WO2022119529A1 - Method for obtaining collagen from extracellular matrix components - Google Patents

Method for obtaining collagen from extracellular matrix components Download PDF

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Publication number
WO2022119529A1
WO2022119529A1 PCT/TR2021/051174 TR2021051174W WO2022119529A1 WO 2022119529 A1 WO2022119529 A1 WO 2022119529A1 TR 2021051174 W TR2021051174 W TR 2021051174W WO 2022119529 A1 WO2022119529 A1 WO 2022119529A1
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Prior art keywords
collagen
minutes
rpm
centrifuging
precipitate
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PCT/TR2021/051174
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French (fr)
Inventor
Şebnem Tavman
Özge ATA
Seher Kumcuoğlu
Original Assignee
Ege Üni̇versi̇tesi̇
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Priority claimed from TR2021/01707A external-priority patent/TR202101707A2/en
Application filed by Ege Üni̇versi̇tesi̇ filed Critical Ege Üni̇versi̇tesi̇
Publication of WO2022119529A1 publication Critical patent/WO2022119529A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

Definitions

  • the invention relates to the method of obtaining collagen, which can be used in the health, food, cosmetics, and medical equipment sectors, from the extracellular matrix components in the lamb’s foot.
  • ESM extracellular matrix
  • Collagen properties vary depending on the raw material and extraction conditions.
  • the most commonly used extraction methods are based on the solubility of collagen, which is a classical extraction method, in neutral saline solutions, acid solutions, and acid solutions to which enzymes are added. It is seen with the classical extraction method that both the amount of solvent used, and the extraction times are long.
  • the functional properties of collagen vary depending on the process conditions of the raw material from which it is obtained. It is important for this reason to determine the conditions suitable for the raw material to obtain high efficiency and best collagen properties. It is seen in the state-of-the-art acid hydrolysis process or enzymatic hydrolysis methods are generally used in collagen extraction.
  • Present invention relates to the method of obtaining collagen, from the extracellular matrix components in the lamb’s foot, which meets the aforementioned needs, eliminates all the disadvantages, and provides some additional advantages.
  • the primary object of the invention is to develop an innovative and environmentally friendly method and to shorten the time of obtaining collagen by applying ultrasound treatment after 6 hours of treatment instead of 24 hours and more used in traditional extraction methods.
  • the object of the invention is to produce high yield, purity, and high added value collagen by utilizing lamb foot wastes.
  • Ultrasound application is seen as an effective method in increasing product purity and quality. It was observed in studies on collagen extraction that ultrasound application improved the product properties by preserving the collagen structure.
  • the extraction method of the invention includes acid and enzymatic hydrolysis methods as well as ultrasound application.
  • the method of the invention is not limited to the use of lamb’s foot as a source of collagen.
  • the method of the invention can also be used effectively for collagen raw materials of animal origin such as feet, tendons, skins or trachea of bovine, ovine or poultry; chicken sternum or cartilage; fish skin, scales, fins or bones.
  • Figure 1 Flow chart of the pre-treatments applied to the lamb’s foot
  • Figure 2 Flow chart of collagen production from lamb’s feet
  • Figure 3 The SDS-PAGE bands of the collagen samples obtained under different extraction conditions (M: molecular weight marker. 1- 20°C-20 min.; 2- 20°C-40 min.; 3-20°C-60 min.;
  • Figure 7 SEM images of collagen samples obtained under different extraction conditions.
  • the method of obtaining collagen of the invention includes the following process steps: a) Cleaning and grinding of collagen raw materials of animal origin, b) Homogenizing the cleaned and downsized collagen raw materials in 0,05 M Tris-HCl at a ratio of 1 :20 (w/v) and having a pH value of 7.5 for 50 minutes c) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm, d) Treating the precipitate taken after centrifugation with 50% ethanol at a ratio of 1 : 10 (w/v) for 3 hours for fat removal, e) Taking the precipitate by centrifuging the mixture at 4°C for 20-30 minutes at 9000-15000 rpm, f) Mixing the precipitate taken after centrifugation with 0.5 M EDTA-2Na at a ratio of 1 : 10 (w/v) for 6 hours at 4°C for decalcification, g) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm, h
  • the collagen raw material of animal origin may be feet, tendons, skins, or trachea of bovine, ovine or poultry; chicken sternum or cartilage; fish skin, scales, fins or bones. Lamb’s foot was preferred as a collagen raw material of animal origin in the invention.
  • composition of the lamb’s foot was considered when determining the pre-treatment steps.
  • the precipitate was kept in 0.1 M NaOH for overnight at a ratio of 1 : 10 (w/v) in order to remove non-collagen proteins. The precipitate was then taken by centrifuging at 4°C for 25 minutes at 9000 rpm.
  • Dialysis was performed for removal of excess salt. The samples were first dialyzed against 0.1 M lactic acid for 24 hours and then against distilled water for 48 hours.
  • Collagen concentrations were found between 715,13 ⁇ 70,94 and 5061,75 ⁇ 171,41 pg/pl with the applied method. Collagen concentrations are given in Table 1.
  • Table 1 Collagen concentration of the samples obtained under different extraction conditions a-b-c-d-e jjffgj-g ce between the groups marked with different letters in the same column is statistically significant (p ⁇ 0,05).
  • the extraction condition with the highest collagen concentration is determined as 25°C-60 min.
  • ultrasound application 5061,75 pg/pl.
  • An increase in collagen concentrations was also observed depending on the increased ultrasound time at the same temperature. However, the duration of 60 minutes should not be exceeded to prevent the structure from denaturation. It was observed that collagen samples obtained at different ultrasound times at 25°C had the highest collagen concentration. It has been observed that the collagen concentration has started to decrease at temperatures and times above 25°C. It is seen in general that the ultrasound time affects the collagen concentration more than the temperature.
  • the results of the collagen analysis have shown that lamb’s feet can be used as an alternative source of collagen.
  • the surface morphology and particle sizes of the collagen samples were determined by scanning electron microscopy. SEM images were taken with 5000x magnification. The SEM images of the collagen samples obtained under different extraction conditions are given in Figure 7.
  • the gap between the nested layers gives the collagen porosity. Spiral-like fibers were observed as an interconnected layer, as reported in the art. Porosity, the size of the pores, and the material surface area are considered to be important biomedical parameters of biomaterials.
  • the homogeneous and regular mesh structure in the collagen obtained from the lamb’s foot makes collagen suitable as a drug carrier.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the method of obtaining collagen, which can be used in the health, food, cosmetics, and medical sectors, from the extracellular matrix components in the lamb's foot.

