KR20150136802A - Recovery of collagen peptide derived from fish bone and skin using pressurized hydrothermal hydrolysis - Google Patents

Abstract

The present invention relates to a method for obtaining and retrieving collagen hydrolysate from fish bones and shells through a reaction with high-temperature high-pressure water. According to the present invention, the method ensures efficiency in a process and economicality by selecting a simple process while having eco-friendliness and safety as well. The method also enables rapid production of collagen which is applied as a functional ingredient in food, medicine, and cosmetics, thereby creating added value by being applied to medical fields, bioindustries, cosmetic industries, and food industries.

Description

COLLAGEN PEPTIDES FROM FISH BONE AND SKIN USING PRESSURE SEPTIC HEAT HYDROPOLYY [0002] RECOVERY OF COLLAGEN PEPTIDE DERIVED FROM FISH BONE AND SKIN USING PRESSURIZED HYDROTHERMAL HYDROLYSIS [0003]

The present invention relates to a method for recovering a functional ingredient from a fish, and more particularly, to a method for recovering and obtaining a functional ingredient, collagen, from bone and skin of a fish using a pressurized hydrothermal reaction.

Collagen is a major component of cell matrix matrix of animal and connective tissue including humans. Collagen is present in various forms. Typical collagen type, Collagen I, forms a triple helical structure in which three polypeptide chains with a molecular weight of about 100,000 are twisted (tropo collagen). Specifically, collagen has a structure in which "glycine-proline-X" or "glycine-X-hydroxyproline" (X is an arbitrary amino acid) is repeated. In the biotissue, an organic solvent is used for extraction, followed by acid or alkali treatment, and then an appropriate enzyme such as trypsin or hyaluronidase is applied to obtain insoluble collagen.

Gelatin obtained by extracting collagen with respect to food can be used as a coagulant such as used in jelly. In addition, collagen improves immune function, especially strengthens joints by promoting the regeneration of bone cells through absorption of calcium through bone tissue, and exerts excellent effects on skin beauty through activation of metabolism of skin and maintenance of moisture. It is utilized as a functional ingredient of medicines and cosmetics. In particular, a large amount of a component for promoting the synthesis of collagen is contained as a component of functional cosmetics for improving wrinkles of skin caused by aging or ultraviolet rays. In addition, collagen has excellent bio-compatibility and bio-degradability and is widely used as biomaterial for tissue regeneration.

As such, collagen required in various industries such as food, cosmetics, and biologic transplantation and other functional ingredients and materials is currently being used mainly from bones and shells of land animals such as cows and pigs. However, in recent years there have been worldwide epidemics caused by terrestrial animals and birds, such as foot-and-mouth disease (FMD), avian influenza (AI) and mad cow disease There is a problem of safety. Since the pig is an animal that is disgusted by Muslim origin, collagen obtained from pigs is a material that can not be used in certain religions, so it is required to develop a technology to produce alternative collagen.

On the other hand, many consumers are interested in health functional foods or health supplements derived from natural products that enhance immunity. As described above, collagen is involved in various physiological activities such as enhancing immunity in the human body. Collagen having a relatively high molecular weight can not be absorbed in the living body because it can not penetrate deeply into living body. Therefore, in order to efficiently infiltrate the collagen into the living body, it is required to develop a technique capable of producing a collagen peptide having a relatively small molecular weight.

It is an object of the present invention to provide a method for recovering and obtaining a collagen peptide and a hydrolyzate containing an amino acid from a fish such as sea anemone .

Another object of the present invention is to provide a method for recovering collagen hydrolyzate derived from fish, which can be economically achieved through a simple process and is environmentally friendly and safe.

It is still another object of the present invention to provide a method for producing a collagen hydrolyzate derived from fish which is easy to absorb and penetrate into the human body.

The present invention having the above-mentioned object is characterized by comprising the steps of: drying at least one sample out of bones and shells of prepared mackerel; Pulverizing the dried sample; Adding a proteolytic enzyme to the pulverized sample to separate collagen; And recovering the collagen hydrolyzate obtained by reacting the powder of the separated collagen with water at a high temperature and high pressure, to obtain a collagen hydrolyzate derived from a fish.

Illustratively, in the step of recovering the collagen hydrolyzate, water at a temperature of 150 to 350 ° C and a pressure of 5 to 400 bar may be used.

The collagen hydrolyzate obtained according to the present invention may have a molecular weight of 3000 Da or less.

In one exemplary embodiment of the present invention, separating the collagen comprises grinding the bone of the mackerel, diluting the bone of the mackerel in a ratio of 1: 5 to 1:20 (w / v) Treating the mackerel bone with an alkaline solution and adding a decalcifying agent in a ratio of 1: 5 to 1:20 (w / v) to the insoluble mackerel bones obtained by treatment with an alkali solution, Degreasing treatment with 1: 5 to 1:20 (w / v) of alcohol to calcified mackerel bones, and hydrolyzing pepsin by adding pepsin to the degreased mackerel bone residue.

In another exemplary embodiment of the present invention, separating the collagen comprises grinding the shell of the mackerel, diluting the crushed shell of the mackerel at a ratio of 1:20 to 1:50 (w / v) A step of treating the insoluble mackerel bark obtained by treatment with an alkaline solution with an alcohol at a ratio of 1:20 to 1:50 (w / v) to degrease the treated mackerel bark, Followed by hydrolysis by adding pepsin.

The collagen hydrolyzate obtained according to the present invention is characterized by having antioxidative activity.

In the present invention, collagen peptide hydrolyzate having a low molecular weight was isolated using press hydrothermal hydrolysis using high temperature and high pressure water from fish bones and shells. The method of the present invention is a safe method that is simple in process and can provide sufficient economical efficiency, is environment-friendly, and has no harmful effect on human body.

Since the collagen peptide hydrolyzate finally recovered and obtained according to the present invention has a small molecular weight of 3 KDa or less and can be easily absorbed into the human body, the collagen peptide hydrolyzate can be efficiently absorbed by various physiological activity functions, for example, Is possible.

Therefore, the low molecular weight collagen hydrolyzate recovered through the method of the present invention can be added to foods, medicines, cosmetics, etc. as a functional ingredient for immunity enhancement, prevention of skin aging, bone cell regeneration, etc., .

