Salmon-derived peptide and preparation method therefor

Abstract

The present invention relates to a salmon-derived peptide and a preparation method therefor and, more specifically, to a salmon-derived peptide and a preparation method therefor, in which salmon nasal cartilage, which is a waste by-product, is utilized and a biocompatible material can be obtained through acetic acid extraction.

Description

Salmon-derived peptide and method for preparing the same

[0001] The present invention relates to a salmon-derived peptide and a method for producing the same, and more specifically, to a salmon-derived peptide and a method for producing the same that utilizes salmon nasal cartilage, which is a waste byproduct, and can secure a biocompatible material through acetic acid extraction.

[0002] Proteoglycans are major components of the human body distributed in the skin, joints, cartilage, blood vessels, etc. They are mucinous mucopolysaccharides (protein polysaccharides) in which glycosaminoglycans (GAGs) are covalently bonded to a core protein in a specific form, like stems and branches. Proteoglycans are classified into dicorin, biglycan, versican, testican, perecan, agrican, neurocan, brevican, CD44, CSPG4, CSPG5, platelet factor 4, SMC3, fibromodulin, lumican, keratocan, syndecan, etc.

[0003] A process for extracting proteoglycans from raw salmon cartilage is being developed. Korean Registered Patent No. 10-2464344 discloses a cosmetic composition for preventing personalized pigmentation of the dtc gene containing salmon-derived proteoglycans as an active ingredient, and Korean Published Patent No. 10-2021-0110669 discloses a method for manufacturing a proteoglycan-containing composition and a proteoglycan-containing composition.

[0004] However, since hot water extraction processes at high temperatures of 80°C or higher or NaOH-containing alkaline extraction processes are currently being applied, there is a need to develop an extraction process that can provide high-quality, high-purity, and powdered proteoglycans while utilizing components that are harmless to the human body and increasing the economic efficiency and effectiveness of the extraction process.

[0005] The present invention aims to provide a new manufacturing method for extracting peptides (i.e., proteoglycans) from salmon nasal cartilage using a low-temperature process and a harmful substance-free process, and to expand the application fields by pulverizing the extracted peptides and forming them into complexes.

[0006] As a means to achieve the above-mentioned objective, the composition of the present invention comprises the steps of: obtaining a dried powder of salmon nasal cartilage; immersing the dried powder in a 3.0 to 6.0 wt% acetic acid solution and soaking it at room temperature for 2 to 3 days; neutralizing the cartilage, which has an acidic pH, by washing it several times with purified water after the soaking step; immersing the cartilage in a 5 wt% saline solution at a weight ratio of 5 to 30 times (based on the mass of the cartilage), grinding it with a grinder, and then extracting proteoglycans by stirring at 3 to 4 ℃ for 2 to 5 days; obtaining a filtrate by vacuum filtration of the proteoglycan extract through a diatomaceous earth filter; and obtaining a primary purified solution by purifying the filtrate using a TFF system (Tangential Flow Filtration (TFF) system). It is preferable to include the following steps: removing salt by dialyzing the above primary purified solution with stirring in deionized water at 3 to 4 ℃ for 2 to 5 days using a cellulose membrane (SpectraPor Dry Standard RC); obtaining a dried product by freeze-drying the solution after dialyzing is completed; redissolving the dried product in deionized water and adding ammonium sulfate to saturate 30 to 80 wt% to form an insoluble precipitate and remove salt; obtaining a secondary purified solution of the supernatant from which the precipitate has been removed by centrifugation; removing salt by dialyzing with stirring in deionized water at 3 to 4 ℃ for 2 to 5 days using a cellulose membrane; obtaining a proteoglycan concentrate by concentrating the solution after dialyzing under reduced pressure; and freeze-drying the concentrate.

[0007] The composition of the present invention is such that the peptide is a proteoglycan with a molecular weight (Mw) of 300 to 700 kDa, and the step of obtaining the dried powder of the salmon nasal cartilage comprises: a step of extracting the nasal cartilage from a frozen salmon head and grinding it using a grinder; a step of immersing the ground cartilage in a 70 to 99 wt% acetone solution at a weight ratio of 5 to 20 times for 2 to 5 days and performing a first drying step at room temperature; and a step of pouring a mixture of a 70 to 99 wt% acetone solution, a 70 to 99 wt% ethanol solution, and a 3.0 to 6.0 wt% acetic acid solution (7 : 1 to 2 : 0.5 to 1 (v / v)) onto the first dried ground cartilage and immersing it at a temperature of 5 to 10 ℃ and performing a second drying step at room temperature. The step of obtaining the concentrate may include [the following], and may involve concentrating under reduced pressure at 40°C or lower.

