WO2018211311A1

WO2018211311A1 - Method of isolating collagen

Info

Publication number: WO2018211311A1
Application number: PCT/IB2017/052905
Authority: WO
Inventors: Markus Depfenhart
Original assignee: Anderson David Warwick
Current assignee: Anderson David Warwick
Priority date: 2017-05-17
Filing date: 2017-05-17
Publication date: 2018-11-22
Anticipated expiration: 2019-11-17

Abstract

This invention relates to a method for isolating collagen from animal material, including jellyfish, the method comprising (a) drying collagen containing material and processing the dried material to produce a powder, (b) dissolving the powder in an acid solution, (c) adding riboflavin to the acid solution, (d) subjecting the acid solution to UV radiation or blue light having a wavelength in the range of about 350 nm to about 500 nm to produce a collagen precipitate, and (e) separating the collagen precipitate of step (d) from the solution and optionally drying the precipitate.

Description

METHOD OF ISOLATING COLLAGEN

INTRODUCTION

This invention relates to a method of isolating collagen comprising irradiation in the presence of riboflavin, and to a collagen product produced by the method of the invention, !n particular, but not exclusively, the invention further relates to a method for producing pH neutral liquid collagen. In one aspect of the invention the collagen may be derived from jellyfish.

BACKGROUND

Collagen is a major component of extracellular matrix distributed in skin, bone, and cartilage protein. Collagen is a fibrous, high-molecular weight protein with a triple helix structure. The physical and biological stability of the collagen structure results from the cross-linking between the tropocoilagen molecules, which are considered the basic molecules of fibrous protein. Generally the peptide structure of collagen is composed of about 1/3 (Gly-X- Y)_n, wherein X is proline and Y is hydroxyproline, the remaining 2/3 of the structure comprising other amino acids.

Collagen is a functional material that is widely used in a number of industries including the food, medicine, cosmetics, and cell culture industries. In the food industry, for example, collagen may be used as an edible casing, a carrier, or as an additive to improve the taste of certain foods, for example processed meats such as sausage or ham.

Animal based collagen is widely used in the medical industry. However, there remains a relatively high risk animal based collagen may expose a recipient thereof to infectious pathogens including bovine spongiform encephalopathy, avian influenza, transmissible spongiform encephalopathy, and others. Attempts have been made to reduce these risks through the use of human based collagen. However, the use of human collagen is still limited and has problems associated with insufficiently low yields due to extraction challenges. In addition, human based collagen remains commercially unattractive due to high processing costs.

Jellyfish is known to comprise attractively high levels of collagen. As a result, there has been much investigation into methods for extracting collagen from jellyfish. In addition, factors such as a reduction in its natural predators as a result of international fishing activities, together with the effects of global warming, has led to an increase in jellyfish numbers, typically encountered as a bycatch in commercial fishing operations.

To date conventional methods for extracting collagen from jellyfish has depended on basic wet chemistry methods which includes treatment with acid, alkali, and salt solutions. However, these methods are known to suffer from undesirable yields as well as the negative environmental impact associated with the use of the particular reagents used in these methods. Previous studies on marine organism based collagen, including acid soluble collagen extracted from jellyfish and fish skin and bone, have shown that collagen from these sources compare favourably to other animal based collagen based on amino acid composition, denaturation temperature, and solubility. In particular, jellyfish collagen has been shown to be effective in increasing skin elasticity, in regulating blood circulation, and in the treatment several medical conditions including arthritis, hypertension, bronchitis, and asthma, for example.

United States patent application publication number US 2016/0052962 discloses a method for isolating collagen from jellyfish using radiation. The method includes irradiating an acid solution of dried jellyfish material with gamma rays or electron beams, with the irradiation typically having a dose in the range of 5 kGy - 200 kGy. The disadvantage associated with the method disclosed in US 2016/0052962 is that Gamma- Radiation facilities are prohibitively expensive (installation, safety regulations, etc.), while there is a relatively small window wherein the relationship between crosslinking and denaturation is optimal. Other alterations that could be mutagen through ionization radiation are not predictable.

Therefore, there remains a need for an improved method for the extraction of collagen from sources that are difficult to extract including jellyfish (low melting point), and for improved collagen based products.