Brief Description of the Drawings Fig. 1 is a schematic view showing a process for recovering collagen peptide hydrolyzate from bone and skin of a fish such as mackerel, which is excellent in absorption to the human body, according to an exemplary embodiment of the present invention, A flow chart schematically showing various analysis methods for examining functions.
2 is a schematic view of an apparatus used in a high temperature, high pressure hydrothermal hydrolysis process to recover collagen peptide hydrolyzate from fish bones and shells according to an exemplary embodiment of the present invention.
FIG. 3 is a photograph showing SDS-PAGE measurement results of pepsin-soluble collagen separated from mackerel bones and shells according to an exemplary embodiment of the present invention. In Fig. 3, MMP represents a molecular weight marker protein, CSC represents calf skin collagen, MSC represents mackerel skin collagen, MBC represents mackerel bone collagen, and BATC represents Achilles tendon collagen.
FIGS. 4A and 4B are graphs showing FT-IR spectrum analysis results of pepsin-soluble collagen isolated from mackerel bones and shells, respectively, according to an exemplary embodiment of the present invention.
FIG. 5 is a graph showing the results of measurement of thermal denaturation for pepsin-soluble collagen isolated from mackerel bones and shells according to an exemplary embodiment of the present invention.
6A to 6C are graphs showing MALDI-TOF mass spectrum analysis results of collagen hydrolyzate obtained by pressurized hydrothermal hydrolysis of pepsin-soluble collagen isolated from mackerel bones and shells according to an exemplary embodiment of the present invention . 6a is a collagen hydrolyzate obtained by hydrothermal hydrolysis of pepsin-soluble collagen separated from mackerel bone at 200 ° C and 30 bar, FIG. 6b shows hydrolysis of pepsin-soluble collagen separated from mackerel shell by pressurized hydrothermal decomposition at 200 ° C and 30 bar, FIG. 6C is a photograph of collagen hydrolyzate obtained by hydrothermal hydrolysis of pepsin-soluble collagen isolated from mackerel shell at 250 DEG C and 70 bar.
7A to 7D are graphs showing the results of antioxidant measurement on collagen hydrolyzate obtained by hydrothermal hydrolysis of pepsin-soluble collagen separated from bone and bark of mackerel according to an exemplary embodiment of the present invention. FIG. 7A is a DPPH free radical scavenging activity, FIG. 7B is an ABTS free radical scavenging activity, FIG. 7C is a ferric reducing power assay, and FIG. 7D is a graph showing Fe ²⁺ chelating activity.

DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies to solve the problems of the prior art by separating collagen from a sample of at least one of bones and bark of a fish and hydrolyzing the collagen by pressurized hydrothermal treatment using high temperature and high pressure water to form collagen peptide and amino acid The present inventors have completed the present invention in view of the fact that the hydrolyzate containing the hydrolyzate can be obtained and used as various industries having high potential. Hereinafter, the present invention will be described with reference to the accompanying drawings where necessary.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart schematically illustrating the process of collecting and obtaining collagen hydrolysates from fishes according to an exemplary embodiment of the present invention and analyzing them; FIG. As shown, at least one fish sample is prepared from the bones and shells of the mackerel. Mackerel ( Scomber japonicus ) is an important food ingredient consumed worldwide, is consumed through various cooking methods, and a large amount of mackerel is consumed every day. When mackerel is consumed as food, bones, bark, and viscera are treated as by-products as non-edible parts. Improper treatment of these wastes can cause environmental pollution by odor and nutrients. However, considering the high nutritional value and other industrial applicability of the functional materials contained in these by-products, the problem of environmental pollution due to these currently discarded materials is solved, and when appropriate processing techniques are introduced, It is possible to retrieve and utilize it.

In addition, as described above, recently, local and global outbreaks of foot-and-mouth disease have caused safety problems for collagen extracted from terrestrial animals. However, this problem can be solved by using collagen that exists in fish bones and shells. In particular, if the collagen hydrolyzate can be extracted from the shells and bones of the mackerel, which is currently abundantly abundant in the domestic geographical environment surrounded by the sea on the three sides, it may be preferable in terms of efficient utilization of fishery resources. Furthermore, the collagen hydrolyzate recovered from bones and shells of fish according to the present invention has the advantage of being applicable to high value-added industries as a functional material such as cosmetics, medicines and foods.

The bones and skin of prepared mackerel are thoroughly washed with water, and then the prepared samples are dried. As a method for drying the washed fish sample, a method of naturally drying in an airy shade may be used, but a method of freeze-drying the bones and bark of mackerel at a low temperature may be adopted in particular have. If the freeze-drying method is adopted, collagen contained in bones and bark of mackerel can be particularly useful because it is not deformed by heat. When the bones and bark of mackerel as a fish sample are to be dried by the freeze-drying method, the freeze-drying temperature is approximately -20 to 10 ° C, preferably -5 to 10 ° C, and the drying time is 48 to 96 hours . When lyophilized under these conditions, fish samples can be completely dried to obtain bones and skin samples of moisture-free mackerel. However, the freeze-drying temperature and the drying time may vary depending on the state of the bones and shells of the prepared mackerel.

The bone and skin samples of the dried mackerel are then cut and ground to the appropriate size. In an exemplary embodiment, the dried mackerel meat can be cut to a size of about 0.1 to 3 cm using a mechanical blend, and then pulverized into a powder form. Alternatively, the bones and skin of dried mackerel can be ground into powder form using a homogenizer.

It is then possible to separate the collagen from the bone and skin samples of the ground mackerel, which is exemplified by a suitable protease, for example a proteinase other than collagenase which completely degrades the collagen Pepsin and the like. In this exemplary embodiment, pepsin-solubilized collagen (PSC) can be isolated from the fish sample (bone and / or skin) through this step and recovered in powder form.

For example, when a fish bone such as a bone of a mackerel is used as a sample, an alkali such as crushed mackerel bones is diluted with a ratio of 1: 5 to 1:20 (w / v) Treating the solution with an alkali solution, adding a demineralizing agent to the insoluble mackerel bones obtained by treatment with an alkali solution in a ratio of 1: 5 to 1:20 (w / v), decalcifying the degreased calcined mackerel bone at a ratio of 1: 5 Deg.] C to 1:20 (w / v) alcohol, and adding pepsin to the degreased mackerel bone residue to hydrolyze it. For example, a sodium hydroxide or calcium hydroxide solution having a concentration of 0.01 to 1.0 M can be used as an alkali solution used for removing a portion without collagen. As the demineralization agent used for calcination, a demineralization method using an acid solution such as formic acid, acetic acid, picric acid or trichloroacetic acid at a concentration of 1 to 20% or a buffer solution such as a citric acid-citric acid buffer It is possible to use a histochemical deliming method such as using a complex forming a chelate with a metal, for example, a complex salt using 0.1 to 1.0 M concentration of ethylene diamine tetra acetic acid (EDTA). In the degreasing step, alcohols having 1 to 4 carbon atoms such as butyl alcohol can be used, and pepsin can be used in a form diluted with an acid such as acetic acid.

