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Title:
A COLLAGEN PRODUCTION PROCESS
Document Type and Number:
WIPO Patent Application WO/2021/076079
Kind Code:
A1
Abstract:
In terms of the high capacity stocking and the stocking duration of the (cattle and/or small cattle) animal tendons used while obtaining collagen, the industrial production by means of the developed method is more advantageous when compared with similar productions. The advantage of stocking is that the degradation of protein-based product is prevented, and the usage of the tendon in fragmenting and powdering processes is facilitated and the shelf lifetime is extended. The obtained collagen will be a bio-technologic raw substance which can be used in medicine, food and medical sectors.

Inventors:
KARA MAGDEN GAMZE (TR)
ERDOGAN AYKUT (TR)
Application Number:
PCT/TR2020/050887
Publication Date:
April 22, 2021
Filing Date:
September 25, 2020
Export Citation:
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Assignee:
BUGAMED BIYOTEKNOLOJI SANAYI VE TICARET ANONIM SIRKETI (TR)
International Classes:
C07K14/78; A23L13/20; A61L27/38
Attorney, Agent or Firm:
KAYA, Erdem (TR)
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Claims:
CLAIMS

1. A process for obtaining collagen with high efficiency from tendons of cattle and/or small cattle animals, wherein the problem of fragmenting the tendons into smaller dimensions in moist form and for eliminating the problem of short duration stocking are eliminated and said process is characterized by comprising application of the following steps: i. Washing achilles and flexor tendons of cattle and/or small cattle animals by means of alcohol and separating thereof from the fascia tissue, ii. Applying 1 st incubator process to the tendons, iii. Cutting tendons taken from the incubator and freezing thereof at specific temperature ranges, iv. Drying by keeping in vacuum medium and separating the dried tendons to sarcostyles, v. Washing by means of buffer solution for sterilizing the grinded tendon parts, vi. Taking collagens by means of enzyme-acid solution in the 2nd incubator process from the washed tendon parts and applying centrifuge process after filtering, vii. Dissolving the obtained collagens in the acid solution and realizing the purification process, viii. Drying by keeping the collagens under vacuum after the purification process and obtaining the product.

2. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (i), said alcohol is ethyl alcohol or isopropyl alcohol with purity between 50% and 99.9%.

3. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (ii), the tendons are incubated at temperatures between +2eC and +24eC.

4. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (ii), the tendons are incubated between 10 minutes and 24 hours.

5. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (ii), the speed of the incubator is at a value between 50 rpm and 1000 rpm.

6. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (iii), the process of freezing of the cut tendons is realized at a value between - 10eC and -80eC.

7. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (iii), the frozen tendons are subjected to vacuuming process for duration between 12 hours and 24 hours.

8. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (iv), the vacuum process is applied at a value between -40eC and -110eC.

9. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (v), said buffer solution is peracetic acid-water solution.

10. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vi), the enzyme, provided in said enzyme-acid solution, is one of or mixtures of pepsin, papain, trypsin, pronase or collagenase chemical compounds at specific weight proportions.

11. The process of obtaining collagen with high efficiency according to claim 1 , wherein the enzyme mentioned in step (vi) is at a value between 0.1 :100 and 1 :10 by weight in the acid solution.

12. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vi), the process speed of the 2nd incubator is at a value between 50 rpm and 1000 rpm.

13. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vi), the process duration of the 2nd incubator is at a value between 12 hours and 72 hours.

14. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vi), said filter pore size is at a value between 10 microns and 10000 microns.

15. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vi), the centrifuge process speed is between 1500 rpm and 22000 rpm.

16. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vi), the duration of the centrifuge process is between 15 minutes and 45 minutes.

17. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vii), the tendon parts are kept for duration between 1 minute and 120 minutes in buffer solution for providing sterilization.

18. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vii), said acid is one of hydrochloric acid or acetic acid.

19. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (vii), the purification process is realized by using one of or a number of the microfiltration, ultra-filtration, dialysis or flow filtration methods.

20. The process of obtaining collagen with high efficiency according to claim 1 , wherein in step (viii), sterilization processes are applied to the obtained product.

21. The process of obtaining collagen with high efficiency according to claim 20, wherein said sterilization process is applied by means of gamma sterilization.

