structure comparable in size to collagen types I and II However, unlike these collagens, its N-terminal propeptides remain attached to the molecule, integrating it into the fiber (Smith and Birk, 2012).
Structures of collagen
Collagen proteins are unique due to their triple helix structure, composed of three polypeptide chains and a molecular mass of approximately 360 kDa, which significantly contributes to the strength of the collagen molecule.
Figure 1.2 Structure of collagen (Lin et al., 2018)
Collagen exists in two primary forms: heterotrimer and homotrimer A heterotrimer is a trimer made up of two or more different, yet similar, biologically active monomers In contrast, a homotrimer consists of three identical protein units.
Collagen is uniquely structured with three α chains, each composed of thousands of amino acids, including approximately 35% glycine (Gly), 11% alanine (Ala), 21% proline (Pro), and hydroxyproline The amino acid sequence forms polypeptide chains through a repeating Gly-X-Y pattern, where glycine appears at every third position, facilitating tight molecular packaging The X and Y positions are typically filled by proline and hydroxyproline, contributing to collagen's structural integrity.
Application and origin of collagen types
Collagen fibers have different specific biochemical properties, they have been widely used in important industries, especially food, medicine, pharmaceuticals, and cosmetics
Collagen has become a sought-after ingredient in the food industry, playing a significant role in the creation of healthy foods The body's collagen production is influenced by diet and decreases with age Instead of relying on injections, dietary sources are the best way to maintain adequate collagen levels Consequently, collagen is now found in various food and beverage products The market offers numerous commercial collagen products from diverse sources, including food-grade bovine collagen brands like Colageno, produced by JBS in Brazil, and Cosen.
Jiangxi Cosen Biochemical in China produces various collagen products, including Ovinex, a type I and III food-grade ovine collagen made through an enzymatic process by Hollista CollTech, Australia Additionally, marine collagens such as Peptan by Rousselot SAS, France, and Ni-Kollagen by Bionic Life Science, Malaysia, are recommended for use in dietary supplements, functional foods, beverages, confectionery, and desserts Collagen serves as a food additive, enhancing the color, texture, taste, and overall quality of food products In sausage production, collagen improves rheological properties and nutrient retention from meat, while in the dairy industry, collagen hydrolysate acts as a structural additive Furthermore, collagen is utilized in the clarification of wine, beer, and juice due to its ability to coagulate cloudy substances in solutions.
Figure 1.3 Collagen sausage casings (Amazon.com )
Collagen, known for its film-forming properties, is utilized in sausage casings and confectionery wrappers It serves as an effective barrier that protects food, prevents oxygen movement, maintains product structure, and extends shelf life Additionally, collagen is a key ingredient in the production of certain functional foods and various types of marshmallows.
Collagen supplements have become a popular trend in the global market, with various forms available, including soy collagen, cocoa collagen, and collagen-infused juices These supplements are designed to stimulate the body's natural collagen production, helping to improve skin elasticity and reduce the appearance of wrinkles and sagging skin.
1.4.2 Applications in medicine and pharmaceuticals
Collagen, a natural polymer, is extensively utilized in plastic surgery for various applications It serves as an effective filler for enhancing lip volume and improving skin contours, while also reducing wrinkles and filling in imperfections Additionally, collagen plays a significant role in wound healing, making it a versatile component in cosmetic procedures.
Collagen drinks, such as those available on AliExpress, may help reduce the risk of scarring in burn patients The majority of medical collagen is sourced from animal tissues, including cows, horses, and pigs, ensuring that it is free from Bovine Spongiform Encephalopathy (BSE) Collagen derived from donor animals raised in closed herds or from regions where BSE has never been reported is considered safe for use.
Collagen plays a crucial role in creating artificial replacement skin for patients suffering from severe burns The sources of collagen utilized in reconstructive surgery can be animal-based, such as from cows, pigs, and horses, or human-derived from placentas and organ donors Often, collagen is combined with other materials like silicones, glycosaminoglycans, fibroblasts, and growth factors to enhance its effectiveness.
Collagen hydrolysate is used as a lubricant during endoscopy, making it easy to insert the endoscopes into the body without causing pain to the patient
In drug production, collagen hydrolyzes to form gelatin, which is the main component of softgel capsules and hard capsules It serves as the polymer that
Collagen peptide products, such as those available on Amazon.com, contribute to the formation of the shell membrane When gelatin is dissolved in water, it produces viscosity, making it an effective stabilizer in emulsion dosage forms.