[0008] The composition of the present invention further comprises, after the step of obtaining the concentrate, the step of adding and mixing an additive comprising water-soluble silicon; PEG-60 hydrogenated castor oil, caprylyl / capryl glucoside and Tween 80; and a compound represented by the following chemical formulas 1 and 2; and hyaluronic acid to the concentrate; the step of adding purified water and ultrasonically stirring; and the step of concentrating under reduced pressure; wherein the mixing ratio of the water-soluble silicon; PEG-60 hydrogenated castor oil, caprylyl / capryl glucoside and Tween 80; the compound represented by chemical formulas 1 and 2; and hyaluronic acid is 0.5~1 : 0.1~1 : 0.2~0.5 : 1~2 (w / w), and the mixing ratio of the concentrate and the additive is 1 : 0.1~0.2 (w / w).

[0009] [Chemical Formula 1]

[0010]

[0011] [Chemical Formula 2]

[0012]

[0013] The composition of the present invention is such that the water-soluble silicon is silicic acid derived from an anisotropic fossil, igneous rock, or rock; silicate, and Y x Si(OH) 4-x (Y is (Cl-C4)-alkyl, (C2-C5)-alkenyl, (Cl-C4)-alkoxy, or amino, and x is 0 to 2) may include at least one of these.

[0014] The present invention provides a new method for extracting peptides from salmon nasal cartilage, and has the effect of expanding the fields of application by pulverizing the extracted peptides (i.e., proteoglycans) and forming them into complexes.

[0015] The present invention can provide a method for producing salmon-derived peptides, comprising an extraction process using acetic acid harmless to the human body for the development of biocompatible materials, a process for removing impurities and extracting a target molecular weight peptide (i.e., proteoglycan) through an ultrafiltration process, and a process for extracting a peptide (i.e., proteoglycan) of high purity and concentration through rotary evaporation concentration.

[0016] The present invention can provide a method for producing salmon-derived peptides using an extraction solvent or treatment solvent that is safe for the human body and room temperature or low temperature.

[0017] The present invention can provide a method for producing a salmon-derived peptide (i.e., proteoglycan) that utilizes salmon nasal cartilage, a waste byproduct, and secures a biocompatible material through acetic acid extraction.

[0018] Figure 1 shows the results of a component analysis of a salmon-derived peptide obtained in an embodiment of the present invention according to one embodiment.

[0019] Figure 2 shows the cytotoxicity of a salmon-derived peptide obtained in an embodiment of the present invention according to one embodiment.

[0020] Figure 3 shows the collagen expression effect of a salmon-derived peptide obtained in an embodiment of the present invention according to one embodiment.

[0021] Hereinafter, the method for preparing salmon-derived peptides according to the present invention will be described in detail with reference to the examples and drawings. However, the present invention is not limited to these examples and drawings.

[0022] The method for producing salmon-derived peptides according to the present invention may include a raw material pretreatment process, a soft tissue process, an extraction process, a primary purification process of the extract through ultrafiltration and dialysis, and a process for obtaining a final extract through secondary purification, concentration, and freeze-drying. That is, the present invention can provide a high-purity and high-concentration peptide extract (i.e., proteoglycan extract) through a soft tissue process using acetic acid immersion, an extraction process, primary purification through ultrafiltration and dialysis, secondary purification, and concentration / freeze-drying.

[0023] Example 1

[0024] Nasal cartilage is extracted from a salmon head in a frozen state (-20 ℃ rapid freezing) and finely ground using a grinder. Additionally, the extraction was performed in the aforementioned low-temperature environment (approx. 4 ℃), and the weight of the extracted raw cartilage was measured. The grinding was carried out using a rotary stator type homogenizer (approx. 4 ℃, 1000 rpm, 1 min homogenization). The finely ground cartilage is immersed in a 99 wt% acetone solution at a 10-fold weight ratio for 3 days to remove oil from the cartilage, and then completely dried at room temperature (approx. 25 ℃). The weight of the dried cartilage is measured. The dried cartilage is immersed in a 3.0–6.0 wt% (preferably 4.5 wt%) acetic acid solution and left at room temperature (approx. 25 ℃) for 3 days. After 3 days, the cartilage, which has an acidic pH, is neutralized by washing it several times with purified water. Specifically, a first wash with deionized water, a second wash, and a third wash with weakly alkaline water were performed. Soft cartilage was immersed in a 10 wt% saline solution at a 20-fold weight ratio, and proteoglycans were extracted by stirring at 4°C for 3 days. Since smaller cartilage particle sizes are advantageous, the particles may be further finely ground using a grinder within the saline solution. After 3 days, the extracted solution is translucent and has a slight viscosity. The proteoglycan extract was vacuum-filtered through a diatomaceous earth filter to obtain a filtrate (a translucent filtrate). The filtrate was purified using a Tangential Flow Filtration (TFF) system to obtain a primary purified solution. Salts were removed by dialyzing using a cellulose membrane (SpectraPor Dry Standard RC) in deionized water at approximately 4°C for 3 days while stirring. The dialysate was exchanged three times a day. The solution after dialysis is freeze-dried (for 40 hours) to obtain a dried product and weigh it. The dried product is redissolved in deionized water, and ammonium sulfate is added to saturate it to about 70 wt% to form an insoluble precipitate and remove the salt.A secondary purified solution is obtained by centrifuging at 10,000 rpm for 20 minutes in a high-speed centrifuge to remove the precipitate and obtain the supernatant. Salts are removed by dialyzing with stirring in deionized water at approximately 4°C using a cellulose membrane for 3 days. The dialysate is exchanged three times a day. The solution after dialyzing is concentrated under reduced pressure using a rotary evaporator to obtain a proteoglycan concentrate. At this time, the temperature of the water bath is maintained at approximately 40°C or lower (approx. 37°C) to prevent denaturation of the proteoglycans. The concentrate is freeze-dried to obtain the final product.

