WO2015012682A2

WO2015012682A2 - A method for extracting collagen from aquatic animals, collagen and products containing it

Info

Publication number: WO2015012682A2
Application number: PCT/MY2014/000190
Authority: WO
Inventors: Fatimah Md. Yusoff; Jamilah Bakar; Mahiran Basri; Maznah Ismail; Mun Hoe Nicholas KHONG
Original assignee: Universiti Putra Malaysia (UPM)
Current assignee: Universiti Putra Malaysia (UPM)
Priority date: 2013-07-22
Filing date: 2014-06-25
Publication date: 2015-01-29
Anticipated expiration: 2016-01-22
Also published as: WO2015012682A3

Abstract

The present invention provides an improved process for extracting collagen from aquatic animals, comprising alkaline treatment, followed by acidic treatment in combination with an orderly sequence of physical and/or mechanical treatments and precipitation of collagen using a salt solution. The process increases the yield and quality of the collagen while decreasing the production time and is more cost-effective than the processes known heretofore. The invention further includes the obtained collagen and products containing it.

Description

A METHOD FOR EXTRACTING COLLAGEN FROM AQUATIC ANIMALS, COLLAGEN AND PRODUCTS CONTAINING IT

Field of the Invention

The present invention provides a process for extracting collagen from aquatic animals with improved efficiency.

Background Art

Collagen is one of the most abundant forms of animal tissues. Collagen products find numerous medical and bioengineering uses. Collagen, as the chief structural protein of the body, is used in medicine and dentistry for many purposes, including wound dressings, matrices for tissue growth, as well as biomaterials for cosmetic surgery, reconstructive surgery, drug delivery system and scientific researches. Most of the collagen products used is derived from young beef cattle (bovine) as well as porcine (pig) tissue.

Collagen is used widely in cosmetic surgery and as wound healing aids in burn patients. These are used widely for reconstruction of bone and a wide variety of dental, orthopedic and surgical purposes. Collagen is used as sheets, dermal filler and functional films in cosmetic procedures. Although it cannot be absorbed through the skin, collagen is now being used as a main ingredient for some cosmetic makeup.

Collagen can be used in the construction of artificial skin substitutes used in the management of severe bums. This may be derived from bovine (cows), procine (pigs), equine (horses) or even human sources and are sometimes used in combination with silicones, glycosaminoglycans, fibroblasts, growth factors and other substances. Human collagen is derived from donor cadavers, placentas and aborted foetuses which pose possibilities of immune reactions. In cardiological procedures, the heart valves are made up of collagen tissue. Individual valvular leaflets are held in shape by collagen under variable pressure. In addition, with aging there is calcium deposit on the collagen leading to hardening of the valves.

Collagen is also used widely as barrier films and sheets/discs. The main application of collagen films is as barrier membrane. Films with the thickness of 0.01 - 0.5 mm and made of biodegradable materials. The drugs can be loaded into collagen membranes by hydrogen bonding, covalent bonding or simple entrapment. They can be sterilized and become made flexible. This are used in ophthalmology in delivering drugs to the eyes and also used over wounds to make healing rapid and uniform. Also in ophthalmological applications, collagen shields are used as bandage contact lenses that gradually dissolve in cornea. This idea has led to the development of more drug delivery systems for ophthalmic applications. One of the advantages is ease of application and self administration.

Collagen sponges are very useful in the treatment of severe burns and as a dressing for many types of wounds, such as pressure sores or bed sores, donor sites from where skin grafts have been taken, surgical sites, leg ulcers etc. These have the capacity to absorb large amounts of tissue secretions, lead to smooth adherence to the wet wound and maintain a low-moisture climate in the wound and shield against mechanical harm and secondary bacterial infection.

Collagen is useful as bone substitutes in the form of implantable carriers for bone inducing proteins and has been used as bone substitutes due to its osteo-inductive activity. Collagen film may be used as gene delivery carriers for osteoinduction and collagen sponge may be used for bone related protein carriers.