If necessary, washing with an alkaline solution, washing / neutralizing with distilled water or the like to remove alkali, washing / neutralizing with distilled water or the like to remove the demineralizer after decarcification, And a step of washing with distilled water to remove alcohol and the like after the degreasing step.

In the case of using pepsin diluted with an acid such as acetic acid, for example, thereafter, a salt such as NaCl is added to adjust the concentration of the solution, and a step of centrifuging the precipitate and dissolving the precipitate in an acid such as acetic acid , Dialyzing with acetic acid / distilled water, and lyophilizing the sample obtained by dialysis (for example, -20 to 10 ° C).

On the other hand, when a fish shell such as a mackerel's skin is used as a sample, the step of separating the collagen is carried out in such a manner that the crushed mackerel bark is diluted to a concentration of 0.01 to 1.0 M at a ratio of 1:20 to 1:50 (w / v) Of an alkali solution such as sodium hydroxide or calcium hydroxide solution, adding an alcohol having a carbon number of 1 to 4 such as butyl alcohol of 1:20 to 1:50 (w / v) to the insoluble mackerel shell obtained by treatment with an alkali solution Deg.] C, and then adding pepsin to the degreased mackerel bark residue to hydrolyze it. Like fish bones, pepsin can be used in diluted form, for example, in acids such as acetic acid.

If necessary, washing with an alkaline solution, washing / neutralizing with distilled water or the like to remove alkali, and washing with distilled water to remove alcohol and the like after the degreasing step. In the case of using pepsin diluted with an acid such as acetic acid, for example, thereafter, a salt such as NaCl is added to adjust the concentration of the solution, and a step of centrifuging the precipitate and dissolving the precipitate in an acid such as acetic acid , Dialyzing with acetic acid / distilled water, and lyophilizing the sample obtained by dialysis (for example, -20 to 10 ° C).

Collagen containing collagen type I is composed of three chains of three polypeptide chains forming a helix structure and has a structure called a telopeptide having no helical structure at both ends of the molecule. In the collagen molecule structure, the telopeptide is a part where intra-molecule and inter-molecule cross-linking are introduced, and the molecule is cross-linked into the telopeptide part of collagen in vivo to insolubilize the insulator. Treatment of this insoluble collagen with a protease such as pepsin removes the telopeptide in the cross-linked portion and obtains the digested and solubilized atelocollagen.

In the exemplary embodiment of the present invention, SDS-PAGE analysis of pepsin-soluble collagen isolated from fish samples such as bones and / or bark of mackerel through the above procedure revealed three chains (two α-chains and one β -Chain) were combined (SDS-PAGE of Example 3 and FIG. 3; FT-IR of Example 4 and FIGS. 4A and 4B), and the heat denaturation properties and viscosity were also good See Example 5). Regarding thermal denaturation, it may be related to the content of proline and hydroxyproline, which are the major amino acids of collagen, as well as imino acid, which is involved in the thermal stability of collagen.

Then, the collagen hydrolyzate is recovered and obtained by pressurized hydrothermal reaction in which high-temperature and high-pressure water is added to collagen derived from fish. Conventionally, as a method for decomposing a macromolecule derived from a living body, there are a chemical treatment using an acid, an alkali or a catalyst, and a hydrolysis using an enzyme. However, the chemical treatment requires a severe reaction condition and causes severe environmental pollution, And the quality of the product may be deteriorated. The enzymatic treatment method is complicated in the reaction process, takes a long time to complete the production cycle, and has a problem in economy.

Accordingly, in the present invention, a pressurized hydrothermal decomposition reaction using high temperature and high pressure water is used. Pure water has a boiling point of 100 ° C at atmospheric pressure, and hot water extraction is performed in this region, but the test range for temperature changes is limited without changing the pressure. However, the critical point of water is the intrinsic region present at a temperature of 374 ° C and a pressure of 22 MPa, below which the water maintains the subcritical state, while in the subcritical state the water retains the liquid state due to the high pressure. In other words, the water in this condition can be formed in the absence of the gas and liquid regions, and the conditions depending on various temperature and pressure changes can be formed, and the asymptotic coefficients in various regions exist in different physical properties.

When hydrolyzing a polymer material by using the unique physical properties such as high solubility, low dielectric constant, small interfacial tension, fast permeability, and low viscosity by converting water to a subcritical state, the hydrolysis reaction can be efficiently performed Lt; / RTI > For example, a hydrothermal reaction using sub-critical water under various conditions has much more ions than at room temperature, and these ions catalyze an acid or base. In addition, for example, due to the low dielectric constant of subcritical water, it can function as a solvent suitable for dissolving these organic compounds by reacting with a wide range of organic compounds. Thus, due to the physical and chemical properties of the subcritical condition, that is, the critical temperature and the subcritical condition, the subcriterion functions as a new reaction medium in many chemical reactions.

In particular, the hydrothermal hydrolysis process in the subcritical state is recognized as an alternative to the conventional hydrolysis process due to short reaction time, high hydrolysis rate, stability of the hydrolyzate, simplicity of the process, and environmentally friendly process have. Thus, in the present invention, by using hydrothermal hydrolysis reaction using hydrothermal hydrolysis using high-temperature and high-pressure water in the subcritical state, when the subcritical water is removed from the saturated gas region and becomes liquid, the polymeric substance is not carbonized, A pressurized hydrothermal hydrolysis reaction proceeds to obtain a product having functional properties. The submerged hydrolysis process requires no pre-treatment, has a relatively short reaction time, produces less corrosion or residue by reaction, does not require the use of a toxic solvent, Is a method for the hydrolysis of environmentally friendly and rapid biomass that can be used as an alternative to chemical or catalytic processes using existing acids or bases, due to its small size.

Illustratively, high-temperature, high-pressure water used for recovering and obtaining fish-derived collagen, for example, hydrolysates of pepsin-soluble collagen, according to the present invention is heated at a temperature of 150 to 350 ° C, preferably 220 to 260 ° C Such as a temperature of 200 to 300 캜, such as a temperature, and a pressure of 20 to 100 bar, such as a pressure of 4 to 400 bar, preferably a pressure of 30 to 70 bar.