[0025] In each process, freeze-drying was performed by first freeze-drying at -50 ℃ (below zero) for 20 hours under atmospheric pressure, second freeze-drying at -40 ℃ (below zero) for 10 hours under vacuum, and third freeze-drying at -20 ℃ (below zero) for 10 hours under vacuum. The total drying time is 40 hours.

[0026] For characteristic analysis, a total of 5 batch extractions and molecular weight analysis were performed.

[0027] The molecular weight and composition of the peptide obtained in Example 1 were analyzed. The results are shown in Table 1 and Figure 1. In Figure 1, the proteoglycan extract components were identified as in the standard sample (STD, commercially available proteoglycan product).

[0028] The molecular weight of the proteoglycan obtained in Example 1 was measured by GPC, and it was confirmed that the low molecular weight extraction had an average molecular weight of 300 to 700 kDa for 5 batches of proteoglycan.

[0029] Mn (g / mol) Number average molecular weight Mw (g / mol) Mass average molecular weight Mz (g / mol) Volume average molecular weight Mz+1 (g / mol) Volume+1 average molecular weight PD Polydispersity 1385,864500,640800,0591,487,3991.29

[0030] The cytotoxicity of the peptide obtained in Example 1 (based on ISO 10993-5) was evaluated. The results are shown in Figure 2. The results of batch 3 and batch 4 confirmed no cytotoxicity in 1% proteoglycan.

[0031] The pro-collagen expression effect of the peptide obtained in Example 1 was evaluated. The results are shown in Figure 3. Maximum efficacy was confirmed at 0.1 wt% and 0.25 wt%, which can be seen as superior to commercial products.

[0032] Example 2

[0033] The procedure was carried out in the same manner as in Example 1, except that an additive containing water-soluble silicon 1; PEG-60 hydrogenated castor oil, Caprylyl / Capryl Glucoside and Tween 80; a compound represented by Chemical Formula 1 and Chemical Formula 2 (1:1 (w / w)); and hyaluronic acid in a mixing ratio of 0.5 : 0.1 : 0.2 : 2 (w / w) and purified water were added to the concentrate and ultrasonically stirred to obtain a concentrate. A freeze-dried peptide complex powder was obtained. The mixing ratio of the concentrate, the additive, and the purified water is 1 : 0.1 : 0.3 (w / w).

[0034] Water-soluble silicon 1

[0035] Choline chloride was treated with dry hydrochloric acid. Silicon tetrachloride (IV) was added to the formed choline solution (SiCl4 to choline chloride ratio: 1 mol per 1–5 mol of SiCl4). The resulting solution was hydrolyzed by adding water (ice / ice water) while cooling within a temperature range of -10 to -30°C. Sodium hydroxide was added to the solution to neutralize it, and the temperature was maintained below 0°C. The final pH was 1–1.5. The pH was measured using a Knick pH analyzer, MS A405 type with a Memosens pH electrode, commercially available from Stratos, equipped with an Ag / AgCl2 reference system and a liquid KCl electrolyte. After purification with activated carbon, the precipitate was removed by filtration along with the activated carbon. The water concentration was reduced by distillation under vacuum until a formulation containing 2.0–4% silicon by volume, 60–80 wt% choline chloride by weight, and 15–30 wt% water by weight was obtained. The precipitate formed during distillation was removed by filtration.

[0036] [Chemical Formula 1]

[0037]

[0038] [Chemical Formula 2]

[0039]

[0040] Example 3

[0041] The procedure was carried out in the same manner as in Example 1, except that an additive containing water-soluble silicon 2 (silicate (Na2SiO3 10H2O)); PEG-60 hydrogenated castor oil, caprylyl / capryl glucoside and Tween 80; a compound represented by Chemical Formula 1 and Chemical Formula 2 (1:1 (w / w)); and hyaluronic acid in a mixing ratio of 0.5 : 0.1 : 0.2 : 2 (w / w) and purified water were added to the concentrate and ultrasonically stirred to obtain a concentrate. A freeze-dried peptide complex powder was obtained. The mixing ratio of the concentrate, additive, and purified water is 1 : 0.1 : 0.3 (w / w).