Collagen foam is widely utilized in dentistry and surgical procedures to control bleeding and enhance wound healing Additionally, collagen fibers can be engineered into elongated structures that serve as tendons and ligaments in medical applications Furthermore, collagen tubes are employed to reconstruct esophageal structures, peripheral nerves, ureters, and more.
Collagen constitutes 3.5 – 5.5% of plasma expanders, serving as a vital alternative for severe blood loss when blood reserves are unavailable These expanders effectively replace lost blood, restoring volume and pressure Additionally, in cell culture, collagen acts as a supportive substrate, promoting cell growth and stimulating wound healing.
1.4.3 Application in the cosmetic industry
Collagen is a key ingredient in cosmetic formulations due to its exceptional moisturizing, regenerating, and film-forming properties Its excellent water-binding ability helps to maintain optimal hydration levels in the skin.
Collagen, a natural humectant, is renowned for its film-forming properties that minimize transepidermal water loss, making it essential in both skin and hair care As a primary ingredient in certain hydrogels, collagen serves as a "beauty mask" that enhances the skin's mechanical properties, including tension and elasticity It plays a crucial role in maintaining skin moisture, ensuring pigmentation, and promoting a smooth, fresh, and youthful appearance.
Collagen is essential for skin repair and regeneration, especially when the skin is damaged or healing from conditions like melasma, acne, scars, or excess skin following weight loss.
The cosmetic industry prioritizes innovative and effective products, highlighting the significance of collagen sources These sources can vary, often including by-products from mammals such as cows.
Collagen used in beauty masks can be derived from various sources, including bovine and porcine, as well as marine sources like fish and jellyfish However, collagen from bovine and porcine origins poses risks of zoonotic diseases such as BSE, TSE, and FMD In contrast, marine collagen presents a significant opportunity, offering a safer alternative and highlighting its vast potential in the beauty industry.
Research situation on collagen extraction in the country and in the world 13
The demand for type I collagen in health care, food, and cosmetics is rapidly increasing Two primary technologies for collagen production are the extraction from animal sources and recombinant DNA technology However, the extraction method is favored in industry due to lower costs, higher yields, and better control over bioactivity and post-translational modifications Various animal tissues, including those from mammals like cows and pigs, as well as poultry and fish, are commonly used for collagen extraction due to their biocompatibility, safety, and availability (Gallo et al., 2020).
Mammals provide the highest sequence similarity to human collagen, making them a prime source for extraction (Stover and Verrelli, 2011) The abundance of waste materials from meat processing, including skin, tendons, bones, and adipose tissue, presents an opportunity for low-cost extraction of valuable byproducts like collagen This approach not only optimizes resource utilization but also minimizes disposal costs and environmental impact.
Over the past 50 years, the application of heterologous collagen in medicine has advanced significantly, leading to refined extraction methods, effective allergen removal, and enhanced sterilization processes (J A Ramshaw, 2016) Type I collagen, primarily sourced from areas like skin and tendons, is particularly valued due to its high protein yield In tendons, collagen constitutes approximately 60% - 85% of the dry weight, with Type I collagen making up 90% - 95% of the total collagen present.
Buffalo and pigs are the primary sources of collagen extraction among mammals, largely due to their high consumption rates in the United States and globally While pig and cow-derived foods dominate the market, their collagen extraction is constrained by immune responses, infectious disease risks, and religious restrictions Although bovine and porcine collagens share similarities with human collagen, immunological differences in the telopeptide regions can trigger immune responses in 2%–4% of the global population Additionally, the risk of zoonotic diseases, such as foot and mouth disease and bovine spongiform encephalopathy, poses significant health concerns While pig collagen is associated with fewer allergic reactions, its use is also limited by cultural and religious beliefs, particularly among Jewish, Muslim, and Sikh communities.