22. The process of obtaining collagen with high efficiency according to claim 21 , wherein sterilization process is applied by keeping in ethylene oxide, hydrogen peroxide or peracetic acid solutions.

Description:
A COLLAGEN PRODUCTION PROCESS

TECHNICAL FIELD

The present invention relates to the process of obtaining collagen with high efficiency from achilles and/or flexor tendons of cattle and/or small cattle animals.

PRIOR ART

Collagen is the main protein which forms the structure stones of the movement system, particularly bones, cartilages, sarcostyles and articulars.

The main structure of collagen is formed by three alpha (a) chains wrapped onto each other. Polypeptides, which exist in triple a-chains, rotate in a mutual axis and they form triple helix structure which has length of approximately 3000 A e (Angstrom) and which has diameter of approximately 15 A e . The molecular weight of the triple polypeptide chain is approximately 300000 grams/mole. Each a-chain has amino acid array which continues in a repetitive manner in the form of Gly-X-Y, and 1014 amino acids must be brought together for forming polypeptide chains. 19 different types of collagen have been defined and have been named as type I and type II, and this variety results from the molecular structure.

Approximately 60% of the total protein which exists in livings is formed by different collagen types, and the 80%-90% of these collagens is formed by type-1 collagen. The robust and stable structure of type-1 collagen is provided thanks to covalent cross bindings formed between the fibrils. Pluralities of collagen fibrils are brought together thanks to these cross bindings and they form the basic structure of tissues like skin, bone and tendon.

Collagen is frequently used as raw substance while developing tissue engineering products for treating various clinical complications thanks to the distribution and characteristics (for instance, cell diagnosis signals, 3-dimensional extracellular matrix formation ability for the cells, controllable mechanical characteristics, bio-compliancy and bio-degradability) in human tissues. The basic reason of preferring collagen as bio-material is that collagen comprises extra-cellular matrix components which exist in plenty of amounts in the body and thus collagen is sensed not as a foreign substance, and collagen is sensed as an endogen component of the body. In the tendon, pure type-1 collagen is provided with proportion of approximately 90%. Although it is difficult to fragment the tendon into smaller dimensions due to the stiff and robust structure of the tendon, the collagen obtaining efficiency is relatively lower when compared with the collagen obtaining efficiency of the other tissues even after the tendon is fragmented. Therefore, for obtaining collagen from the tendons with high efficiency, improvements must be made in the standard collagen extraction process. In the present applications, the disadvantages are as follows: (i) the process duration is long, (ii) the process for the extraction is not efficient and (iii) collagen efficiency is low.

As a result, because of all of the abovementioned problems, an improvement is required in the related technical field.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to development of collagen obtaining methods with high efficiency from achilles and flexor tendons of cattle and/or small cattle animals, for eliminating the above mentioned disadvantages and for bringing new advantages to the related technical field.

The main object of the present invention is to provide a process where collagen is obtained while the achilles and/or flexor tendons of cattle and/or small cattle animals are in dry form.

Another object of the present invention is to provide a process where collagen is obtained from the achilles and/or flexor tendons of cattle and/or small cattle animals with high efficiency.

Another object of the present invention is to provide a process where collagen, which can be stocked with long lifetime, is obtained from the achilles and/or flexor tendons of cattle and/or small cattle animals.

In order to realize the abovementioned objects and the objects which are to be deducted from the detailed description below, the present invention relates to development of collagen obtaining processes with high efficiency from the achilles and flexor tendons of cattle and/or small cattle animals. Accordingly, the greatest advantage provided by the process is that collagen can be obtained from the tendons while the tendons are in dry form and not in moist form. By means of this, the problem of fragmenting the tendons into smaller dimensions in moist form and the problem of short duration stocking is eliminated. By means of the present invention, less amount of pepsin, papain, trypsin, pronase or collagenase chemical compounds are used as enzyme in obtaining collagen.