Collagen has also been increasingly used for enhanced drug delivery systems. Gel, hydrogel, liposomes-collagen are used as vehicles for drug delivery. They are often combined with synthetic polymers for drug delivery. The combination of natural and synthetic polymers may provide mechanical stability and biological acceptability, acquiring from synergistic properties of both materials. Minipellets made of collagen have been developed. These are used in drug delivery systems. The minipellet is small enough to be injected into the subcutaneous space through a syringe needle and large enough to contain large molecular weight protein drugs, such as interferon. Collagen nanoparticles/nanospheres also act as drug delivery systems as colloidal drug delivery carriers.

Collagen is also frequently used in scientific research applications for cell culture, studying cell behavior and cellular interactions with the extracellular environment. Recent studies also uses collagen in 3D drug study/screening models.

Many procedures describing extraction of collagen from vertebrates, such as cattle, pigs, horses, sheep, poultry, whales, sharks, fish are known.

Fresh jellyfish contain approximately 95 to 98 % water by weight, depending on the particular species and approximately 2 to 3 % salt by weight, which is in approximate osmotic equilibrium with salt water. The protein content is approximately 1.3 %. The lipid content of jellyfish is very low, up to 0.2 %. Jellyfish proteins consist almost entirely of collagen.

Analysis of the amino acid composition of mesogloea hydrolysate showed that glycine is the most abundant amino acid, and that hydroxyproline and hydroxylysine, which are

characteristic of collagen, are present. Tryptophan is almost totally absent. Thus, mesogloea contain proteins belonging to the collagen group. Wolfinbarger (US 7,241,871) describes a process for obtaining collagen from invertebrate marine animals, in particular jellyfish, which includes the steps of mild mechanical disruption of the marine animal material, followed by mild acid solubilization and precipitation of the collagens by salts in combination with mild stirring or slow dialysis. The acid solubilization step alone takes several days (3 days in most presented examples).

Allard et al. (US 6,660,280) patented a method of extracting collagen or collagen hydrolyzate with improved quality from marine animals using oxidizing substance such as sulfite, hydrogen peroxide or ozone. Many studies extract collagen using proteases, in particular pepsin and/or plant-based proteases such as papain (e.g., US 2012/114570), however, the use of proteases is costly and can pose halal or kosher issues. If may further affect the ultrastructure of the collagen, which negatively affects the quality of the collagen produced.

The prior art collagen producing methods provide for a long production time, low purity collagen, low extraction yield, high ash content, requirements for expensive equipment.

Disclosure of the Invention The present invention provides a process for extracting collagen from aquatic animals, comprising alkaline treatment of the aquatic animal tissue, followed by acidic treatment in combination with an orderly sequence of physical and/or mechanical treatments including but not limited to pH adjustments, homogenization, mixing as well as sonication; and

precipitation of collagen using a salt solution.

The aquatic animal tissue can be obtained by, e.g., maceration, homogenization, cutting and/or slicing fresh, processed or frozen biomass.

The present invention provides, more specifically, a process for extracting collagen from aquatic animals, comprising the steps of:

a) removing excess moisture from the tissues by mild drying techniques such as centrifugation and freeze thawing method,

b) optionally homogenizing the cell biomass with alcohol solution for at least 1 hour and then washing with a neutral solution,

c) mixing the tissues with alkaline solution for at least 6 hours;

d) washing the tissues with water to eliminate residual alkaline solution;

e) adjusting the pH until solution containing the tissues are maintained at pH below 8; f) treating the tissues by continuous mixing and/or homogenization in an acidic solution at a working temperature ranges from 4° C to room temperature for less than 2 hours; g) sonicating the mixture for at least 10 min;

h) performing the process of step (f) to step (g) for at least 1 repeat; i) centrifuging the mixture at 4-5° C;

j) optionally precipitation of collagen by introducing the pooled supernatant from the previous step into a buffer exchange system, and then centrifuging the mixture at 4-5° C and redissolving the pellet using an acidic solution,

k) precipitation of collagen by introducing the collagen solution into a sodium chloride solution to precipitate collagen fibers,

1) collecting the collagen fibers and centrifuging the collagen for at least 20 minutes; m) obtaining collagen pellet;

n) dissolving the collagen pellet into an acidic solution and dialysing the solution against water;

o) drying the dialysed suspension with mild drying procedures including but not limited to freeze-drying, spray-drying and vacuum-drying.