Buddhist) collagen, which further limits their potential application (Gallo et al.,
Equine-derived collagen is recognized for its minimal risk of shingles transmission and lack of immune response (Silverstein et al., 2014) While horses can contract alphavirus-associated equine encephalitis (AEE), a zoonotic disease spread by mosquitoes, the overall risk of transmission to humans is low, with few cases of encephalitis and a very low mortality rate This makes equine by-products a safer source for collagen extracts in medical applications Notably, horse collagen exhibits the highest sequence similarity to human collagen, second only to bovine collagen, with a similarity rate of up to 95% due to the close taxonomic relationship between horses, cows, and humans Consequently, equine collagen is considered a valid alternative to bovine collagen in terms of composition.
In recent years, type I collagen extracted from equine tissues has garnered significant attention in life sciences and bioengineering as a viable alternative to traditional bovine collagen for manufacturing implantable medical devices The shift from using collagen sourced from cows and pigs is driven by the need for safer options, leading to the rising popularity of equine collagen derived from horse tendons This collagen is increasingly marketed by various companies due to its unique properties, including a favorable structure, low risk of animal disease transmission, and minimal potential to provoke immune responses, making it particularly appealing for applications in medicine, cosmetics, and pharmaceuticals.
In the last 15 years, advancements in regenerative medicine and tissue engineering have established collagen as a premier biocompatible, biodegradable, and bioactive scaffolding material that is easy to produce The remarkable benefits of this ancient natural protein ensure a consistent demand for collagen and collagen-based products, which continues to rise alongside the development of innovative therapies (Sbricoli et al., 2020).
Vietnam's extensive coastline offers significant potential for fishing and aquaculture, particularly in the extraction and recovery of collagen The country's seafood processing industry generates waste materials, such as fish bones and skins, which can be utilized for collagen extraction Numerous studies and methods for extracting collagen from these waste products have been developed and implemented in Vietnam, showcasing the country's advancements in this field.
In 2012, Vinh Hoan Joint Stock Company in Dong Thap launched a project to extract collagen from pangasius skin, achieving a production scale of 7.2 tons of powder per year The Vietnam Academy of Science and Technology has also conducted significant research on collagen extraction, including a study by Dr Nguyen Thuy Chinh in 2022, which successfully isolated hydrolyzed collagen from carp scales for biomedical applications This collagen can be combined with ginsenoside Rb1 and yellow tea polyphenols for hemostatic and wound treatment purposes Utilizing innovative technology and local raw materials, these research efforts have led to the development of cost-effective biomedical products Additionally, studies on collagen recovery from jellyfish have further demonstrated Vietnam's commitment to leveraging its marine resources The ongoing exploration and application of modern processes in Vietnamese science aim to produce high-quality products at lower costs.
MATERIALS AND METHODS OF RESEARCH
Materials
Horse skins are sourced from slaughterhouses located in Hanoi, Vinh Phuc, and Phu Tho provinces The skins, particularly from horses aged 1.5 years and older, are collected immediately post-slaughter and preserved at a temperature of -20°C.
Chemicals, machinery and equipment
The chemicals used are supplied by reputable companies around the world and are presented in Table 2.2.1, while the equipment of the key genetic engineering laboratory is presented in Table 2.2.2
AR Acetic acid (CH3COOH)
Sigma (St Louis MO, U.S) Chemicals for protein electrophoresis, acrylamide, bis – acrylamide, TEMED, APS, SDS
BRNAstead Ultrapure water system Nanopure
Bio – Rad Mini- protein tetra vertical electrophoresis cell Hewlett Packard (California, U.S) NIR spectrometer
Janke & Kunkel (Germany) Magnet Stirrer IKAMAG
Kendro (U.S) Centrifuge system Sorvall Bio fuge pico, Sorvall Bio fuge fresco, Sorvall Legend TM T/RT
OSI Vortex sharker, Heating block
Precisa (Switzerland) Annalytical balance XT 220A,
Precision balance XB 1200C Sanyo (Japan) Refrigerator -20 o C, 4 o C
Freeze dryer Virtis sentry, SN 206588
FE – SEM (Hitachi Japan) Scanning electron microscope (SEM)
Methods of research
Collagen can be produced through chemical and enzymatic hydrolysis, with current methods including hydrolysis in neutral salts, acids, and with pepsin (pepsin-solubilized collagen, PSC) While chemical methods are prevalent in industry, enzymatic processes are more advantageous for enhancing the nutritional value and functional properties of collagen Enzymatic hydrolysis provides improved reaction selectivity and minimizes damage to collagen, making it a promising approach for optimizing collagen production and ensuring the purity of the extracted product.