For example, the pressurized hydrothermal hydrolysis process using high temperature, high pressure water according to the present invention can be performed using the hydrolysis apparatus exemplarily shown in FIG. 2, a pressure gauge 2, a needle valve 3, an electric heater 4, a high-pressure reactor 5, an impeller / stirrer 6, 7 is a stirring speed and temperature controller, and 8 is a sample collector. For example, a collagen sample obtained from fish-derived bones and / or shells such as mackerel is transferred from the sample collector 8 to the reactor 5, and the reactor is filled with water, such as distilled water. In an exemplary embodiment, the collagen sample and the water that can be converted to the subcritical state can be mixed and reacted in the reactor 5 at a ratio of 1: 100 to 1: 300 w / v. Then, the inside of the reactor 5 is operated to reach a predetermined temperature and pressure by using a temperature controller 7 and a pressure gauge 2 for hydrolysis of collagen by a pressurized hot water reaction. At the same time, the stirrer 6 attached inside the reactor 5 is used to maintain the homogeneity of the collagen and submersible mixture injected into the reactor 5, while at the same time inducing the given pressure and heat to remain constant in the mackerel meat .

In the present invention, molecular weight measurement, amino acid composition analysis and antioxidant activity of collagen hydrolysates derived from bones and / or bark of fish recovered through the above-mentioned process were measured by MALDI-TOF-MS analysis. According to the exemplary embodiment, the molecular weight of the collagen hydrolyzate obtained by the pressurized hydrothermal reaction was all 3000 Da or less, which was much smaller than that of a few hundred kDa, which is the molecular weight of common collagen (see Figs. 6a and 6c and Example 7 ). This analysis result means that the collagen hydrolyzate derived from the fish bone and / or the shell, which is finally recovered according to the present invention, can be rapidly introduced into the human body.

In addition, the collagen hydrolyzate obtained according to the exemplary embodiment of the present invention has a relatively large content of lysine, which is an amino acid that is helpful for people suffering from anorexia and anemia, and has an important function for the formation of connective tissue of the body (See Table 7 in Example 8), which is a glycine, proline and hydroxyproline. In addition, according to an exemplary embodiment of the present invention, the collagen hydrolyzate recovered according to the process of the present invention has antioxidant activity (see Table 8 and Fig. 7a-7d of Example 9).

Therefore, according to the present invention, the collagen is separated from the bones and / or the bark of the fish, and the collagen peptide and the hydrolyzate containing the amino acid can be obtained through the hydrothermal reaction. Since the collagen hydrolyzate recovered in accordance with the present invention has antioxidative activity and can be easily administered to the human body, the collagen hydrolyzate can be added as a functional ingredient of cosmetics, drugs, and / or foods, .

Hereinafter, the present invention will be described in more detail by way of illustrative examples, but the present invention is by no means limited to the invention described in the following examples.

material

The mackerel (Scomber japonicus ) was received from a seafood processing company (Dongwonhae Love Inc.) located in Busan. The reagents used (pepsin, protein marker, terrestrial animal collagen, 2,2-diphenyl-1-picrylhydrzyl (DPPH), 2,2-azino-bis (3-ethylbeznothiazoline-6-sulfonic acid) diammonium salt ferricyanide, 3- (2-pyridyl) -5,6-bis (4-phenylsulfonic acid) -1,2,4-triazine (ferrozine), 6-hydroxy-2,5,7,8-tetramethylchroman- -carboxylic acid (trolox) was purchased from Sigma Aldrich (St. Louis, Mo., USA), and other reagents and solvents were used for analytical or HPLC grades.

Example  1: Analysis of components of bones and shells of mackerel

The bones and bark of the unprocessed mackerel were separated, washed with cold water, and the sample was lyophilized for 72 hours. The dried samples were crushed in a homogenizer and stored at -20 ° C. Moisture, ash, crude fat and protein contents were measured according to AOAC method to analyze the general composition of mackerel bones and shells. The non-protein content was calculated by subtracting moisture, ash, and total fat and protein content. The samples used in this example were lyophilized mackerel bones and bark, and the results of analysis of the content of major constituents in dried mackerel bones and bark are shown in Table 1 below. The dried mackerel bark was found to have a higher content of protein and lipid than the dried mackerel bones.

Main constituents of dried mackerel bones and shells Sample name moisture (%) Ashes (%) Crude fat (%) Crude protein (%) Non-protein (%) Dried mackerel bone 6.68 + 0.07 6.89 ± 0.06 17.59 ± 1.10 47.25 + 1.25 21.59 ± 1.28 Dried mackerel shell 4.60 + 0.06 4.50 ± 0.05 29.47 ± 1.26 49.49 1.52 11.94 + 0.76

Example  2: Separation of pepsin-soluble collagen from bones and shells of mackerel

Pepsin-solubilized collagen (PSC) in mackerel bones and shells was isolated as follows, and the procedure was carried out at -4 ° C. First, crushed mackerel bones were treated with 0.1 M NaOH (alkali solution) diluted 1:10 (w / v) to remove collagen-free parts. The mixture was continuously stirred at 250 rpm for 24 hours using a magnetic stirrer. At this time, the alkali solution was changed every 6 hours. The sample washed with cold distilled water was then washed with water until it reached neutrality and lyophilized (EYELA FDV-2100, Rikakikai Co. Ltd., Tokyo, Japan). Insoluble bones were then decarcified for 4 days with 0.5 M ethylenediaminetetraacetic acid (EDTA, pH 7.5) diluted 1:10 (w / v). At this time, the EDTA solution was changed every day. The residues were washed with cold distilled water and then treated with 10% (v / v) butyl alcohol diluted 1:10 (w / v) for 24 hours to remove fat. The remaining residues were treated with 0.57 M acetic acid diluted in a ratio of 1:10 (w / v) containing 0.1% (w / v) pepsin for 3 days and centrifuged at 12,000 rpm for 50 minutes. The remaining residue was again extracted with the same solution for 3 days and centrifuged under the same conditions. Each viscous solution was adjusted to a final concentration of 2.0 M by the addition of NaCl. The solution was incubated for 24 hours and the precipitate was measured. The precipitate was centrifuged at 12,000 rpm for 20 minutes and then dissolved in 0.57 M acetic acid. This solution was dialyzed with 0.1 M acetic acid and distilled water and stored in a dialysis bag for 2 hours and lyophilized.