In particular, the process of extracting collagen from whole tissues of large zooplanktons comprises the steps of:

b) optionally homogenizing the cell biomass with alcohol solution for at least 1 hour followed by washing the biomass with a neutral solution;

c) mixing the tissues with alkaline solution for at least 6 hours;

d) washing the tissues with water to eliminate residual alkaline solution;

e) adjusting the pH until solution containing the tissues are maintained at pH below 8; f) treating the tissues from step (c) by continuous mixing and/or homogenization in an acidic solution at a working temperature ranges from 4° C to room temperature for less than 2 hours;

g) sonicating the mixture from step (e) for at least 10 min;

h) performing the process of step (f) to step (g) for at least 1 repeat;

i) centrifuging the mixture from step (f) at 4-5° C;

j) optionally precipitation of collagen by introducing the pooled supernatant from step (h) into a buffer exchange system,

k) centrifuging the mixture from step (f) at 4-5° C, 1) redisolving the pellet obtained from step (j) using an acidic solution, m) reprecipitation of collagen by introducing the collagen solution into a sodium chloride solution to precipitate collagen fibers,

n) collecting the collagen fibers from step (i) and centrifuging the collagen for at least 20 minutes;

o) obtaining collagen pellet;

p) dissolving the collagen pellet into an acidic solution and dialysing the solution against water;

q) drying the dialysed suspension with mild drying procedures including but not limited to freeze-drying, spray-drying and vacuum-drying.

In particular, the process of extracting collagen from biomass of smaller zooplanktons comprises the steps of:

d) mixing the biomass with alkaline solution for at least 6 hours;

e) washing the biomass with water to eliminate residual alkaline solution followed by adjusting the pH until solution containing the tissues are maintained at pH below 8; f) treating the biomass from step (e) by continuous mixing and/or homogenization in an acidic solution at a working temperature ranges from 4° C to room temperature for less than 2 hours;

g) sonicating the mixture from step (e) for at least 10 min;

h) the process of step (e) to step (f) should be repeated at least 1 time;

i) centrifuging the mixture from step (h) at 4-5° C;

j) optionally precipitation of collagen by introducing the pooled supernatant from step (h) into a buffer exchange system followed by centrifugation of the mixture at 4-5° C, k) redisolving the pellet obtained from step (j) and reprecipitation of the collagen by introducing the collagen solution into a sodium chloride solution;

1) obtaining collagen fibers from step (j); m) collecting the collagen fibers from step (k) and centrifuging the collagen for at least 20 minutes;

n) obtaining collagen pellet and dialysing the collagen obtained against water;

The extraction can optionally be followed by analysis of amino acid content in the collagen, in particular of the content of glycine, proline, hydroxyproline, alanine, arginine,

hydroxylysine and serine.

The aquatic animals are preferably zooplankton including cnidarians from the species of hydrozoa, siphonophora, scyphozoa, ctenophore, rotatoria, pennatulacea and polychaeta with preference to edible gelatinous zooplanktons and/or jellyfish. Also, the said aquatic animals include soft bodied organisms/ animals especially invertebartes such as cephalopods, oligochaetes, annelides, nematodes, hirudineans (leeches) and aquatic snails.

The present invention allows the production of zooplankton and/or jellyfish collagen in 6 days with high yield (more than 50% protein basis), good sensory properties (snowy white) and protein content of more than 78g/ lOOg.

The obtained collagen contains more than 50 % protein basis, preferably more than 80 % protein basis, and the apparent molecular weight of the peptide between 30 and 300 kilo Dalton, and has an increased content of hydroxyproline. The present invention further encompasses a pharmaceutical and/or cosmetic composition comprising the collagen obtained by the method of the present invention, which preferably is in the form of cream, lotion eye cream, ointment or gel, sun-screen, oral administration, face mask cream, nano-particulate collagen, starting materials for encapsulation, drug-delivery, antioxidant and/or health promoting supplements. In a preferred embodiment, the composition is in the form suitable for topical, intravenous, intramuscular or oral applications, antiinflammatory medicine, and/or anti-irritant medicine. The present invention further encompasses a food composition and/or product comprising the collagen obtained by the method of the present invention. Preferably, the food composition and/or product include beverages, dairy products, confectionaries, chocolates, and functional food.

