Denatured collagen solution and the preparation method thereof

Abstract

The invention relates to a denatured collagen solution obtained from fish waste, preferably sea bass, salmon, sea bream, and the preparation method thereof. By means of this production method, fish wastes that cause environmental pollution are evaluated, thus preventing environmental pollution. The denatured collagen solution with green content produced at low cost, which is the subject of the invention, is used in cosmetic products, preferably hair and skin care products.

Description

[0001] DENATURED COLLAGEN SOLUTION AND THE PREPARATION METHOD THEREOF

[0002] Technical Field of the Invention

[0003] The invention relates to a denatured collagen solution obtained from fish waste and the preparation method thereof. Utilizing this preparation method, fish wastes that cause environmental pollution are evaluated, thus preventing environmental pollution. The denatured collagen solution produced at low cost, which is the subject of the invention, is used in cosmetic products.

[0004] The State of the Art

[0005] Collagen, as a tissue composition, is a water-insoluble protein with high tensile strength and is known as tendons, fibroblasts, odontoblasts in teeth, and specialised cells that synthesise collagen. Collagen, the main component of connective tissue in the body, constitutes approximately 1 / 3 of the total body proteins, and due to its high tensile strength, this protein is often used in the body to make ligaments and tendons

[0001] . Collagen primarily produces protein membranes around tissues and organisms, and degradation of the protein causes wrinkles with ageing due to its role in the strength and elasticity of the skin [2], Although the human body can naturally produce collagen, there is a decrease in this production due to advancing age, therefore, it is possible to benefit from the advantages it provides to the body by adding foods containing the materials necessary for the production of collagen that the body needs to the diet or by using collagen-containing cosmetic products due to their moisturising, regenerating and film-forming properties.

[0006] Collagen is a primary ingredient in many cosmetic formulations because it is a natural moisturiser. In addition to being found in a wide range of sources, from bovine and porcine by-products to marine sources, bovine and porcine collagen is associated with the risk of zoonotic diseases such as TSE (transmissible spongiform encephalopathy), BSE (bovine spongiform encephalopathy) and FMD (Foot Mouth Disease) [3]. However, marine collagen represents a crucial distinction from other animal sources of this protein. Fish, one of the marine collagen sources, is considered the most valuable collagen source, and the main reason for this is that the collagen content of a fish constitutes 75% of its weight [4], Using fish in collagen production requires first mincing the fish skin and stirring it in alkaline solution for 24 hours, the aim being to remove non-collagenous protein. In the next step, the mixture is filtered through a sieve as necessary to remove mechanical impurities, or alternatively, homogenised with a suitable acidic solution, followed by stirring for another 24 hours. Afterwards, centrifugation is performed, and the resulting supernatant is collected. After the supernatant is extracted once again with a suitable acidic solution, acid-soluble collagen is obtained. There are two additional different approaches required for collagen in this form to be applicable. One of these is homogenisation, 24-hour stirring and pepsin application, followed by 24-hour stirring, centrifugation and collection of the supernatant, followed by application of alkaline solution and repeating the stirring process. Next, there is the need to suspend the precipitate in the correct buffer to obtain the appropriate pH value, which should be equal to 7.4, and the need to dialyse the prepared sample against the same buffer and centrifuge it. If pure collagen powder is the final product after centrifugation, the sample must undergo a drying process, for example lyophilisation. The second method comprises directly adding the alkaline solution from the primarily collected supernatant [5]. However, as mentioned, these techniques require treatment with more than one chemical and, accordingly, the production time and cost increase.

[0007] In the state of the art, the use of collagen derived from fish waste (e.g., skin, scales) is rapidly expanding due to its distinct advantages over mammal-based collagen, including reduced risk of zoonotic transmission, lack of cultural-religious limitations, cost-effectiveness of the production process, and superior bioavailability. In addition, the low cost of by-products and the need to minimise the environmental impact of industrial wastes paves the way for the use of fish waste in the development of collagen-based products with significant added value [6].

[0008] The patent application numbered CN103725738A in the state of the art relates to the method of preparing collagen polypeptides using residues of Larimichthys crocea, a fish species. In this method, fish scales, fish skins and internal organs are used. To obtain fish waste collagen, activated Bacillus coagulants is inoculated into fermentation broth for 24 to 48 hours at 37-45 °C. Fermentation broth comprises 2-5% fish scale, 1 - 4% fish skin, 3-6% fish internal organs, 0.5% yeast extract, 0.2% dipotassium hydrogen phosphate, 0.2% ammonium citrate, 0.5% sodium acetate, 2% glucose, magnesium sulphate (MgSC ■ 7H2O) 0.058%, manganese sulphate (MnSO4'4H2O) 0.025%, Tween 80 1.0 ml / L, and pH 6.2-6.4 water. Although said method describes the production of collagen from fish residues, the final product obtained in said method is not environmentally friendly due to the use of too many chemicals, and the cost of the method is also high.