Description

METHOD FOR OBTAINING COLLAGEN FROM EXTRACELLULAR MATRIX COMPONENTS
Technical Field of the Invention
The invention relates to the method of obtaining collagen, which can be used in the health, food, cosmetics, and medical equipment sectors, from the extracellular matrix components in the lamb’s foot.
State of the Art of the Invention (Prior Art)
The extracellular matrix (ESM) is a complex and dynamic structure that fills the intercellular spaces and connects the cells, contains most proteins, hormones, proteoglycans, and growth factors, and allows cells to interact with intracellular signaling pathways to perform special functions. ESM, often called connective tissue, is found in large amounts in the cartilage and bone. Collagen is one of the basic extracellular matrix components that make up a significant portion of ESM.
Collagen properties vary depending on the raw material and extraction conditions. The most commonly used extraction methods are based on the solubility of collagen, which is a classical extraction method, in neutral saline solutions, acid solutions, and acid solutions to which enzymes are added. It is seen with the classical extraction method that both the amount of solvent used, and the extraction times are long. The functional properties of collagen vary depending on the process conditions of the raw material from which it is obtained. It is important for this reason to determine the conditions suitable for the raw material to obtain high efficiency and best collagen properties. It is seen in the state-of-the-art acid hydrolysis process or enzymatic hydrolysis methods are generally used in collagen extraction.
Recent studies have shown that the use of ultrasound with these methods is effective in increasing the extraction efficiency. Ultrasound technology plays an important role in achieving the goal of sustainable ‘green’ chemistry. Additionally, it has many advantages such as being safe, cheap, repeatable, and environmentally friendly compared to other methods. Studies with the use of ultrasound in collagen extraction are limited. This reveals the need for new studies to evaluate the effect of ultrasound on enzyme activity and the benefits of its use in combination with other methods. However, extraction times and thus energy consumption are also fairly high in extraction with collagen extraction methods in the prior art.
Brief Description and Objectives of the Invention
Present invention relates to the method of obtaining collagen, from the extracellular matrix components in the lamb’s foot, which meets the aforementioned needs, eliminates all the disadvantages, and provides some additional advantages.
The primary object of the invention is to develop an innovative and environmentally friendly method and to shorten the time of obtaining collagen by applying ultrasound treatment after 6 hours of treatment instead of 24 hours and more used in traditional extraction methods.
The object of the invention is to produce high yield, purity, and high added value collagen by utilizing lamb foot wastes. Ultrasound application is seen as an effective method in increasing product purity and quality. It was observed in studies on collagen extraction that ultrasound application improved the product properties by preserving the collagen structure.
The extraction method of the invention includes acid and enzymatic hydrolysis methods as well as ultrasound application.
High added value collagen production was carried out from lamb feet, which are animal byproducts, in shorter extraction times compared to the methods known in the art with the invention method. The production of collagen, which is considered to be one of the most important biomaterials by utilizing lamb’s feet, is especially important for the meat industry in terms of waste evaluation.
The effects of ultrasound on collagen structure were examined in detail with the invention method. Use of many commercial collagens not preferred, as many of them are derived from mammalian animals such as pigs in religious aspects. Halal collagen will be obtained as an alternative to pig collagen by using lamb feet in this context.
The method of the invention is not limited to the use of lamb’s foot as a source of collagen. The method of the invention can also be used effectively for collagen raw materials of animal origin such as feet, tendons, skins or trachea of bovine, ovine or poultry; chicken sternum or cartilage; fish skin, scales, fins or bones.
Definitions of Figures Describing the Invention
The figures and related descriptions required to better understand the inventive method of obtaining collagen from extracellular matrix components and the characteristics of the resulting collagen are as follows.
Figure 1: Flow chart of the pre-treatments applied to the lamb’s foot
Figure 2: Flow chart of collagen production from lamb’s feet