Enzymatic treatment offers several advantages over chemical methods, including reduced corrosion of processing equipment, lower energy consumption, decreased waste production, and better control over hydrolysis levels Additionally, the collagen produced through this method has a lower salt content and is of higher quality.
Figure 2.1 Schematic diagram of the method of extracting collagen from horse skin
Recent research has focused on utilizing pepsin proteolysis for various raw materials, including skin, tendons, nails, feathers, and slaughterhouse waste This study specifically examines the extraction of hydrolyzed collagen from horse tendon skin using pepsin, which effectively breaks down collagen into peptides like gelatin.
To effectively extract collagen, it is essential to first eliminate various covalent bonds within and between molecules This involves pretreating raw materials to remove non-collagenous substances, which leads to higher yields when using neutral saline or acid solutions In horse skin by-products, non-collagen components such as lipids, minerals, pigments, and non-collagen proteins must be addressed The processing method varies based on the chemical composition of the raw materials, with diluted NaOH solutions or mixtures of NaOH and non-polar solvents like alcohol or n-hexane commonly employed to remove fats.
In this study, we eliminated fat from skin samples by soaking them in NaOH and NaCl solutions for 24 hours Subsequently, we used NaCl and HCl solutions to extract mineral salts from the skin samples for an additional 24 hours After this pretreatment process, the skin samples were prepared for further analysis.
Figure 2.2 Skin samples before and after pretreatment
A Original skin sample, B Pretreated skin samples samples were washed again with distilled water and weighed to prepare for collagen extraction
After alkali and acid treatment, horse skin was neutralized by washing with distilled water and then extracted with 2000 U/g pepsin in varying ratios (1:25, 1:50, 1:100 w:v) for 48 hours at 25°C in a 0.7 M acetic acid medium The extract solution was collected using medical gauze and filtered in two steps with decreasing pore sizes The collagen dissolved in the extraction solution was precipitated with 2.5 M NaCl for 24 hours, followed by centrifugation at 12,000 rpm for 25 minutes at 4°C The supernatant was removed to obtain collagen pellets, which were then weighed and prepared for dialysis.
Ultrasonic waves at 47 kHz are utilized in collagen extraction to boost yield and reduce extraction time These waves generate significant turbulence in the solution, which enhances mass transfer and accelerates chemical reactions The ultrasonic treatment is conducted concurrently with enzyme treatment at 25 °C, using different concentrations of pepsin.
Figure 2.3 Pepsin 2000 U/g of Novaco(Vietnam) is used in the collagen extraction process
To prevent collagen destruction during the ultrasonic process, the machine operates for approximately one hour at concentrations of 1%, 2%, and 4% Regularly adding ice helps maintain a safe temperature in the ultrasound machine, ensuring the sample solution does not overheat.
Raw collagen extraction typically contains neutral salts and non-collagen proteins, necessitating a purification process to isolate collagen fractions with varying molecular weights This purification involves multiple steps, such as filtration or centrifugation, where a high concentration of salt is added to precipitate the target protein from the solution Factors like temperature, pH, ion concentration, and salt concentration influence collagen precipitation, making it essential to optimize "salting out" conditions to reduce the precipitation of other proteins (Matinong et al., 2022) In this study, sodium chloride (NaCl) was employed for collagen precipitation due to its strong precipitating effect and minimal impact on the solution's pH.
To precipitate collagen, a neutral salt (NaCl) is added to an extract at a concentration of 2.5 M, followed by magnetic stirring for 24 hours at 4°C After stirring, collagen pellets are collected through centrifugation at the same temperature For further purification, these pellets are then dissolved in acetic acid at a 1:9 w/v ratio, achieving a concentration of 0.7 M.
After precipitation, pepsin soluble collagen is left with a significant amount of neutral salt To purify acid-soluble collagen obtained from enzymatic extraction, dialysis is employed The extract is placed in Thermo's snakeskin dialysis membrane and dialyzed with a 0.1 M acetic acid solution for 24 hours, followed by dialysis with distilled water for another 24 hours To enhance ion migration during dialysis, the dialysate solution is changed every 2 hours.