On the other hand, the crushed mackerel bark was treated with a 0.1 M NaOH alkali solution diluted 1:35 (w / v) ratio for 24 hours and stirred using a magnetic stirrer. At this time, the solution was changed four times a day. And washed with cold distilled water. The lyophilized samples were then degreased in 10% (w / v) butyl alcohol diluted 1:35 (w / v) for 24 h. The degreased residues were washed with cold distilled water and mixed with 0.57 M acetic acid diluted 1:35 (w / v) containing 0.1% (w / v) pepsin for 3 days. Repeated procedures were performed following the process of separating collagen from the mackerel bones described above.

The content of pepsin-soluble collagen was determined by quantifying hydroxyproline according to the method described in DE Goll et al., 1963 J. Food Science , 28 (5): 503-509, and then multiplying by the conversion factor, The content of proline was determined by slightly modifying the method described in the literature (I. Bergman et al., 1963, Analytical Chemistry , 35 (12): 1961-65). Table 2 shows the analysis results of the pepsin-soluble collagen and the general composition obtained from the mackerel bones and shells. Pepsin soluble collagen showed higher contents in shell than mackerel bone. The protein contents of mackerel bones and shells were 90.05 ± 2.34 and 86.89 ± 2.48%, respectively. Moisture contents (6.28 ± 0.12 and 7.48 ± 0.10%), ash (3.48 ± 0.09 and 5.37 ± 0.06%), and fat content (0.19 ± 0.02 and 0.26 ± 0.03%) were also observed in the mackerel bone and skin.

Collagen content and general composition of dried mackerel bones and shells Furtherance(%) Collagen bone skin yield 1.75 + 0.07 8.10 ± 0.12 protein 90.05 + - 2.34 86.89 + - 2.48 moisture 6.28 ± 0.12 7.48 ± 0.10 Ash 3.48 ± 0.09 5.37 ± 0.06 Lipid 0.19 + 0.02 0.26 + 0.03

Example  3: Measurement of molecular weight of pepsin soluble collagen

The pepsin-soluble collagen powder obtained in Example 2 was subjected to SDS-PAGE and the molecular weight thereof was measured. Proteins were analyzed according to modified Laemmli method using 3.0% stacking gel and 5.0% resolving gel. 2 g of the PSC sample obtained in Example 2 was dissolved in 1.0 mL of 0.02 M sodium phosphate buffer (pH 7.2) and mixed with the sample buffer. Then, 20 μL of each sample (20 μg protein) was prepared in polyacrylamide gels and separated by continuous electrophoresis at 30 mA current. Collagen separated from calf skin and bovine tendon by the same method and procedure was used as a reference material and compared with collagen separated from bone and bark of mackerel in Example 2. Markers with high molecular weight (MW) were also used to determine the molecular weight of the protein. The SDS-PAGE pattern results for the molecular weight magnitude of pepsin soluble collagen (PSC) isolated from mackerel bones and shells according to this example are shown in FIG. Collagen containing _two α-chains (α ₁ and α ₂ ) has a molecular weight of 116 kDa (α ₁ ) and 126 kDa (α ₂ ), similar to the molecular weight of standard collagen. In addition, the collagen of β-chain isolated from bone and bark of mackerel has a molecular weight of 205 kDa. This means that the collagen separated from the bones and shells of mackerel has a molecular weight similar to that of animal-derived collagen.

Example  4: Pepsin soluble collagen FT - IR  analysis

Fet-IR analysis was performed on pepsin-soluble collagen powder obtained from bones and bark of mackerel in Example 2, respectively. The PSCs FT-IR spectra of bone and shell of lyophilized mackerel were obtained using Perkin Elmer (USA), Spectrum X, and the spectra were measured at a resolution of 4000-650 cm ^-1 and 4 cm ^-1 . Results of FT-IR spectral analysis of collagen separated from mackerel bones and shells are shown in FIGS. 4A and 4B, respectively. As shown in these figures, the bands of collagen separated from the mackerel bones and shells were 3283 cm ^-1 and 3285 cm < ^{-1 &} gt ;. The wavelengths of the amide Ⅰ, amide Ⅱ and amide Ⅲ bands shown in these figures are directly related to the properties of the collagen, and the amide Ⅰ band (1600 to 1660 cm ^-1 ) shows the oscillation of the carbonyl groups (C═O) in the polypeptide Lt; / RTI > Amide Ⅱ band (~ 1550 cm ^-1 ) is related to NH bending and CN stretching vibration, and amide Ⅲ band (1220 ~ 1320 cm ^-1 ) is related to CN stretching vibration and NH strain. Through the FT-IR spectrum analysis according to this example, it was confirmed that the substance isolated and obtained in Example 2 was collagen.

Example  5: Measurement of viscosity and denaturation temperature of pepsin soluble collagen

The viscosity and denaturation temperature of the pepsin soluble collagen powder obtained in Example 2 were measured. The viscosities of the mackerel bones and bark were measured by the following method using a Brookfield DVII + Pro viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA 02346 USA). 8 mL of 0.1% (w / v) PSC was added to 0.1 M acetic acid, incubated at 10 ° C for 20 minutes, and placed in a container. The data obtained by rotating the sample at 150 rpm using SC4-18 were then used for viscosity measurement of the sample and the viscosity was expressed in centipose (cP) units.

The high viscosity of collagen is important. The viscosity of collagen (PSC) isolated from mackerel bones and shells according to this example is shown in Table 3. The viscosity range was 18.34 ± 0.25 and 20.26 ± 0.21 cP.

The denaturation temperature (Td) was measured according to the following method. Add 8 mL of 0.1% (w / v) PSC to 0.1 M acetic acid and heat at 10 ° C to 40 ° C at 3 ° C intervals. This solution was stored at the specified temperature for 20 minutes before measuring the viscosity. The thermodynamic curve of the PSC showed a slight change in viscosity with temperature at the coordinates. The fractional viscosity was calculated for each temperature as follows. The fractional viscosity is the maximum viscosity divided by the measured viscosity. The denaturation temperature was determined as the temperature when the fractional viscosity was close to 0.5. Table 3 and Figure 5 below show the conversion curves for the denaturation temperature of the collagen obtained from the mackerel bones and shells.