The present invention further encompasses a biomedical composition and/or product comprising the collagen obtained by the method of the present invention. Preferably, the biomedical composition and/or product include biomaterial, scaffolds, implants and matrix for tissue engineering, biomaterials fabrication, bioactive and/or drug delivery, gene therapy, biomimetic materials, artificial cells and tissues, and 3-D cell culture applications.

The present invention avoids the use of proteases, thereby achieving cost-effectivity and overcoming the halal and kosher problems. Furthermore, the present invention provides for a much faster extraction of an increased yielding, higher purity collagen from aquatic animals than the prior art. Another advantage is that aquatic animal such as zooplankton have not been reported to be sources of contaminating pathogens especially BSE and could be potentially produced and up-scaled in controlled environments.

Brief Description of Drawings

Figure 1. Yield (dry weight basis) of collagen extracted using different methods from the bell and oral arms of jellyfish.

Abbreviations: AB: acid-soluble collagen from jellyfish bell; AOA: acid-soluble collagen from jellyfish oral arms; PB: pepsin-soluble collagen from jellyfish bell; POA: pepsin-soluble collagen from jellyfish oral arms; IB: improved extraction of collagen from jellyfish bell; IOA: improved extraction of collagen from jellyfish oral arms.

Figure 2. Proximate composition of collagen extracted using different methods.

Figure 3. Improved extraction method increases hydroxyproline content in the collagen extracted.

Abbreviations: AB: acid-soluble collagen from jellyfish bell; AOA: acid-soluble collagen from jellyfish oral arms; PB: pepsin-soluble collagen from jellyfish bell; POA: pepsin-soluble collagen from jellyfish oral arms; IB: improved extraction of collagen from jellyfish bell; IOA: improved extraction of collagen from jellyfish oral arms. Figure 4. SDS-PAGE patterns of collagen from the bell and oral arms of jellyfish using different methods

Figure 5. The improved method enhanced amino acids content of collagen extracted from the jellyfish bell. Figure 6. The improved method enhanced amino acids content of collagen extracted from the jellyfish oral arms.

Figure 7. Collagens extracted using the improved procedure decreases LDH release (bell: dark green; oral arms: purple). 3T3 fibroblast cells were treated with 500 ppm of different collagens. Bovine collagen (Merck KGaA, Darmstadt, Germany) is used as standard collagen. Abbreviations: AB: acid-soluble collagen from jellyfish bell; AOA: acid-soluble collagen from jellyfish oral arms; PB: pepsin-soluble collagen from jellyfish bell; POA: pepsin-soluble collagen from jellyfish oral arms; IB: improved extraction of collagen from jellyfish bell; IOA: improved extraction of collagen from jellyfish oral arms. Examples Materials

River jellyfish (Acromitus hardenbergi) is an edible jellyfish caught extensively from a local fisherman village in Bagan Datoh, Perak. Freshly caught jellyfish weighed between 400 g to 800 g (bell) and 150 g to 350 g (oral arms) per animal. The jellyfish were stored at -20°C until used.

Chemicals

Pepsin (crystallized and lyophilized, EC 3.4.23.1, from porcine stomach mucosa) with a declared activity of 3460 Units/ mg protein was obtained from Sigma-Aldrich. All other chemicals used were of analytical grade.

Conventional extraction of collagen

Extraction of acid soluble collagen and pepsin soluble collagen was carried out according to the methods of Miura & Kimura, 1985, Nagai et al., 2000, and Hsieh et al., 2005.