A novel technique has been developed to determine the alpha chain of various collagens using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at concentrations of 10% and 7.5% The process involves dialysis and centrifugation to eliminate acetic acid while collecting the collagen pellet, which is then washed with distilled water to neutralize acidity and reduce its impact on analysis results Following this, soluble collagen is mixed with a 5x sample buffer and denatured at 95°C for 10 minutes, facilitating SDS denaturation, breaking disulfide bonds, and enhancing reactions with reducing agents Finally, the samples undergo centrifugation at 13,000 rpm for 8 minutes at room temperature.
The sample extract was transferred into a new Eppendorf tube, followed by the addition of 10–15 µL of distilled water The mixture was then denatured for 5 minutes before being loaded into the electrophoresis wells.
Scanning electron microscopy (SEM) is a widely used technique for imaging the microstructure and morphology of materials It involves directing a low-energy electron beam at the sample's surface, leading to various interactions that produce emitted photons and electrons (Sampath Kumar and Nazeer, 2013) The signals generated from these interactions are captured by different detectors, depending on the SEM mode employed Various SEM modes, including X-ray mapping, secondary electron imaging, backscattered electron imaging, and electron channels, are utilized to effectively characterize materials.
5 Add 30àl sample buffer 5x (sb5x)
6 Denature at 95 o C for 10 minutes 1.Processed sample
2 Pour sample in the electrophoresis wells
1 Draw the chemicals according to the formula into a tube and mix well.
2 Pour the solution into the mold and wait for the gel to solidify.
The morphological characteristics of PSC were analyzed using a Hitachi S-800 high-resolution scanning electron microscope (FE-SEM Japan) Samples were fixed on a standard SEM holder, wrapped in conductive carbon tape, dried, and coated with conductive platinum They were then placed in the sample chamber and examined for surface morphology at an accelerating voltage of 2 kV, utilizing magnifications of 5.00x and 20.0x.
RESULTS AND DISCUSSION
Effect of enzymes on the concentration of soluble collagen in pepsin
In previous studies on collagen extraction using acid-soluble extraction methods, collagen yield was not high Collagen has untwisted sections on the
Figure 3 1 Pepsin-soluble collagen (PSC) is extracted from horse skin A: The horse skin is cut into small pieces; B: PSC before lyophilization;
C: PSC after lyophilization telopeptide region at both the N-terminus and the C-terminus, which is a cross- linked 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
During SDS-PAGE preparation, collagen is boiled, resulting in the loss of its triple helix structure and the formation of random coil configurations in the polypeptide sequences Covalent bonds between polypeptide chains can link two or all three chains together SDS-PAGE analysis of PSC samples extracted from horse skin with varying pepsin enzyme ratios (1%, 2%, and 4% w/w) revealed three distinct bands at sizes of 250, 140, and 120 kDa, corresponding to beta chains (dimer), α1, and α2 chains of collagen, respectively Additionally, UV-visible spectroscopic analysis indicated the presence of helical polypeptide chains in the triple helix structure, confirming that the collagen extracted from horse skin is type I collagen, consisting of two α1 chains and one α2 chain This type of collagen is prevalent in skin, tendons, bones, ligaments, and interstitial tissue of animals and has also been isolated from fish species such as Aluterus monocerous, Scomberomorous niphonius, and Istiophorus platypterus.
Figure 3.2 illustrates the SDS-PAGE analysis of PSC extracted from horse skin using varying pepsin concentrations of 1%, 2%, and 4% (w/w) The samples were derived from the body wall of sea cucumbers, specifically Holothuria cinerascens and Bohadschia bivitatta, as well as from human placenta, bovine, and pig sources.
Besides, the protein bands β, α1, α2 in track 2 are darker than those in tracks
The absorbance of PSC at a 2% pepsin ratio is 0.67 times higher than at a 1% ratio, indicating a greater concentration of PSC in the extract solution SDS-PAGE results show that the extraction efficiency is consistent between 2% and 4% pepsin ratios, as both yield bands of the same density This study aims to optimize the extraction process and production costs, with a 2% pepsin concentration providing efficient collagen quality at a lower cost These findings align with the research of Weiwei Feng et al (2013) on collagen extraction from Chinese sturgeon skin.