Viscosity and denaturation temperature of shells separated from bark of mackerel and mackerel shell Properties Collagen bone skin Viscosity (cP) 18.34 ± 0.25 20.26 ± 0.21 Denaturation temperature (캜) 27 30

Example  6: Collagen using pressure hydrothermal hydrolysis Hydrolyzate  Recovery

A high temperature, high pressure batch reactor as shown in FIG. 2 was used to hydrolyze the pepsin soluble collagen obtained in Example 2. The hydrolysis reactor was run with a 200 mL batch reactor consisting of Hastelloy 276 with a temperature controller. The collagen (0.5 g) obtained in Example 2 and water were mixed at a ratio of 1: 200 (w / v) and injected into the reactor. The hydrolysis reaction conditions were heated at the desired temperature (200-250 ° C) and pressure (30-70 bar) using an electric heater. The temperature and pressure of the reactor were measured during the experiment using a temperature controller and a pressure gauge. The sample was stirred at 150 rpm using a magnetic stirrer. The heating time was reached after 26 to 54 minutes and the desired temperature was maintained for 3 minutes. After cooling the reactor by connecting a cooling circulation jacket, the hydrolyzate sample was recovered from the reactor and filtered using filter paper.

Example  7: Collagen Hydrolyzate  About MALDI - TOF  Mass analysis

MALDI-TOF-MS (Matrix Assisted Laser Desorption / Ionization-Time of Flight-Mass Spectrometry) analysis was performed on the recovered collagen hydrolyzate in order to measure the molecular weight of the collagen hydrolyzate obtained in Example 7. 1 μL of a pepsin soluble collagen (PSC) hydrolyzate sample isolated from the bones and skins of mackerel through Example 6 was attached to a glossy steel 384 target plate. The matrix solution containing 1 μL of 2,5-dihydroxybenzoic acid (DHB) and 1% THF was mixed and dried. Each sample was analyzed using a MALDI TOF MS from an Ultraflex Ⅲ mass spectrometer (Bruker Daltonics, Germany) equipped with a pulsed nitrogen laser at 377 nm. The measurement range was obtained by accumulating data from 200 laser shots in cationization and reflection mode at 700-6000 m / z.

The results of the analysis according to the present embodiment are shown in Figs. 6A to 6C and Tables 4 to 6 below. As shown in these figures and tables, when the collagen in the recovered hydrolyzate was hydrolyzed at a temperature of 200 ° C and 30 bar, collagen isolated from mackerel bone had a molecular weight of about 759-988 Da , The peptide size of the collagen hydrolyzate of the mackerel bark, which was treated under the same conditions, ranged from 789 to 1632 Da. Hydrolysis conditions Peptide size in the collagen hydrolyzate of mackerel shell treated at 250 ℃ and 70 bar was in the range of 952-1638 Da. These results show that the molecular weight of collagen is significantly reduced from 116 to 126 kDa. The peptides present in the hydrolyzate were identified using the Mascot Database, and the expected peptides were also shown in Tables 4-6.

MALDI-TOF mass spectrometry on mackerel bone collagen hydrolyzate (200 ° C, 30 bar) m / z Molecular weight (dalton) Predicted peptide ^* Intensity 760.13 759.12 K.KATLAR.M 144.83 762.12 761.11 K.RMDLAR.I 212.60 764.14 763.14 K.VTFNRK.Q 129.75 889.63 888.62 K.ATLARMAR.G 181.04 891.62 890.61 R.MARGAMVR.F 178.46 953.29 952.29 R.FVFIYQH.- 282.55 985.22 984.21 -.MKIIIAPAK.K 3335.74 987.21 986.21 M.ADAELEAIR.Q 2814.04 989.21 988.20 K.TLWHCSDK.L 987.93

MALDI-TOF mass spectrometry (200 ° C, 30 bar) on collagen hydrolysates of mackerel bark m / z Molecular weight (dalton) Predicted peptide ^* Intensity 790.36 789.36 R.SDGSRIR.F 114.43 1121.27 1120.26 -.MIQMQTKLK.- 91.13 1133.11 1132.10 K.DAAADKAEDVK.D 150.02 1146.10 1145.09 K.EGLGKLTGNEK.L 138.77 1148.09 1147.08 R.RYANIGDVIK.Y 70.98 1217.12 1216.11 K.QLDTLGNDKGR.L 2173.88 1219.12 1218.11 K.DAVEDKVEDAK.E 1036.45 1221.11 1220.10 K.KGDVYDAVVVR.T 169.52 1633.92 1632.92 R. SAQFMKIVSLAPEVL.- 120.54

MALDI-TOF mass spectrometry on mackerel bone collagen hydrolyzate (250 ° C, 70 bar) m / z Molecular weight (dalton) Predicted peptide ^* Intensity 953.30 952.30 R.HAEVVASIK.A 5328.47 955.30 954.29 R.SVDPGSPAAR.S 1027.45 976.27 975.27 -.MKTAQELR.V 398.92 1369.13 1368.13 K.NLLTGSASESVYK.A 163.41

The abbreviations in Tables 4 to 6 include: A- alanine, C-cysteine, D-aspartic acid, E-glutamic acid, F- phenylalanine, G- glycine, H- histidine, I- isoleucine, K- lysine, M- Asparagine, P-proline, Q-glutamine, R-arginine, S-serine, T-threonine, V-valine, W-tryptophan, Y-tyrosine; ^{* The} expected peptides are from the mascot database (available at http://www.matrixscience.com)

Example  8: Collagen Hydrolyzate  Analysis of amino acid composition

In order to measure the molecular weight of the collagen hydrolyzate obtained in Example 7, the amino acid composition of the recovered hydrolyzate was analyzed. An amino acid analyzer (S430 (SYKAM)) was used for the analysis of amino acid content. Column temperature of 37-74 ℃ and buffer pH range of 2.90-7.95 were used for cation separation column LCA K07 / Li (4.6 × 150 mm). The mobile phase flow rate of 1 per minute was used 5 mM p-toluenesulphonic acid solution in 0.45mL, 5 mM p and mixtures -toluenesulphonicacid, 20 mM of Bis-Tris, EDTA 100 m is a post-column reagent at a flow rate of 0.25 mL per minute Respectively. The emission wavelengths were operating at 440 and 570 nm. Table 7 shows the composition and content of amino acids produced through hydrothermal reaction of collagen isolated from bone and bark of mackerel.