[0009] Due to reasons such as the limitations and deficiencies of existing technical solutions, the methods used in collagen production being not environmentally friendly and requiring high costs, causing the formation of chemical waste, as well as the length of the traditional production process and the very low efficiency (5-10%), it has become necessary to make an improvement in the collagen production method.

[0010] Brief Description and Aims of the Invention

[0011] In the invention, a denatured collagen solution obtained from fish waste and the preparation method thereof are explained. By means of this preparation method, fish wastes that cause environmental pollution are evaluated, thus preventing environmental pollution. The denatured collagen solution with green content produced at low cost, which is the subject of the invention, is used in cosmetic products.

[0012] The aim of the invention is to provide denatured collagen solution with green content at low cost. The denatured collagen solution of the invention has a clean content, as it does not use any enzymes or chemicals in its preparation method, and can be produced at low cost, as it eliminates the need for the use of such high-cost components.

[0013] Another aim of the invention is to prevent environmental pollution. In the invention, denatured collagen solution is produced using fish waste, thus preventing these wastes from causing environmental pollution.

[0014] Another aim of the invention is to increase the efficiency of denatured collagen production. In the method of the invention, the efficiency reaches 30% by means of the green technology production systems - high pressure and ultrasound - used. Detailed Description of the Invention

[0015] The invention relates to a denatured collagen solution obtained from fish waste, preferably sea bass, salmon, sea bream, and the preparation method thereof. By means of this preparation method, fish wastes that cause environmental pollution are evaluated, thus preventing environmental pollution. The denatured collagen solution with green content produced at low cost, which is the subject of the invention, is used in cosmetic products, preferably hair and skin care products, as well as nutritional supplements.

[0016] The denatured collagen solution with green content, which is the subject to the invention, contains 10% (g / ml) collagen and 90% water. Said collagen protein contains the amino acids glycine, hydroxyproline and proline.

[0017] The method of preparing the denatured collagen solution that is the subject of the invention comprises the process steps of; i. pre-treating fish skin, bones and / or head wastes in pure water and ultrasound bath, ii. drying the pre-treated wastes, iii. keeping the dried wastes in the supercritical extraction device, iv. removing the waste kept in the device from the device and collecting the oil, v. keeping the waste remaining in the device in pure water, vi. reducing the pressure inside the device and keeping it when the temperature reaches 150-180°C, vii. collecting the collagen solution by taking flow from the device at a constant speed, and viii. drying the collagen solution.

[0018] In another embodiment of the invention, the method of preparing the denatured collagen solution that is the subject of the invention comprises the process steps of; i. pre-treating fish skin, bone and / or head wastes in pure water and 10-50 Hz ultrasound bath at 30-40 °C for 15-60 minutes, with a ratio of 1 gram: 10 ml to water, ii. drying the pre-treated wastes at 40-70°C for 30-120 minutes, iii. keeping the dried wastes in a supercritical extraction device for 2-3 hours at 40-70 °C and 250-350 bar pressure, iv. removing the wastes kept in the device from the device for 1 -3 hours and collecting the oil, v. Keeping the waste remaining in the device for 1 -2 hours at 40-70 °C and 300-400 bar pressure with pure water such that the ratio of the remains to water is 1 gram: 10 ml or 1 gram: 5 ml, vi. reducing the pressure inside the device to 60-80 bar and keeping it for 3-10 minutes when the temperature reaches 150-180 °C, vii. collecting the collagen solution by taking flow from the device at a constant speed, and viii. drying the collagen solution.

[0019] In another embodiment of the invention, the method of preparing the denatured collagen solution that is the subject of the invention comprises the process steps of; i. pre-treating fish skin, bone and / or head wastes in pure water and 20 Hz ultrasound bath at 40 °C for 1 -60 minutes, with a ratio of 1 gram: 10 ml to water, ii. drying the pre-treated wastes at 60 °C for 2 hours, iii. keeping the dried wastes in a supercritical extraction device for 2.5 hours at 60 °C and 250 bar pressure, iv. removing the wastes kept in the device from the device for 1 hour and collecting the oil, v. Keeping the waste remaining in the device for 1 hour at 60 °C and 320 bar pressure with pure water such that the ratio of the remains to water is 1 gram: 10 ml or 1 gram: 5 ml, vi. reducing the pressure inside the device to 80 bar and keeping it for 5 minutes when the temperature reaches 180 °C, vii. collecting the collagen solution by taking flow from the device at a constant speed, and viii. drying the collagen solution. In the invention, sea bass, salmon or sea bream can be used as a collagen source in process step (i).

[0020] In the method that is the subject of the invention, in the process step (ii), the wastes are dried in a vacuum dehydrator. Additionally, the wastes can be pulverised after the process step (ii) and before the process step (iii). In addition, in the process step (viii) of the method that is the subject of the invention, the drying process is carried out with a lyophiliser or spray dryer.