Improved extraction of collagen

Jellyfish bell and oral arms tissue (herein collectively known as jellyfish tissues) were rinsed to remove superfluous mucus before storage in -20°C. Dewatering of jellyfish tissues were performed using freeze-thaw method followed by drip drying whereby the process of freeze- thaw would not exceed two times. Dewatered jellyfish tissues were cut into small 1 to 1.5 cm strips. Jellyfish tissue strips were then stirred with 1: 1 (w:v) of 0.1 M NaOH to remove non- collagenous proteins especially proteases which would affect yield quality. This procedure was repeated at least 3 times. The insoluble jellyfish tissues were thoroughly washed with tap water until faintly neutral before stirring with 1:2 (w:v) of distilled water to further aid swelling of tissue as well as to remove water soluble proteins. Upon removal of distilled water, jellyfish tissues were blended together with 1: 1 (w/v) 0.5M acetic acid using a Waring blender (Waring Laboratory Supplies, Waring Laboratory and Science, Torrington, CT). The suspension were further subjected to mechanical treatments i.e. homogenization and sonication using a stirring homogenizer (IKA, Germany) and a high frequency sonicator (PowerSonic, Korea) respectively. Insoluble tissues were re-extracted using fresh 0.5M acetic acid and this process was repeated 3 times. Soluble extracts were pooled and extensively dialysed against 1 :10 (v:v) 0.02M phosphate buffer, pH 7.2, until the pH nears neutral. The resultant precipitate was redissolved into 0.5M acetic acid and collagen was salted out by adding NaCl to a final concentration of 0.9M. Precipitate separated were further purified by redissolving into 0.5M acetic acid and subsequent dialysis against 0.1M acetic acid followed by 0.05M acetic acid and 0.025M acetic acid. Pure collagen was obtained upon lyophilization of the dialysed collagen slurry. Analyses of collagen

Yield of collagen (% w/w)

Yield was determined on dry weight basis, the results are shown in Fig. 1 Proximate analyses, visual observations and colour

Proximate analyses were determined using the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC, 2000). Ash content was determined using Method 938.08; Total lipid content using Method 948.15; Total protein content using Method 976.05; Moisture content using Method 950.01 ; whereas total carbohydrate were determined by difference. All analyses were carried out in triplicates. Visual observations for colour and texture were also noted. The results are shown in Fig. 2 and Table 1.

Table 1. Visual observation of jellyfish collagen as extracted by different methods.

Collagen samples Appearance

Acid-soluble from bell, AB Light yellow and light textured

Pepsin soluble from bell, PB Yellowish and light textured

Improved extraction from bell, IB Snowy white and fluffy

Acid-soluble from oral arms, AOA Yellowish and light textured

Pepsin soluble from oral arms, POA Yellowish and light textured

Improved extraction from oral arms, Snowy white and fluffy IOA

Amino acids composition

Amino acid composition in collagen was determined as phenylisotiocyanate (PITC) derivatives using Waters-Pico Tag Amino Acid Analyzer High Performance Liquid

Chromatography, Model: Waters 501 Millipore Corporation, USA with column size 3.9x 150 mm. Each sample was hydrolyzed with 6N HC1 at 1 10° C. for 24 hrs prior to derivatization. The amino acids compostion of collagens extracted using the conventional and improved procedure are shown in Fig. 3, 5 and 6. The extraction method of the present invention increases hydroxyproline content in the collagen.

Determination of molecular weight

Molecular weight was determined by SDS-PAGE (Laemmli, 1970) and the run was made in a 5% T gel containing 0.1% SDS. Molecular weight marker SDS-6H (Sigma) was used as the standard. Samples (4-50 μg/well) were applied to the gel and the gel was stained for protein with Coomassie Brilliant Blue R-250. The results are shown in Fig. 4.

Purity

Purity of collagen was evaluated by means of protein content, hydroxyproline content and distribution of molecular weight.

Denatu ration temperature

Denaturation temperature of collagen was determined using Differential Scanning Calorimetry as recommended by ASTM, 2008 (F 2212-08). Table 2 shows the results.

Table 2. Denaturation temperature (Tmax) of jellyfish collagens revealed by differential scanning calorimetry (DSC).

Collagen samples Extrapol. peak (°C)

Acid-soluble from bell, AB 36.11 Pepsin soluble from bell, PB 37.11

Improved extraction from bell, IB 50.86

Acid-soluble from oral arms, AOA 39.56

Pepsin soluble from oral arms, POA 42.44

Improved extraction from oral arms, 44.77

IOA

Toxicity assay

Collagens were assayed for possible toxicity effect by the evaluation of lactate dehydrogenase (LDH) release (Sigma-Aldrich, MO, USA). The results are shown in Fig. 7.