Figure 3: The SDS-PAGE bands of the collagen samples obtained under different extraction conditions (M: molecular weight marker. 1- 20°C-20 min.; 2- 20°C-40 min.; 3-20°C-60 min.;
4- 25°C-20 min.; 5, 25°C-40 min.; 6-25°C-60 min.; 7- 30°C-20 min.; 8- 30°C-40 min.; 9- 30°C-60 min. ultrasound application)
Figure 4: FTIR spectra of collagen obtained at different ultrasound times at 20°C
Figure 5: FTIR spectra of collagen obtained at different ultrasound times at 25°C
Figure 6: FTIR spectra of collagen obtained at different ultrasound times at 30°C
Figure 7: SEM images of collagen samples obtained under different extraction conditions.
(Al : 20°C-20 min.; A2: 20°C-40 min.; A3: 20°C-60 min.; Bl : 25°C-20 min.; B2: 25°C-40 min.; B3: 25°C-60 min.; Cl : 30°C-20 min.; C2: 30°C-40 min.; C3: 30°C-60 min.)
Detailed Description of the Invention The necessary pre-treatments and extraction process steps for obtaining collagen from the extracellular matrix components in lamb feet are described only for clarifying the subject matter in a manner such that no limiting effect is created in this detailed description.
The method of obtaining collagen of the invention includes the following process steps: a) Cleaning and grinding of collagen raw materials of animal origin, b) Homogenizing the cleaned and downsized collagen raw materials in 0,05 M Tris-HCl at a ratio of 1 :20 (w/v) and having a pH value of 7.5 for 50 minutes c) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm, d) Treating the precipitate taken after centrifugation with 50% ethanol at a ratio of 1 : 10 (w/v) for 3 hours for fat removal, e) Taking the precipitate by centrifuging the mixture at 4°C for 20-30 minutes at 9000-15000 rpm, f) Mixing the precipitate taken after centrifugation with 0.5 M EDTA-2Na at a ratio of 1 : 10 (w/v) for 6 hours at 4°C for decalcification, g) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm, h) Keeping the precipitate taken after centrifugation in 0.1 M NaOH for overnight at a ratio of 1 : 10 (w/v) to remove non-collagen proteins, i) Taking the precipitate by centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm, j) Storing the final precipitate at -18°C until extraction k) Mixing the precipitate with 5% lactic acid at a ratio of 1 :20 (w/v) containing 0.1% pepsin (w/v) for 6 hours at 4°C, l) Then, keeping it in an ultrasonic bath at 20-30°C temperature and for 20-60 minutes, m) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm and taking the supernatant, n) Treating the supernatant taken after centrifugation with 2.5 M NaCl for overnight for precipitation of proteins, o) Centrifuging at 4°C for 25-30 minutes at 9000-15000 rpm, p) Dialysis for removal of excess salt, q) Lyophilization of samples at the end of dialysis.
It contains the process steps. The collagen raw material of animal origin may be feet, tendons, skins, or trachea of bovine, ovine or poultry; chicken sternum or cartilage; fish skin, scales, fins or bones. Lamb’s foot was preferred as a collagen raw material of animal origin in the invention.
Component analyses were performed on lamb feet to effectively determine the pre-treatments to be performed. The composition of the lamb’s foot was considered when determining the pre-treatment steps.
Pre-Treatments:
(1) The lamb’s feet were washed for the purpose of cleaning contaminants and then ground in an industrial grinding machine for size reduction.
(2) The cleaned and downsized lamb feet were homogenized using a waring blender for 50 minutes in 0,05 M Tris-HCl (pH 7.5) at a ratio of 1 :20 (w/v). The mixture was centrifuged at 4°C for 25 minutes at 9000 rpm at the end of the treatment. The precipitate was taken for the next process after centrifugation.
(3) The precipitate taken after centrifugation was treated for 3 hours with 50% ethanol at a ratio of 1 : 10 (w/v) for fat removal. The precipitate was taken by centrifuging the mixture at 4°C for 20 minutes at 9000 rpm at the end of the treatment.
(4) The precipitate taken after centrifugation was mixed with 0.5 M EDTA-2Na at a ratio of 1 : 10 (w/v) for 6 hours at 4°C for decalcification. The precipitate was taken by centrifuging the mixture at 4°C for 25 minutes at 9000 rpm at the end of the treatment.
(5) The precipitate was kept in 0.1 M NaOH for overnight at a ratio of 1 : 10 (w/v) in order to remove non-collagen proteins. The precipitate was then taken by centrifuging at 4°C for 25 minutes at 9000 rpm.
(6) Pre-treated lamb feet were stored at -18°C until extraction.
Solvents and ratios used in the extraction process and ultrasound conditions were determined as a result of preliminary tests. Ultrasound application was performed at three different temperatures and times; the production continued in the parameter with the highest collagen concentration. Extraction phase:
(1) The samples were stirred with 5% lactic acid at a ratio of 1 :20 (w/v) containing 0.1% pepsin (w/v) for extraction for 6 hours at 4°C after pre-treatments.