The inventor of the present invention has surprisingly found that a method combining photosensitized light irradiation and chemical treatment on animal tissue like jellyfish is advantageous in producing collagen with relatively low costs but high yield. Compared with state of the art methods that depend on chemical treatment only, and the method described in US 2016/0052962 the method of the present invention reduces the costs with an increase in yield, leads to a reduction of pathogens in the resultant product as a result of the combination of riboflavin and light irradiation, and simultaneously reduces environmental pollution and contamination of the collagen end product. In addition, use of the method provides and effective mechanism for reducing harmful excessive jellyfish. Further, the method of the invention can be efficiently used as a separation technique usable for different proteins including collagen.

SUMMARY OF THE INVENTION

According to a first aspect to the present invention there is provided a method for isolating collagen, the method comprising the steps of:

(a) drying collagen containing material and processing the dried material to produce a powder,

(b) dissolving the powder in an acid solution,

(c) adding riboflavin to the acid solution,

(d) subjecting the acid solution to UV radiation or blue light having a wavelength in the range of about 350nm to about 500nm to produce a collagen precipitate,

(e) separating the collagen precipitate of step (d) from the solution and optionally drying the precipitate.

In one embodiment of the invention, the coliagen containing material is jellyfish material.

In one embodiment of the invention, prior to step (c) the solution was placed in a container flushed with nitrogen and exposed to UV radiation at 254nm.

In one embodiment of the invention, oxygen is flush into the system at step (d). In one embodiment of the invention the method further comprises dialysing the collagen by reconstituting the precipitate in an acid solution and dialysing the solution against a chelating agent. in a preferred embodiment the chelating agent is a weak acid or EDTA.

Preferably, the weak acid is citric acid.

In a preferred embodiment the dialysed collagen solution has a pH after treatment in the range of about 6.5 to about 7.5.

Preferably, the EDTA is disodium EDTA, and the concentration is between about 5mM and about 75mM.

Preferably, the weak acid has a pKa value similar to disodium EDTA.

In one embodiment of the invention the riboflavin is added to the acid solution to a concentration of about 0.01 % to about 1% w/v.

Preferably, the riboflavin is added to the acid solution to a concentration of about 0.05 % to about 0.5 % w/v.

More preferably, the riboflavin is added to the acid solution to a concentration of about 0.075 % to about 0.125 % w/v.

In a preferred embodiment of the invention the applied light of step (d) has a wavelength in the range of about 350nm to about 380nm, or about 400 nm to about 460nm.

In a particularly preferred embodiment, the applied light of step (d) has a wavelength of about 365nm or about 450nm.

Preferably, the energy dose of the applied light is between 2,8J/cm² and 5,4 J/cm². The acid solution in step (b) may be selected from the group consisting of acetic acid, citric acid, and formic acid.

Preferably, the acid solution in step (b) is acetic acid.

In one embodiment of the invention the concentration of the acid solution in step (b) is in the range of about 0.01 M to about 2.0 .

Preferably, the concentration of the acid solution in step (b) is about 1.0 .

In a preferred embodiment, the drying in step (a), and/or the optional drying in step (e) is freeze-drytng.

According to a second aspect to the present invention there is provided a jellyfish derived collagen preparation produced by the method according to the method of the invention.

According to a third aspect to the present invention there is provided a neutralized jellyfish derived liquid collagen preparation produced according to the method of the invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter with reference to the accompanying figures, in which some of the non-limiting embodiments of the invention are shown.

The invention as described hereinafter should not be construed to be limited to the specific embodiments disclosed, with slight modifications and other embodiments intended to be included within the scope of the invention.

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. As used herein, throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.

The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having", "including", and variations thereof used herein, are meant to encompass the items listed thereafter, and equivalents thereof as well as additional items.

The present invention provides for a method for isolating collagen from collagen containing animal material, including jellyfish. The isolation method comprises the step of UV irradiation in the presence of riboflavin, and to a collagen product produced by the method of the invention.

In one aspect of the invention, the method produces a jellyfish based pH neutral liquid collagen product.

The method of the present invention comprising the steps of drying collagen containing animal material, including jellyfish, and processing the dried animal material to produce a collagen containing powder, dissolving the powder in an acid solution, adding riboflavin to the acid solution, subjecting the acid solution to light with a wavelength in the range of about 350nm to about 500nm to produce a collagen precipitate, and separating the collagen precipitate from the solution.

The obtained precipitate may optionally be subjected to further drying steps. The method may include further optional steps known in the art such as reconstitution in an acid solution and salting out of the resultant acid solution.