The present invention is a process for obtaining collagen with high efficiency from tendons of cattle and/or small cattle animals and for eliminating the problem of fragmenting the tendons into smaller dimensions in moist form and for eliminating the problem of stocking the tendons of cattle and/or small cattle animals, said process is characterized by comprising application of the following steps: i. Washing achilles and flexor tendons of cattle and/or small cattle animals by means of alcohol and separating thereof from the fascia tissue, ii. Applying 1 st incubator process to the tendons, iii. Cutting tendons taken from the incubator and freezing thereof at specific temperature ranges, iv. Drying by keeping in vacuum medium and separating the dried tendons to sarcostyles, v. Washing by means of buffer solution for sterilizing the grinded tendon parts, vi. Taking collagens by means of enzyme-acid solution in the 2 nd incubator process from the washed tendon parts and applying centrifuge process after filtering, vii. Dissolving the obtained collagens in the acid solution and realizing the purification process, viii. Drying by keeping the collagens under vacuum after the purification process and obtaining the product.

BRIEF DESCRIPTION OF THE FIGURES

In Figure 1 , the view of the collagen obtained from tendons of cattle and/or small cattle animals by means of scanned electron microscope (SEM) is given.

In Figure 2, the thermogram of differential scanned calorimeter (it can be mentioned as DSC) of collagen obtained from tendons of cattle and/or small cattle animals is given.

In Figure 3, the view of the test result of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (it can be mentioned as SDS-PAGE) of collagen obtained from tendons of cattle and/or small cattle animals is given.

In Figure 4, the cytotoxicity results of collagen obtained from tendons of cattle and/or small cattle animals in accordance with 3-(4,5-Dimethyl-thiazole-2-yl)-2,5-diphenyltetrazolium bromide (it can be mentioned as MTT) method are given. DETAILED DESCRIPTION OF THE INVENTION

In this detailed description, the subject matter relates to development of collagen obtaining methods with high efficiency from achilles and flexor tendons of cattle and/or small cattle animals and is explained with references to examples without forming any restrictive effect only in order to make the subject more understandable.

Collagens support extracellular matrix structure of tissues and provide suitable environmental conditions for development of cells. Collagens provide flexibility and resistance to the tissue and they help renewal and development of tissues and organs. Collagens prevent passage of toxins and pathogens into the skin tissue and protect the skin. Collagens play role in augmenting and differentiating the cells and help improvement of bones or blood veins and at the same time, they protect structural integrity. Collagens have a wide usage area essentially in health, cosmetic, medicine and food sectors because of their bio-compliancy, bio-degradability and low immunogenity characteristics.

The molecular weight of collagens used in foodstuffs is recommended to be approximately 2000 Daltons. The reason for recommending low molecular weight is that absorption and bio utilization are greater since the particle dimension is smaller. In accordance with the clinical studies made, in order to utilize the favorable effects of collagen, collagen must be consumed at the recommended daily usage dose (grams/day) regularly. The daily dose recommended by R. Moskowitz has been mentioned as 10 grams. It has been experimentally detected that the reception of this amount of collagen has beneficial effects on the health of articular, bone and skin, and collagen synthesis increases as a result of increase of the hydroxy-pyroline concentration in blood.

In the known state of the art, obtaining of collagens from tendons of cattle and/or small cattle animals is applied while the tendons are in moist form as in a traditional method. While said moist tendons are being cut, they are affected by the medium temperature and become soft and protein degradation occurs.

Because of the rigid and resistant structure of tendons, the fragmenting of tendons is difficult and the collagen efficiency of tendons is lower when compared with the other tissues. For the oil removing and extraction processes applied after cutting the tendons, applications which last for up to 72 hours are needed. In the present applications, some of the disadvantages are as follows: (i) the process duration is long, (ii) the process for extraction is not efficient and (ii) collagen efficiency is low. There are some advantages of the usage of the tendons of cattle and/or small cattle animals in obtaining collagen. Collagen production with telopeptide and without telopeptide can be realized by means of the tendons of cattle and/or small cattle animals. In terms of the high capacity stocking and the stocking duration of the cattle and/or small cattle animal tendons used in obtaining collagen, the industrial production thereof is more advantageous when compared with similar productions. The advantage of stocking is that the degradation of protein-based product is prevented, and the usage of the tendon in fragmenting and powdering processes is facilitated.

The most important advantage in the recommended system in the invention is to provide obtaining of collagen while the tendons are in dry form, not in moist form.