(2) Afterward, three different temperatures (20 ± 2°C, 25 ± 2°C, 30 ± 2°C) and times (20, 40, 60 min.) were applied in ultrasonic bath (Bandelin Sonorex Digitec, Germany) ultrasound (frequency: 35 kHz; power: 140/560 W).
(3) The mixture was centrifuged at 4°C for 30 minutes at 9000 rpm at the end of the extraction process. The supernatant was taken at the end of the centrifugation.
(4) The supernatant taken after centrifugation was treated with 2.5 M NaCl for overnight for precipitation of proteins.
(5) The final precipitate was collected by centrifuging at 4°C for 30 minutes at 9000 rpm after the precipitation process.
(6) Dialysis was performed for removal of excess salt. The samples were first dialyzed against 0.1 M lactic acid for 24 hours and then against distilled water for 48 hours.
(7) The samples were lyophilized at the end of dialysis and stored at -18°C until analysis.
Collagen concentrations were found between 715,13±70,94 and 5061,75±171,41 pg/pl with the applied method. Collagen concentrations are given in Table 1.
Table 1: Collagen concentration of the samples obtained under different extraction conditions
Figure imgf000008_0001
a-b-c-d-e jjffgj-g ce between the groups marked with different letters in the same column is statistically significant (p<0,05). The extraction condition with the highest collagen concentration is determined as 25°C-60 min. ultrasound application (5061,75 pg/pl). An increase in collagen concentrations was also observed depending on the increased ultrasound time at the same temperature. However, the duration of 60 minutes should not be exceeded to prevent the structure from denaturation. It was observed that collagen samples obtained at different ultrasound times at 25°C had the highest collagen concentration. It has been observed that the collagen concentration has started to decrease at temperatures and times above 25°C. It is seen in general that the ultrasound time affects the collagen concentration more than the temperature. The results of the collagen analysis have shown that lamb’s feet can be used as an alternative source of collagen.
SDS-PAGE bands and subunit compositions of collagen samples are shown in Figure 3. Protein distributions of collagen samples are seen in this way. The absence of any other protein band not related to collagen in protein gel electrophoresis results indicates that the sample is >90% purity. The a, P, and y chains forming the collagen structure are shown on the figure.
It was observed that all collagens obtained under different extraction conditions consisted of al, a2, P, and y chains (~130 kDa/ al; ~ 116 kDa/ a2; 250 kDa/ P; ~300 kDa/ y). Electrophoresis results showed that all collagens obtained under different extraction conditions preserve their structural integrity and Type I collagen [al(I)]2 a2(I)] is the main collagen found in the lamb feet. The FTIR spectra of collagen samples obtained at different temperatures and times are shown in Figures 4, 5, and 6.
Amide A, amide B, amide I, amide II, and amide III bands were observed in the FTIR spectrum of all collagen samples. CH2 bending was observed in the range of " 1451-1453cm_1" wave number in all collagen samples. The density ratio between the Amid III band and the " 1450-1454cm_1" band is close to 1, indicating that the triple helical structure of collagen is not disrupted. This ratio was found to be 1.17 for all collagen samples. The FTIR results support that the ultrasound performed at different temperatures and times does not damage the collagen structure.
The surface morphology and particle sizes of the collagen samples were determined by scanning electron microscopy. SEM images were taken with 5000x magnification. The SEM images of the collagen samples obtained under different extraction conditions are given in Figure 7.
It is seen that all samples have a fibrous and porous structure when the surface morphologies and microstructures of the collagen samples are examined. The mean fiber diameter of the collagen samples was found to vary between 0.1~0.9 pm. Some differences were observed depending on the ultrasound temperature and time. While a more regular and distinct fiber structure was observed in the collagen samples at 20°C, a more irregular, rough and spongy structure with small pores was observed as the temperature increased. It is clearly seen that the temperature and increasing ultrasound times affect the collagen structure. The best results were obtained under extraction conditions of 20°C and 60 minutes, considering both the desired collagen structure (Figure 7: A3) and the collagen concentration.
The gap between the nested layers gives the collagen porosity. Spiral-like fibers were observed as an interconnected layer, as reported in the art. Porosity, the size of the pores, and the material surface area are considered to be important biomedical parameters of biomaterials. The homogeneous and regular mesh structure in the collagen obtained from the lamb’s foot makes collagen suitable as a drug carrier.