[0042] The present invention can provide high-purity and low-molecular-weight proteoglycan extracts by applying a low-temperature environment and process components (i.e., solvents) that are harmless to the human body. In addition, the present invention can provide powdered proteoglycan extracts and complexes of proteoglycan extracts that can be utilized in cosmetics, detergents, pharmaceuticals, etc.

[0043] The present invention utilizes salmon nasal cartilage, a waste byproduct, and is used in industrial fields where biocompatible materials can be secured through acetic acid extraction.

Claims

1. A step of obtaining dry powder of salmon nasal cartilage; A step of immersing the above-mentioned dry powder in a 3.0~6.0 wt% acetic acid solution and then soaking it at room temperature for 2~3 days; A step of neutralizing the pH-acidic cartilage by washing it several times with purified water after the above immersion step; A step of immersing the cartilage in a 5 wt% saline solution at a weight ratio of 5 to 30 times (based on the mass of the cartilage), grinding it with a grinder, and then extracting proteoglycans by stirring at 3 to 4 ℃ for 2 to 5 days; A step of obtaining a filtrate by filtering a proteoglycan extract under reduced pressure in a diatomite filter; A step of obtaining a primary purified solution by purifying the above filtrate using a TFF system (Tangential Flow Filtration (TFF) system); A step of removing salts by dialyzing the above primary purified solution with stirring for 2 to 5 days in deionized water at 3 to 4 ℃ using a cellulose membrane (SpectraPor Dry Standard RC); A step of obtaining a dried product by freeze-drying the dialysis-completed solution; A step of redissolving the above dried material in deionized water, adding ammonium sulfate to a saturated concentration of 30 to 80 wt%, and then forming an insoluble precipitate to remove the salt; A step of obtaining a secondary purified solution of the supernatant from which the precipitate has been removed by centrifugation; A step of removing salt by dialysis with stirring in deionized water at 3–4 ℃ using a cellulose membrane for 2–5 days; A step of obtaining a proteoglycan concentrate by concentrating the dialysis-completed solution under reduced pressure; and A method for preparing a salmon-derived peptide, comprising the step of freeze-drying the above concentrate.

2. In Paragraph 1, The above peptide is a proteoglycan with a molecular weight (Mw) of 300 to 700 kDa, and The step of obtaining the dried powder of the salmon nasal cartilage is, A step of extracting nasal cartilage from a frozen salmon head and grinding it using a grinder; A step of immersing the crushed cartilage in a 70–99 wt% acetone solution at a weight ratio of 5–20 times for 2–5 days and performing primary drying at room temperature; and The method comprises the step of pouring a mixture of 70-99% acetone solution, 70-99 wt% ethanol solution, and 3.0-6.0 wt% acetic acid solution (7 : 1-2 : 0.5-1 (v / v)) onto the primary dried pulverized cartilage, immersing it at a temperature of 5-10 ℃, and secondary drying it at room temperature; A method for producing a salmon-derived peptide, wherein the step of obtaining the above concentrate is to concentrate under reduced pressure at 40°C or lower.

3. In Paragraph 1, After the step of obtaining the above concentrate, A step of adding and mixing an additive comprising water-soluble silicon; PEG-60 hydrogenated castor oil, caprylyl / capryl glucoside and Tween 80; a compound represented by the following chemical formulas 1 and 2; and hyaluronic acid to the concentrate; The method further comprises the steps of: adding purified water and ultrasonically stirring; and concentrating under reduced pressure. The mixing ratio of the above water-soluble silicon; PEG-60 hydrogenated castor oil, caprylyl / capryl glucoside and Tween 80; compounds represented by Chemical Formula 1 and Chemical Formula 2; and hyaluronic acid is 0.5~1 : 0.1~1 : 0.2~0.5 : 1~2 (w / w), and A method for preparing a salmon-derived peptide, wherein the mixing ratio of the above concentrate and additive is 1 : 0.1~0.2 (w / w): [Chemical Formula 1] [Chemical Formula 2] 4. In Paragraph 3, The above water-soluble silicon is silicic acid derived from fossils, igneous rocks, or rocks; silicates, and Y x Si(OH) 4-x A method for preparing a salmon-derived peptide comprising at least one of (Y is (Cl-C4)-alkyl, (C2-C5)-alkenyl, (Cl-C4)-alkoxy, or amino, and x is 0 to 2).

5. A salmon-derived peptide produced by the method of any one of paragraphs 1 to 4.

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