The subject matter system provides elimination of the difficulty of fragmenting the tendon in cold medium and provides realization of said process in room conditions easily. After the stocking and fragmenting process, the other processes are applied at a temperature between +2 e C and +24 e C for preventing deterioration of the protein.

Another advantage of the subject matter system is to present the product in a sterile form in a manner purified from microbial risks by sterilization by means of gamma rays and by means of one or mixtures of peracetic acid, hydrogen peroxide, glutaraldehyde chemical liquids with specific proportions.

The present invention is a process for obtaining collagen with high efficiency from tendons of cattle and/or small cattle animals and for eliminating the problem of fragmenting the tendons into smaller dimensions in moist form and for eliminating the problem of stocking the tendons of cattle and/or small cattle animals, said process is characterized by comprising application of the following steps: i. Washing achilles and flexor tendons of cattle and/or small cattle animals by means of alcohol and separating thereof from the fascia tissue, ii. Applying 1 st incubator process to the tendons, iii. Cutting tendons taken from the incubator and freezing thereof at specific temperature ranges, iv. Drying by keeping in vacuum medium and separating the dried tendons to sarcostyles, v. Washing by means of buffer solution for sterilizing the grinded tendon parts, vi. Taking collagens by means of enzyme-acid solution in the 2 nd incubator process from the washed tendon parts and applying centrifuge process after filtering, vii. Dissolving the obtained collagens in the acid solution and realizing the purification process, viii. Drying by keeping the collagens under vacuum after the purification process and obtaining the product.

In step (i), said alcohol is ethyl alcohol or isopropyl alcohol with purity between 50% and 99.9%.

In step (ii), during the incubator process, the tendons are kept in at least one of or a number of the ethanol and/or acetone with purity between 50% and 99.9% and 0.1 M - 2 M sodium hydroxide or 0.1 M - 2 M sodium carbonate solvents.

In step (ii), the tendons are incubated between 10 minutes and 24 hours.

In step (ii), the tendons are incubated between temperatures +2 e C and +24 e C.

In step (ii), incubation process is applied to the tendons at values between pH 5-7 or pH 8- 14.

In step (ii), the speed of the incubator is at a value between 50 rpm and 1000 rpm.

In step (iii), the tendons are cut at a length between 1 cm and 50 cm and at a thickness between 0.01 cm 2 and 1 cm 2 .

In step (iii), the process of freezing of the cut tendons is realized at a temperature value between -10 e C and -80 e C.

The tendons, frozen in step (iii), are subjected to vacuum process for duration between 12 hours and 24 hours in step (iv).

In step (iv), the vacuum process is applied at a value between -40 e C and -110 e C.

In step (iv), the frozen tendons are reduced to dimensions between 0.01 cm 3 and 10 cm 3 before being separated from the sarcostyles thereof and finally, they are grinded to dimensions between 10 and 10000 microns. In step (v), for providing sterilization, the buffer solution is one of or mixtures of the chemical compounds like peracetic acid, hydrogen peroxide, glutaraldehyde with specific weight proportions.

In step (v), the chemical compounds like peracetic acid, hydrogen peroxide, glutaraldehyde, provided in the buffer solution, are at a value between 0.01% and 5% by weight in the liquid.

In step (v), the tendon parts are kept in the liquid, comprising buffer solution for sterilization, for duration between 1 minute and 120 minutes.

In step (vi), the enzyme, provided in said enzyme-acid solution, is one of or mixtures of pepsin, papain, trypsin, pronase or collagenase chemical compounds at specific weight proportions.

The enzyme which exists in step (vi) is at a value between 0.1 :100 and 1 :10 by weight in the acid solution.

The acid mentioned in step (vi) is one of hydrochloric acid or acetic acid or the mixtures thereof at specific proportions.

The tendon parts mentioned in step (vi) is at a value between 0.1 :100 and 1 :10 by weight in the enzyme-acid solution.

In step (vi), the speed of the 2 nd incubator process is between 50 rpm and 1000 rpm.

In step (vi), the duration of the 2 nd incubator process is at a value between 12 hours and 72 hours.

In step (vi), in the incubator or outside the incubator, the stirring process is applied preferably in one of ultrasonic water bath, portative ultrasonicator, homogenizer device or mechanic stirrers.