Claims

CLAIMS A method of obtaining collagen, characterized in that it comprises the following process steps: a) Cleaning and grinding of collagen raw materials of animal origin, b) Homogenizing the cleaned and downsized collagen raw materials in 0,05 M Tris- HC1 at a ratio of 1 :20 (w/v) and having a pH value of 7.5 for 50 minutes, c) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm, d) Treating the precipitate taken after centrifugation for 3 hours with 50% ethanol at a ratio of 1 : 10 (w/v) for fat removal, e) Taking the precipitate by centrifuging the mixture at 4°C for 20-30 minutes at 9000-15000 rpm, f) Mixing the taken precipitate after centrifugation with 0.5 M EDTA-2Na at a ratio of 1 : 10 (w/v) for 6 hours at 4°C for decalcification, g) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm, h) Keeping the precipitate in 0.1 M NaOH for overnight at a ratio of 1 : 10 (w/v) in order to remove non-collagen proteins, i) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm and taking the precipitate, j) Storing the pre-treated lamb feet at -18°C until extraction k) Mixing the pre-treated lamb feet with 5% lactic acid at a ratio of 1:20 (w/v) containing 0.1% pepsin (w/v) for 6 hours at 4°C, l) Then, keeping mixture in an ultrasonic bath at 20-30°C temperature and for 20-60 minutes, m) Centrifuging the mixture at 4°C for 25-30 minutes at 9000-15000 rpm and taking the supernatant, n) Treating the taken supernatant with 2.5 M NaCl for overnight for precipitation of proteins, o) Centrifuging at 4°C for 25-30 minutes at 9000-15000 rpm, p) Dialysis for removal of excess salt, q) Lyophilization of samples at the end of dialysis.
9 A method according to claim 1, characterized in that the collagen raw material of animal origin is:
• Feet, tendons, skins or trachea of bovine, ovine or poultry,
• Chicken sternum or cartilage • Fish skin, scales, fins, or bones. A method according to claim 2, characterized in that the collagen raw material of animal origin is a lamb’s foot.
PCT/TR2021/051174 2020-12-03 2021-11-10 Method for obtaining collagen from extracellular matrix components WO2022119529A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
TR2020/19613 2020-12-03
TR202019613 2020-12-03
TR2021/01707 2021-02-04
TR2021/01707A TR202101707A2 (en) 2020-12-03 2021-02-04 METHOD OF OBTAINING COLLAGEN FROM EXTRACELLULAR MATRIX COMPONENTS

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ATA, OZGE; ORMANLI, EBRU; KUMCUOGLU, SEHER; TAVMAN, SEBNEM: "Determination of Thermal and Rheological Properties of Collagen Protein", 6TH INTERNATIONAL CONFERENCE: THERMOPHYSICAL AND MECHANICAL PROPERTIES OF ADVANCED MATERIALS (THERMAM), 8TH ROSTOCKER INTERNATIONAL CONFERENCE: THERMOPHYSICAL PROPERTIES FOR TECHNICAL THERMODYNAMICS; CESME, IZMIR, TURKEY; SEPTEMBER 22-24, 2019, 24 September 2019 (2019-09-24) - 24 September 2019 (2019-09-24), pages 47, 201 - 207, XP009538264 *

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