Amino acid composition of collagen hydrolyzate (mg / g) Amino acid composition Bone collagen hydrolyzate Skin collagen hydrolyzate Temperature (° C) / Pressure (bar) 200/30 250/70 200/30 250/70 Phosphoserine 0.32 ± 0.03 ^a 0.27 ± 0.02 ^a 0.20 ± 0.01 ^b 0.16 ± 0.01 ^b Taurine 0.40 + 0.02 ^a 0.48 + 0.03 ^a 0.18 + 0.01 ^b 0.26 ± 0.02 ^b Aspartic acid 2.28 ± 0.05 ^a 1.89 + 0.04 ^a 0.99 + 0.04 ^b 1.01 0.03 ^b Hydroxyproline 5.99 ± 0.09 ^a 5.33 + 0.07 ^b 5.37 + 0.08 ^b 4.71 ± 0.06 ^c Serine 1.67 ± 0.06 ^a ND 1.08 + 0.04 ^b ND Glutamic acid 1.17 + 0.04 ^a 1.24 + 0.04 ^a 0.73 ± 0.02 ^b 0.64 ± 0.03 ^b Proline 13.20 ± 0.12 ^b 11.52 ± 0.17 ^c 15.36 ± 0.11 ^a 11.92 + - 0.15 ^c Glycine 40.86 ± 1.80 ^a 35.26 ± 1.12 ^b 38.56 ± 1.15 ^a 33.59 ± 1.25 ^b Alanine 24.82 ± 0.88 ^a 21.19 ± 0.82 ^b 24.83 + - 0.92 ^a 19.36 ± 0.78 ^c ? -aminobutyric acid 1.09 0.04 ^a 1.15 ± 0.03 ^a 0.59 + 0.03 ^b 0.31 0.02 ^b Balin 4.44 ± 0.15 ^b 4.07 ± 0.18 ^c 5.27 ± 0.16 ^a 3.89 ± 0.11 ^c Cysteine 2.61 ± 0.09 ^a 2.28 0.08 ^a 1.70 + 0.08 ^b 1.36 + 0.07 ^b Methionine 2.29 ± 0.07 ^a 1.35 + 0.08 ^b 0.64 ± 0.03 ^c 0.59 0.02 ^c Cystathione 3.34 + 0.10 ^a 2.80 ± 0.07 ^b 1.42 ± 0.06 ^c 0.88 0.04 ^d Isoleucine 4.36 + 0.15 ^a 3.77 ± 0.12 ^a 1.78 + 0.09 ^b 1.42 ± 0.08 ^b Leucine 8.05 ± 0.18 ^a 4.07 ± 0.11 ^c 5.33 ± 0.15 ^b 4.47 ± 0.12 ^c Tyrosine 5.34 ± 0.16 ^a 4.53 ± 0.11 ^b 1.90 0.08 ^c 1.63 + 0.09 ^c Phenylalanine 6.03 ± 0.13 ^a 5.89 ± 0.11 ^a 2.30 ± 0.10 ^b 2.02 0.08 ^b beta-alanine 0.01 ± 0.01 0.04 0.01 0.02 ± 0.01 0.01 ± 0.01 ? -aminobutyric acid 0.18 + 0.02 0.12 + 0.02 0.10 ± 0.01 0.11 + - 0.01 gamma -amino-n-butyric acid 0.03 ± 0.01 0.05 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 Histidine 5.46 ± 0.11 ^a 3.50 ± 0.10 ^b 1.93 + 0.08 ^c 1.73 + 0.07 ^c Hydroxylysine 0.13 0.01 ^b 0.55 0.02 ^a 0.06 ± 0.01 ^b 0.43 + 0.02 ^a Ornithine 1.09 ± 0.05 ^b 1.01 0.06 ^b 1.91 + 0.06 ^a 0.83 0.04 ^b Lee Sin 7.08 ± 0.12 ^a 6.79 ± 0.10 ^a 4.21 + - 0.11 ^b 3.64 ± 0.09 ^b Ethanolamine 0.79 + 0.03 ^a 0.62 ± 0.02 ^b 0.46 0.04 ^c 0.28 ± 0.01 ^d Arginine 3.42 ± 0.09 ^a ND 2.11 + 0.08 ^b ND

As shown in Table 7, it can be seen that the amino acid composition varies depending on the applied temperature and pressure conditions, and therefore temperature and pressure of the hydrothermal reaction are important variables. Glycine content was high in the amino acids present in the hydrolyzate, 27.90-29.44% in the hydrolyzate of the mackerel bone, and 32.38-35.25% in the hydrolyzate of the mackerel bark.

Example  9: Collagen Hydrolyzate  Antioxidant activity analysis

DPPH free radical scavenging ability, ABTS free radical scavenging ability, ferric reducing power activity and Fe ²⁺ chelating activity method were measured to examine antioxidative activity against the collagen hydrolyzate obtained in Example 6.

DPPH free radical scavenging method

For DPPH free radical scavenging analysis, 3.95 mL of methanol containing 0.1 mM of DPPH solution was added to test tubes containing 0.1 mL of collagen samples at various concentrations before and after hydrolysis. The mixtures were mixed for 10 seconds and incubated at room temperature for 30 minutes in the dark. The absorbance of all samples was measured at 517 nm. The DPPH free radical scavenging activity ratio was calculated by the following equation.

DPPH radical scavenging activity (%) = (1 - [ As / Ac ]) 100

(As is the absorbance of the sample measured at 517 nm; Ac is the absorbance of the sample at 517 nm)

Experimental and laboratory experiments (various concentrations of trolox in methanol) were analyzed using the same procedure as described above.

ABTS + free radical scavenging method

The ABTS ⁺ free radical scavenging assay was performed in the following manner. ABTS was dissolved in water at a concentration of 7 mM / L to prepare ABTS ⁺ . ABTS ⁺ reacted with the same amount of 2.45 nM / L potassium sulfate as the ABTS stock solution and was made by incubating the mixture at room temperature and darkness for 16 hours before use. For sample analysis, the ABTS ⁺ stock solution was 80% methanol with an absorbance of 0.70 ± 0.02 at 734 nm, added 3.95 mL of diluted ABTS and 0.05 mL of the PSC and its hydrolyzate sample (after different conditions before and after hydrolysis ), The mixture is kept at room temperature for 6 minutes in a dark environment. All samples were measured for absorbance at 734 nm. The free radical scavenging activity ABTS + percent was calculated using the formula:

ABTS ⁺ radical scavenging ability (%) = (1 - [ As / Ac ]) 100

(Where As is the absorbance of the sample controlled at 734 nm; Ac is the absorbance of the sample at 734 nm)

Experimental and laboratory experiments (various concentrations of trolox in 80% methanol) were analyzed as described above.