[0021] In the method of the invention, denatured collagen solution is produced without the use of any chemicals or enzymes, thus ensuring that the product obtained has clean (green) content and reducing the cost of the method.

[0022] References

[0023] [1] The predominant role of collagen in the nucleation, growth, structure and orientation of bone apatite. Wang Y, AzaTs T, Robin M, et al. Nat Mater. 2012;11 :724-733.

[0024] [2] Effect of collagen hydrolysate in articular pain: a 6-month randomized, double-blind, placebo controlled study. Bruyere 0, Zegels B, Leonori L, Rabenda V, Janssen A, Bourges C, Reginster JY. Complement Ther Med. 2012;20:124-130.

[0025] [3] Alves A.L., Marques A.L.P., Martins E., Silva T.H., Reis R.L. Cosmetic potential of marine fish skin collagen. Cosmetics. 2017;4:39. doi: 10.3390 / cosmetics4040039.

[0026] [4] Senaratne L., Park P.-J., Kim S.-K. Isolation and characterization of collagen from brown backed toadfish (Lagocephalus gloveri) skin. Bioresour. Technol. 2006;97:191 -197. doi: 10.1016 / j.biortech.2005.02.024.

[0027] [5] Hema G., Shyni K., Mathew S., Anandan R., Ninan G. A simple method for isolation of fish skin collagen- biochemical characterization of skin collagen extracted from Albacore Tuna (Thunnus Alalunga), Dog Shark (Scoliodon Sorrakowah), and Rohu (Labeo Rohita) Ann. Biol. Res. 2013;4:271 -278.

[0028] [6] F;, R. Z. N. L. (n.d.). Collagen derived from fish industry waste: Progresses and challenges. Polymers, https: / / pubmed.ncbi.nlm.nih.gov / 36771844 /

Claims

CLAIMS1. A collagen solution comprising 10% (g / ml) denatured collagen and 90% water.

2. Collagen solution according to Claim 1 , wherein said collagen comprises the amino acids glycine, hydroxyproline and proline.

3. A method of preparing the denatured collagen solution, comprising the process steps of: i. pre-treating fish skin, bones and / or head wastes in pure water and ultrasound bath, ii. drying the pre-treated wastes, iii. keeping the dried wastes in the supercritical extraction device, iv. removing the waste kept in the device from the device and collecting the oil, v. keeping the waste remaining in the device in pure water, vi. reducing the pressure inside the device and keeping it when the temperature reaches 150-180°C, vii. collecting the collagen solution by taking flow from the device at a constant speed, and viii. drying the collagen solution.

4. A method according to claim 3, comprising the process steps of: i. pre-treating fish skin, bone and / or head wastes in pure water and 10-50 Hz ultrasound bath at 30-40 °C for 15-60 minutes, with a ratio of 1 gram: 10 ml to water, ii. drying the pre-treated wastes at 40-70 °C for 30-120 minutes, iii. keeping the dried wastes in a supercritical extraction device for 2-3 hours at 40-70 °C and 250-350 bar pressure, iv. removing the wastes kept in the device from the device for 1 -3 hours and collecting the oil, v. Keeping the waste remaining in the device for 1 -2 hours at 40-70 °C and 300-400 bar pressure with pure water such that the ratio of the remains to water is 1 gram: 10 ml or 1 gram: 5 ml, vi. reducing the pressure inside the device to 60-80 bar and keeping it for 3- 10 minutes when the temperature reaches 150-180 °C,vii. collecting the collagen solution by taking flow from the device at a constant speed, and viii. drying the collagen solution.

5. A method according to claim 4, comprising the process steps of: i. pre-treating fish skin, bone and / or head wastes in pure water and 20 Hz ultrasound bath at 40 °C for 1 -60 minutes, with a ratio of 1 gram: 10 ml to water, ii. drying the pre-treated wastes at 60 °C for 2 hours, iii. keeping the dried wastes in a supercritical extraction device for 2.5 hours at 60 °C and 250 bar pressure, iv. removing the wastes kept in the device from the device for 1 hour and collecting the oil, v. Keeping the waste remaining in the device for 1 hour at 60 °C and 320 bar pressure with pure water such that the ratio of the remains to water is 1 gram: 10 ml or 1 gram: 5 ml, vi. reducing the pressure inside the device to 80 bar and keeping it for 5 minutes when the temperature reaches 180 °C, vii. collecting the collagen solution by taking flow from the device at a constant speed, and viii. drying the collagen solution.

6. A method according to claims 3-5, wherein the fish waste mentioned in the process step (i) is sea bass, salmon or sea bream.

7. A method according to claims 3-5, wherein the drying process mentioned in the process step (viii) is carried out with a lyophiliser or spray dryer.

8. Denatured collagen solution produced by a method according to claim 7.

9. Use of a collagen solution according to claim 2 or 8 in cosmetic products.

10. Hair, skin care product or nutritional supplement composition comprising a collagen solution according to claim 2 or 8.