Claims

CLAIMS:

1. A process for extracting collagen from aquatic animals, comprising alkaline treatment of the aquatic animal tissue, followed by acidic treatment thereof in combination with an orderly sequence of physical and/or mechanical treatments including but not limited to pH

adjustments, homogenization, mixing as well as sonication; and precipitation of collagen using a salt solution.

2. The process of claim 1, comprising the steps of:

c) mixing the tissues with alkaline solution for at least 6 hours;

d) washing the tissues with water to eliminate residual alkaline solution;

h) performing the process of step (f) to step (g) for at least 1 repeat;

i) centrifuging the mixture at 4-5° C;

n) dissolving the collagen pellet into an acidic solution and dialysing the solution against water; o) drying the dialysed suspension with mild drying procedures including but not limited to freeze-drying, spray-drying and vacuum-drying.

3. The process of claim 2, in particular for extracting collagen from whole tissues of large zooplanktons, comprising the steps of:

c) mixing the tissues with alkaline solution for at least 6 hours;

d) washing the tissues with water to eliminate residual alkaline solution;

g) sonicating the mixture from step (e) for at least 10 min;

h) performing the process of step (f) to step (g) for at least 1 repeat;

i) centrifuging the mixture from step (f) at 4-5° C;

k) centrifuging the mixture from step (f) at 4-5° C,

1) redisolving the pellet obtained from step (j) using an acidic solution,

m) reprecipitation of collagen by introducing the collagen solution into a sodium chloride solution to precipitate collagen fibers,

o) obtaining collagen pellet;

p) dissolving the collagen pellet into an acidic solution and dialysing the solution against water; q) drying the dialysed suspension with mild drying procedures including but not limited to freeze-drying, spray-drying and vacuum-drying.

4. The process of claim 2, in particular for extracting collagen from biomass of smaller zooplanktons, comprising the steps of:

d) mixing the biomass with alkaline solution for at least 6 hours;

g) sonicating the mixture from step (e) for at least 10 min;

h) the process of step (e) to step (f) should be repeated at least 1 time;

i) centrifuging the mixture from step (h) at 4-5° C;

1) obtaining collagen fibers from step (j);

m) collecting the collagen fibers from step (k) and centrifuging the collagen for at least 20 minutes;

n) obtaining collagen pellet and dialysing the collagen obtained against water;

5. The process of any of claims 1 to 4, which further contains the step of analysis of amino acid content in the collagen.

6. The process of claim 5, wherein the amino acids are selected from glycine, proline, hydroxyproline, alanine , arginine, hydroxylysine and serine.

7. The process according to any of preceding claims, wherein the aquatic animals are zooplankton including cnidarians from the species of hydrozoa, siphonophora, scyphozoa, ctenophore, rotatoria, pennatulacea, polychaeta with preference to edible gelatinous zooplanktons and/or jellyfish, soft bodied organisms/ animals especially invertebartes such as cephalopods.

8. Collagen obtainable by the process according to any of preceding claims.

9. The collagen according to claim 8, containing more than 50 % protein basis, preferably more than 80 % protein basis.

10. The collagen according to claim 8 or 9, having the apparent molecular weight of the peptide between 30 and 300 kilo Dalton.

11. A pharmaceutical and/or cosmetic composition comprising the collagen according to claim 8.

12. The pharmaceutical and/or cosmetic composition according to claim 1 1 which is in the form of cream, lotion eye cream, ointment or gel, sun-screen, oral administration, face mask cream, nano-particulate collagen, starting materials for encapsulation, drug-delivery, antioxidant and/or health promoting supplements.

13. The pharmaceutical and/or cosmetic composition according to claim 1 1 which is in the form suitable for topical, intravenous, intramuscular or oral applications, anti-inflammatory medicine, and/or anti-irritant medicine.

14. A food composition and/or product comprising the collagen the collagen according to claim 8.

15. The food composition and/or product according to claim 14, which include beverages, dairy products, confectionaries, chocolates, and functional food.

16. Biomedical composition and/or product comprising the collagen according to the process claims 1-7, wherein the biomedical composition and/or product include biomaterial, scaffolds, implants and matrix for tissue engineering, biofabrication, bioactive and/or drug delivery, gene therapy, biomimetic materials, artificial cells and tissues, and 3-D cell culture applications