One example of collagen containing animal material that may be extracted with the method of the present invention is jellyfish material. It will be appreciated that reference to jellyfish may be substituted with any such collagen containing animal material. In the method of the invention the first step, or steps, deals with the initial treatment of the as-obtained jellyfish material. Essentially this part of the method is concerned with creating a collagen containing material that is suitable for further processing, possibly after transportation thereof to another facility.

The drying of the material may involve first subjecting the material to one or more washing steps prior to the drying thereof. These steps are not considered essential and may be varied depending on the nature of the raw material being processed.

After initial washing, the material may be dried by any method known in the art including freeze drying. The dried material may then be subjected to further processing steps, including the pulverisation of the material to a particular size that is considered suitable for further processing.

Typically the dried material may be ground to a particular size of about 50 pm to about 500 pm, preferably about 100 pm to about 400 pm, more preferably about 150 pm to about 350 pm, even more preferably about 200 pm to about 300 pm, and most preferably about 250 pm.

In one example, the processed, dried jellyfish powder is then dissolved in an acid solution. The acid solution may be selected from the group consisting of acetic acid, citric acid, and formic acid. In a preferred embodiment the acid solution is a solution of acetic acid.

The concentration of the acid solution may be selected in the range of about 0.01 M to about 2.0 M, preferably in the range of about 0.1 M to about 1.75 M, more preferably in the range of about 0.5 M to about 1.5 M, and most preferably about 1.0 M. in what is considered a critical step to the method of the present invention, riboflavin is added to the acid solution of dried jellyfish material. The riboflavin is added to the solution as a photosensitizer to aid the cross-linking process in the subsequent light irradiation step of the invention, i.e. a riboflavin/UVA- or blue light cross-linking reaction.

In a preferred embodiment of the invention riboflavin is added to the acid solution to a concentration of about 0.01 % to about 1 % w/v, more preferably about 0.05 % to about 0.5 % w/v, even more preferably about 0.075 % to about 0.125 % w/v, and most preferably about 0.1 % w/v.

The ultimate riboflavin concentration will be a matter of experimental optimization based on the concentration of jellyfish powder in the acid solution, the concentration of the acid solution, and possibly the temperature at which the reaction solution is maintained during the process.

Without thereby wishing to be bound by any particular scientific theory, it is believed that the method of the present invention leads to an increased yield, and collagen products of superior quality, as a result of the photosensitized crosslinking.

Through the light spots and the use of a photosensitizer like riboflavin, collagen fibers are cross-linked according to the lysyl oxidase reaction in live tissue. In other words, the photosensitizer containing agent catalyzes the collagen in the sense of an aldol condensation, similar to the iysyloxidase reaction. This increases the melting temperature and the stability of the collagen precipitate and leads to a higher yield in extraction. The photosensitizer also works as shield to protect the fibres from denaturation by absorbing the energy of the light. An optimal riboflavin concentration will lead to low denaturation yields by high precipitation and extraction rates. Even in an optimal dose range, Gamma Radiation leads to a fragmentation of the fibres of about 20%. The method of the present invention reaches similar quantum yields with fragmentation rates lower than 5%. In addition, the use of riboflavin and light irradiation, such as UV radiation, leads to a product with a reduced pathogen count. The acid solution containing riboflavin and the powder material is then treated by subjecting the solution to light irradiation having with wavelength that is preferably in the range of about 350nm to about 500nm. As discussed above, the riboflavin acts as a photosensitizer which, together with the light irradiation, results in the cross-linking of the material to produce a collagen precipitate.

Based on the absorption spectrum of riboflavin, the wavelength may be varied between about 350nm to about 500nm.

In a preferred embodiment of the invention the wavelength range may be in the region of about 350nm to about 380nm or about 440nm to about 460nm, most preferably the wavelength may be about 365nm or about 450nm.

The two absorption maxima of riboflavin is at about 365nm (UVA) and about 450nm (blue light, lower energy), irradiated riboflavin causes singlet oxygen that leads via a dissemination of the collagen (n-terminal end) to a spontaneous aldolcondensation to a c-treminai end of another fibre (n-to-c- terminal end crosslinking). The optimum dose is about 5,4J/cm² and this leads, with the optimum concentration of riboflavin (0,1 %), to a shielding and a desirable quantum yield. An increase of the riboflavin concentration will lead to an increased shielding effect and a decrease in quantum yield. The riboflavin in different, increasing concentrations in different irradiation rows will lead to a significant reduction of pathogens. Crosslinking of collagen increases the melting point. This is especially very important when avertrebrates tissue like jellyfish is used for a collagen extraction. An extraction of collagen as described in this document with riboflavin leads to more stable collagen, also in terms of temperature stability.