In step (vi), the mixing process changes between 1 hour and 72 hours according to the used mixing device.

In step (vi), the pore size of said filter is at a value between 10 microns and 10000 microns. In step (vi), the speed of the centrifuge process is between 1500 rpm and 22000 rpm.

In step (vi), the duration of the centrifuge process is between 15 minutes and 45 minutes.

In step (vi), said solution is acetic acid or hydrochloric acid or pH buffer.

In step (vii), the purification process is realized by using one of or a number of the microfiltration, ultra-filtration, dialysis or flow filtration methods.

The purification process in step (vii) has duration between 1 hour and 72 hours.

In step (vii), in said purification process, the molecular weighted threshold value of the purification membrane is between 3.5 daltons and 50 kilo-daltons and/or between 0.01 microns and 10 microns.

In step (viii), as said drying process, one of the methods like sprayed freezing drying, sprayed drying or freezing drying under vacuum is applied.

In step (viii), the drying process is realized at a value between -40 e C and -110 e C.

In step (viii), the process of keeping in vacuum is at a value between -40 e C and -110 e C.

In step (viii), the process of keeping in vacuum is applied between 12 hours and 24 hours.

In order to provide product safety before or after packing of the collagen with telopeptide and collagen with atelopeptide obtained after step (viii), the product is sterilized by gamma ray at doses between 1 kgy and 30 kgy.

Thanks to the subject matter process, collagen production with telopeptide and without telopeptide can be realized by means of the tendons of cattle and/or small cattle animals.

TESTS

Specific tests are applied to the collagen types obtained by means of the present invention from the tendons of cattle and/or small cattle animals, in order to make comparison of the present invention with the literature. The test results and interpretations of the collagens obtained by means of the present invention are given in a detailed manner under the titles below. SCANNED ELECTRON MICROSCOPE ANALYSIS OF THE COLLAGEN OBTAINED BY MEANS OF THE PRESENT INVENTION

As shown in Figure 1 , in the scanned electron microscope (SEM) image of the collagen structures obtained by means of the present invention from the cattle animal tendons, the morphological structure of the collagen has been examined in micron dimension and the images are compliant to the literature studies. The collagen sarcostyles in its morphologic structure are seen in a clear manner.

DIFFERENTIAL SCANNED CALORIMETER ANALYSIS OF THE COLLAGEN OBTAINED BY MEANS OF THE PRESENT INVENTION

As shown in Figure 2, it has been determined that the collagen structures, obtained by means of the present invention from the tendons of cattle and/or small cattle animals, deteriorate at temperatures between 43 e C and 44 e C.

SDS-PAGE ANALYSIS OF THE COLLAGEN OBTAINED BY MEANS OF THE PRESENT INVENTION

As shown in Figure 3, according to the SDS PAGE (an electrophoresis method) results of the collagen structures obtained by means of the present invention from the cattle and/or small cattle animal tendons, a-1 and a-2 chains with molecular weight of approximately 130 kDa are observed, and beta and gamma chains with molecular weight over 250 kDa are observed. These values comply with the values of the collagen in the literature.

CYTOTOXICITY TEST OF THE COLLAGEN, OBTAINED BY MEANS OF THE PRESENT INVENTION, ACCORDING TO MTT METHOD

In Figure 4, when the cytotoxicity results of the collagen structures obtained by means of the present invention from the tendons of cattle and/or small cattle animals are evaluated, it is seen in the graphic that the cells continue viability and even a medium is presented where more number of cells can live when compared with the control group.

Cytotoxicity test has been made by using L929 Mouse Fibroblast cells and MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) according to ISO TSE 10993-5 standard. After the collagens are dissolved in acetic acid or in hydrochloric acid, they are subjected to process in cell culture for 24 hours and 48 hours with said cells. The cells, which are processed with MTT at the end of said duration, have been measured at wavelength of 570 nm and bio-compliancy has been proven in the direction of the results. The protection scope of the present invention is set forth in the annexed claims and cannot be restricted to the illustrative disclosures given above, under the detailed description. It is because a person skilled in the relevant art can obviously produce similar embodiments under the light of the foregoing disclosures, without departing from the main principles of the present invention.