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Effect of enzyme on the efficiency of collagen extraction
Figure 3.3 Spectrum investigating the effect of enzyme on collagen extraction efficiency
Effect of ultrasonic on the concentration of soluble collagen in pepsin
Ultrasonic technology utilizes sound waves at frequencies above human hearing (over 16 kHz) to impact fluid systems through cavitation This process leads to the rapid growth and violent collapse of cavitation bubbles, resulting in the generation of intense heat and pressure, which creates a turbulent and severe cavitation zone.
This study utilized ultrasonic treatment alongside pepsin hydrolysis to effectively extract collagen from horse skin The SDS-PAGE results indicated that the protein bands at approximately 250, 140, and 120 kDa in the ultrasonic-treated sample were darker than those in the control, suggesting superior collagen extraction efficiency Additionally, the ultrasonic method resulted in a greater number of smaller protein bands, likely due to prolonged ultrasonic exposure, which increases temperature, shear strength, and pressure from cavitation, leading to the disruption of hydrogen bonds and van der Waals forces in polypeptide chains, ultimately causing protein denaturation Furthermore, UV-visible spectrum analysis revealed that the absorbance during ultrasonic collagen extraction was 0.4 times higher than that of the collagen extract obtained without ultrasound, confirming the enhanced effectiveness of the ultrasonic method.
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Effect of ultrasonic waves on the efficiency of collagen extraction
Figure 3.4 Spectrum investigating the effect of ultrasonic on collagen extraction efficiency concentration in ultrasonic extraction solution is 0.4 times higher than in non- ultrasonic extraction solution
A study by Kim et al (2012) demonstrated that ultrasonic treatment at 20 kHz in 0.5 M acetic acid significantly improved extraction efficiency and reduced extraction time for acid-soluble collagen from Japanese sea bass skin (Lateolabrax japonicus), while the collagen chains remained unaffected Similarly, Ran and Wang (2014) found that using ultrasonic waves with pepsin for 20 seconds enhanced collagen extraction from beef tendons, increasing productivity without compromising collagen integrity Additionally, Li et al (2009) utilized ultrasonic waves at 40 kHz and 120 W to extract collagen from beef tendons, achieving up to a 124% improvement in extraction efficiency This enhancement was attributed to increased substrate activity and solubility, resulting from ultrasound-induced dispersion of pepsin and the opening of collagen fibers, which promoted enzyme activity.
Figure 3.5 SDS-PAGE of PSC extracted from horse skin by ultrasonic
Effect of temperature on the concentration of soluble collagen in pepsin
The collagen extraction process was conducted at two temperatures: a constant 35 °C and room temperature (25-26 °C) SDS-PAGE results indicate that extraction at room temperature yields a clearer band, while the higher temperature of 35 °C fails to show a band line This suggests that elevated temperatures lead to the production of low molecular weight peptide fragments, ultimately reducing the yield of type I collagen (Aukkanit & Garnjanagoonchorn, 2010) Consequently, the extraction temperature significantly influences both the yield and properties of the collagen obtained, with PSC extraction at 25 °C resulting in a higher yield of type I collagen with consistent molecular properties These findings contrast with previous research on collagen extraction from silver-line grunt skin at lower temperatures (4–10 °C) by Aukkanit & Garnjanagoonchorn.
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 at 4 o C, and hydrolysis with pepsin was performed at room temperature (25–
Garnjanagoonchorn, 2010) and from Olatanji's study on collagen extraction from croaker scales (100 o C) (Olatunji & Denloye, 2017) Differences in studies show that collagen extraction temperatures will be right for different materials.
Determination of the efficiency of collagen extraction
The collagen extracted from horse skin was quantified using UV-Visible spectroscopy, revealing a maximum absorption peak at 236 nm, which differs from the typical 280 nm peak associated with amino acids This deviation is attributed to the lack of aromatic amino acids, such as tyrosine and phenylalanine, which absorb at 283 nm and 251 nm Consequently, collagen solubilized by pepsin does not exhibit UV sensitivity at these wavelengths Additionally, the presence of -COOR or -COOH structures in the collagen results in the lowest absorbance at 222 nm These findings align with the research conducted by He et al (2019) on collagen extraction from grass carp.