In the method of the invention, the step of separating and drying the collagen precipitate can be performed by methods known in the art of separation such as filtration and centrifugation, or combinations of centrifugation and filtration steps. The present invention also provides for a method for preparing atelocollagen, the method further comprising a step of treating the acid soluble collagen, prepared according to the present invention as described above, with protease and drying the protease solubilized collagen.

The protease used in the method of preparing atelocollagen may be selected from pepsin or trypsin. Preferably, the protease is pepsin. The protease concentrations is about 1 % to about 10 % (w/w), preferably about 3 % to about 8 % (w/w), and most preferably about 4 % to about 6 % (w/w).

Where a dried collagen product is desired, the obtained precipitate may be dried, preferably by quick freeze drying at temperatures in the range of about - 180 °C to about -70 °C, for example.

In a further aspect of the present invention, the method further comprises reconstituting the collagen precipitate or product in an acid solution and treating the acid soluble collagen with a chelating agent.

The acidic collagen solution prepared by the method of the present invention as described above may be dialyzed against a chelating agent or solution, including a weak acid, an EGTA solution, or a chemical similar solution thereby to prevent collagen fibri!logenesis.

Preferably, the EDTA solution is disodium EDTA. The pH of the collagen solution may be increased from acidic levels to a neutralized pH in the dialysis process by, for example, adjusting the pH of the disodium EDTA dialysis solutions using 1 N sodium hydroxide.

The neutralized pH of the solution may be between about 6.5 and about 7.5, more preferably the pH of the final solution is about 7. At a neutralized pH, the collagen preparation does not undergo typical fibrillogenesis and remains a clear, transparent liquid until it comes into contact with a polarized and ion containing solution or fluid. The EDTA present in the composition is preferably disodium EDTA. The EDTA remaining in the injectable composition after dialysis is preferably present in sufficient concentration to provide anti-coliagenase activity sufficient to inhibit activity of tissue metailoproteinases and to provide bactericidal activity to assure sterility and potentially inhibit biofiim formation.

In certain embodiments, the concentration of the EDTA in the collagen solution is between about 5 mM and about 75 mM, preferably between about 10 mM and about 50 mM, more preferably between about 25 mM and about 40 mM, and most preferably between about 30 mM and about 35 mM.

The neutralized jellyfish derived liquid collagen preparation according to the present invention allows for a direct injectable collagen solution that does not introduce the risk of infectious pathogens associated with bovine based products.

The present invention will now be described in further detail with the aid of the following non-limiting experimental examples.

Example 1

Salted Rhopilema esculenta individuals were first rinsed roughly with tap water and then with highly filtered and purified water (Milli-Q) rinsed and stirred until the salinity in the rinse water after at least one hour or less equal to 0.1 %, respectively.

The washed jellyfish was cut into small pieces in a mixer, and the moisture was eliminated by filtering. The jellyfish was then freeze-dried, and afterwards pulverized in a mixer.

Example 2

The pulverized jellyfish prepared in Example 1 was dipped in 0.5 M acetic acid (Merck, Darmstadt, Germany), Sodium-EDTA 0.1 g per 100ml was added and also riboflavin was added to get a 0,1 % riboflavin solution. The acetic JF and RF solution was then placed under stirring in a UV transparent customized plastic container irradiated with UVA light 365nm (UV LED NCSU033B[T]; Nichia Co., Tokushima, Japan) with 30 mW/cm pulsed light with a 3 seconds on / 3 seconds off duty cycle with an effective dose of UVA dose of 5.4 J/cm², followed by stirring at 4°C for 7 days.

Example 3

The solution prepared in Example 2 was filtered, the filtrate was diluted with 0.02 M Na₂HP0₄ (Sigma, St. Luis Mo., USA) at the ratio of 1 :3 (v/v) and dia!yzed against 10 times volume of at least 0.05% of acetic acid, said membrane having a separation size of about 12-16 kDa. A precipitate was obtained by centrifugation at 2000 rpm for 7 mtn. The precipitated collagen was dissolved in 0.5 M acetic acid and centrifuged at 2000 rpm, 6 min to obtain a supernatant. 0.9 M Sodium chloride (NaCI, Sigma) was added to the supernatant and the gained precipitate was dissolved in 0.5 M acetic acid. The solution was so long diluted until the acetic acid concentration was reduced to 0.1 M. As a result, acid-soluble collagen was achieved.

Example 4

The collagen prepared by the method described above was filtered and centrifuged at 2000 rpm (700*g) for 1 hour and the supernatant was collected and stored at 4°C.

The supernatant containing the isolated collagen was treated with pepsin (0.2 mg/ml) for 24 hours to produce atelopeptide collagen. The collagen solution was subjected to a diafiltration process to remove residual pepsin and low molecular weight components, therefore an Amicon Model DC10L/DC10LA ultrafiltration system with a spiral membrane cartridge (SY0100) with a 100,000 kD molecular weight cut off was used.

Example 5

Sodium chloride was added to the soluble, pepsin-digested collagen solution from Example 4 to a concentration of 0.8M to precipitate collagen. The precipitate was recovered by centrifugation for 60 minutes at 4000 RPM and concentrated to approximately 40mg/mL with filter paper to absorb excess liquid. The concentrated collagen precipitate was placed in dialysis tubing with a molecular weight cut-off of 100,000 daitons and diaiyzed against 0.5M acetic acid for 18 hours and then 0.1M acetic acid for another 18 hours. The collagen concentrate was then diaiyzed against 0.035M (35m ) EDTA (ethylenediaminetetraacetic acid, disodium salt dihydate, SigmaUltra -99%). Dialysis was continued for 7 days with daily adjustment of pH from the starting pH of 4.5 to a final pH of 7.5.

The final collagen concentrate was collected and centrifuged to remove air bubbles. The resulting collagen exhibited a pH of 7.4 and did not undergo fibril formation at room temperature. Collagen fibrillogenesis was not triggered until the collagen was exposed to physiological liquids or liquids containing ions to trigger gelation and polymerizing reactions.

This above description of some of the illustrative embodiments of the invention is to indicate how the invention can be made and carried out. Those of ordinary skill in the art will know that various details may be modified thereby arriving at further embodiments, but that many of these embodiments will remain within the scope of the invention.

Claims

1. A method for isolating collagen, the method comprising the steps of:

(b) dissolving the powder in an acid solution,

(c) adding riboflavin to the acid solution,

(e) separating the collagen precipitate of step (d) from the solution and optionally drying the precipitate,

2. The method according to claim 1 , wherein the collagen containing material is jellyfish material.

3. The method according to claim 1 or claim 2, wherein prior to step (c) the solution is flushed with nitrogen and exposed to UV radiation at 254nm.

4. The method according to any one of the preceding claims, wherein oxygen is flushed into the system at step (d).

5. The method according to any one of the preceding claims, wherein the method further comprises diaiysing the collagen by reconstituting the precipitate in an acid solution, and diaiysing the solution against a chelating agent.

6. The method according to claim 5, wherein the chelating agent is a weak acid, EDTA, or EGTA.

7. The method according to claim 6, wherein the weak acid is citric acid.

8. The method according to any one of claims 5 - 7, wherein the the dialysed collagen solution has a pH after treatment in the range of about 6.5 to about 7.5.

9. The method according to claim 6, wherein the EDTA is disodium EDTA, and the concentration is between about 5mM and about 75m .

10. The method according to any one of the preceding claims, wherein riboflavin is added to the acid solution to a concentration of about 0.01 % to about 1% w/v.

11. The method according to claim 10, wherein the riboflavin is added to the acid solution to a concentration of about 0.05 % to about 0.5 % w/v.

12. The method according to claim 10, wherein the riboflavin is added to the acid solution to a concentration of about 0.075 % to about 0.125 % w/v. 3. The method according to any one of the preceding claims, wherein the applied light of step (d) has a wavelength in the range of about 350nm to about 380nm, or about 440nm to about 460nm.

14. The method according to claim 13, wherein the applied light has a wavelength of about 365nm or about 450nm.

15. The method according to any one of the preceding claims, wherein the solution in step (b) is selected from the group consisting of acetic acid, citric acid, and formic acid.

16. The method according to claim 15, wherein the solution in step (b) is acetic acid.

17. The method according to any one of the preceding claims, wherein the concentration of the solution in step (b) is in the range of about 0.01 M to about 2.0 M.

18. The method according to claim 17, wherein the concentration of the solution in step (b) is about 1.0 M.

19. The method according to any one of the preceding claims, wherein the drying in step (a), or the optional drying in step (e) is freeze-drying.

20. An animal or human tissue derived collagen preparation produced by the method according to any one of the preceding claims.

21. A neutralized tissue derived liquid collagen preparation produced by the method according to any one of claims 5 - 8.