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Quantification of collagen by UV-Vis
Figure 3.7 Quantitative spectrum of collagen by UV-vis method 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 (25-26 o C) using 2% pepsin ratio (w/w) with ultrasonic
Experimental Initial weight of hourse skin
Mass of pepsin-soluble collagen obtained after acid hydrolysis (g)
The efficiency of PSC extraction from horse skin at room temperature (25-
The yield of collagen extracted using ultrasonic waves and 2% (w/w) pepsin at 26°C was 14.88%, which is significantly lower than yields reported from various sources, including Magalaspis cordyla tuna (22.5%), yellow croaker (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 (22.59%–28.30%) (Sampath Kumar & Nazeer, 2013) This discrepancy in collagen yield can be attributed to differences in the source materials used in the studies.
Table 3.2 Results of collagen mass obtained when extracted at room temperature (25-26 o C) using 1% pepsin ratio (w/w)
Experimental Initial weigh of horse skin (g) Mass of pepsin-soluble collagen obtained after acid hydrolysis (g)
The extraction efficiency of PSC from horse skin at room temperature using 1% (w/w) pepsin enzyme was found to be 13.32%, which is 1.56 times lower than the efficiency achieved with 2% (w/w) pepsin under ultrasonic-assisted conditions This indicates that the extraction efficiency of collagen-extracting PSCs at room temperature with ultrasonic support and 2% pepsin is superior to that of the horse skin extract Furthermore, our findings demonstrate that the efficiency of PSC extraction using 2% pepsin at room temperature with ultrasonic assistance surpasses the results reported by Li et al for bullfrog skin collagen extraction.
(2004) (12.6%) and PSC from marine eels (4.7%) This result proves that our method improvement process is quite effective
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
SEM images of pepsin-soluble collagen scaffolds were analyzed to assess their structural features and collagen surface alignment Observations of PSC extracted from horse skin using the Hitachi S-4800 high-resolution scanning electron microscope revealed that PSCs primarily appear as short, thin monofilaments The short fibers, measuring approximately 120-140 kDa, can be identified as alpha 1 or alpha 2 twisted fibers, while the longer fibers, around 250 kDa, are classified as beta twisted fibers These findings are consistent with previous research, confirming the successful isolation of collagen from horse skin.
Figure 3.8 SEM image of lyophilization PSC from horse skin (left, magnification × 5.00k; right magnification × 2.00k
1 α 1 or α 2 twisted fibers 2 β twisted fibers
After 6 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, pepsin- soluble 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
The extracted collagen from horse skin consists of two forms: single strands as polypeptide chains corresponding to α (140 kDa) and β (250 kDa) filaments, confirmed by SEM technique Our research indicates that horse skin is a viable alternative raw material and a promising source of collagen for various applications in food, nutrition, cosmetics, and medicine, addressing the limitations associated with collagen derived from bovine and porcine sources.
Anh, P (Producer) (2021, 7 5) COLLAGEN - Lớp đệm vững chắc cho làn da
Retrieved from https://laura-sunshine.com/collagen-lop-dem-vung-chac- cho-lan-da
Chinh, N T (Producer) (2022, 5 12) TIN KHOA HỌC - CÔNG NGHỆ TRONG
NƯỚC Tách chiết Collagen từ vảy cá nước ngọt Retrieved from https://vast.gov.vn/tin-chi-tiet/-/chi-tiet/tach-chiet-collagen-tu-vay-ca- nuoc-ngot-43905-2.html
Ha, T M (Producer) (2020, 8 17) NGHIÊN CỨU KHOA HỌC VÀ PHÁT
Nghiên cứu này tập trung vào việc chiết xuất collagen từ sứa biển thông qua công nghệ enzyme Phương pháp này hứa hẹn mang lại hiệu quả cao trong việc thu nhận collagen, một thành phần quan trọng trong ngành công nghiệp mỹ phẩm và thực phẩm chức năng Việc ứng dụng công nghệ enzyme không chỉ giúp tối ưu hóa quy trình chiết xuất mà còn bảo toàn các đặc tính sinh học của collagen, từ đó nâng cao giá trị sản phẩm.
Amazon.com: The Sausage Maker - Fresh Collagen Sausage Casings, 22mm
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Amazon.com: Advanced Hydrolyzed Collagen Peptides - Unflavored Protein
Powdered collagen mixes easily into drinks and food, making it a convenient supplement for those following Paleo and Keto diets Sourced from pasture-raised, grass-fed animals, this product supports joint and bone health Each container provides 41 servings of unflavored collagen peptides, ideal for enhancing overall wellness in health-conscious households.
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