Ferric reducing power assay

Reduction power of Fe ³⁺ in PSC sample and hydrolyzate was determined by the following method. 0.125 mL of the PSC sample was mixed with 0.625 mL of 0.2% potassium phosphate buffer, pH 6.6, and 0.625 mL of 1% potassium ferricyanide, at various concentrations before and after hydrolysis. The mixture was incubated at 50 DEG C for 20 minutes. 0.625 mL of 10% trichloroacetic acid was added after centrifugation at 3,000 rpm for 10 minutes. 1.0 mL of the upper portion of the solution was mixed with 1.0 mL of distilled water and 1.0 mL of 0.1% ferric chloride. The absorbance was read at 700 nm. The increase of the absorbance was related to the increase of the reducing power.

Fe ²⁺ Chelation assay

The Fe ²⁺ chelating ability of the sample was determined through the following steps. PSC samples (0.1 mL) were mixed with 3.0 mL of distilled water at various concentrations before and after hydrolysis. Subsequently, 50 μL of 2 mM FeCl ₂ and 0.1 mL of 5 mM ferrozine were incubated at room temperature for 20 minutes. The absorbance was then measured at 562 nm. Various concentrations of EDTA were used as control samples. Distilled water was used as a negative control. The chelating activity (%) was calculated as follows:

Chelating activity (%) = 1 - [ As / Ac ]) 100

(Where As is the absorbance of the sample controlled at 562 nm; Ac is the negative control absorbance at 562 nm)

The results of the analysis according to the present embodiment are shown in Figs. 7A to 7D and Table 8 below. 7A shows DPPH radical scavenging ability of the hydrolyzate recovered from the bark and shell of mackerel by hydrothermal reaction, showing a relatively high antioxidative activity at a concentration of 10 mg / mL. FIG. 7b shows that the antioxidant activity of the hydrolyzate was higher than that of the collagen before the hydrolysis, as a result of comparing the ABTS scavenging ability of collagen (PCS) and hydrolyzate isolated from the mackerel bones and shells. FIGS. 7C and 7D show the results of Fe ³⁺ reducing power and Fe ²⁺ chelating activity of the collagen peptide-containing hydrolyzate after hydrolysis, showing high activity in the hydrolyzate after hydrothermal treatment compared with before hydrolysis of fish collagen It is showing. The hydrolysates of collagen isolated from the mackerel shells obtained at 250 ℃ and 70 bar showed higher Fe3 ⁺ reductivity than the collagen hydrolysates isolated from the mackerel bones at various concentrations. Collagen hydrolysates of mackerel bones and shells show similar Fe ²⁺ chelating effects, indicating that hydrolysates of collagen separated from mackerel bones and shells can function as Fe ²⁺ chelator. The PSCs IC ₅₀ values of the mackerel bones were 8.21 ± 0.15 mg / mL at 200 ℃ and 30 bar, and 7.27 ± 0.14 mg / mL at hydrolyzed samples at 250 ℃ and 70 bar, respectively. In the case of the mackerel shell, the hydrolyzed samples showed a value of 7.01 ± 0.12 mg / mL at a temperature of 7.91 ± 0.11 mg / mL and 250 ° C. and 70 bar at 200 ° C. and 30 bar, respectively. As shown in Table 8.

Collagen hydrolyzate DPPH, ABTS radical scavenging activity and Fe ^{2 +} chelating activity (IC ₅₀₎ Activity Collagen / Reference IC ₅₀ (mg / mL) DPPH Freedom
Radical
Scatters Bone collagen hydrolyzate (200 ° C, 30 bar) 8.71 ± 0.14 ^c Collagen hydrolyzate of bone (250 ° C, 70 bar) 8.38 + - 0.13 ^c Peel collagen hydrolyzate (200 ° C, 30 bar) 9.57 ± 0.12 ^d Peel collagen hydrolyzate (250 ° C, 70 bar) 7.58 ± 0.10 ^b Trolox (reference) 0.35 0.05 ^a ABTS free radical
scavenging Collagen separated from bone 10.77 ± 0.11 ^d Bone collagen hydrolyzate (200 ° C, 30 bar) 4.18 ± 0.07 ^c Collagen hydrolyzate of bone (250 ° C, 70 bar) 2.61 ± 0.05 ^b Collagen separated from the skin 12.12 + - 0.13 ^d Peel collagen hydrolyzate (200 ° C, 30 bar) 4.05 + 0.08 ^c Peel collagen hydrolyzate (250 ° C, 70 bar) 2.50 ± 0.05 ^b Trolox (reference) 0.25 0.03 ^a Fe ^{2 +} chelating Bone collagen hydrolyzate (200 ° C, 30 bar) 8.21 ± 0.15 ^c Collagen hydrolyzate of bone (250 ° C, 70 bar) 7.27 + - 0.14 ^b Peel collagen hydrolyzate (200 ° C, 30 bar) 7.91 + - 0.11 ^c Peel collagen hydrolyzate (250 ° C, 70 bar) 7.01 + - 0.12 ^b EDTA (reference) 0.34 ± 0.06 ^a

Although the present invention has been described based on the preferred embodiments of the present invention, the present invention is not limited to the embodiments described above. Rather, various modifications and changes will readily occur to those skilled in the art to which the present invention pertains based on the above-described embodiments. It will be apparent, however, that such modifications and variations are all within the scope of the present invention.

1: Safety valve 2: Pressure gauge
3: Needle valve 4: Electric heater
5: High Pressure Reactor 6: Impeller / Stirrer
7: stirring speed / temperature controller 8: sample collector

Claims (6)

Drying at least one sample of the bone and skin of the prepared mackerel;
Pulverizing the dried sample;
Adding a proteolytic enzyme to the pulverized sample to separate collagen; And
Recovering the collagen hydrolyzate obtained by reacting the powder of the separated collagen with water at high temperature and high pressure,
To obtain a collagen hydrolyzate derived from a fish.
The method according to claim 1, wherein the step of recovering the collagen hydrolyzate comprises using water at a temperature of 150 to 350 ° C. and a pressure of 5 to 400 bar.
The method for obtaining a collagen hydrolyzate of fish according to claim 1 or 2, wherein the obtained collagen hydrolyzate has a molecular weight of 3,000 Da or less.
The method according to claim 1 or 2, wherein the step of separating the collagen comprises the steps of crushing the bone of the mackerel, diluting the bone of the crushed mackerel at a ratio of 1: 5 to 1:20 (w / v) Treating the treated mackerel bone with an alkaline solution, adding an acid in a ratio of 1: 5 to 1:20 (w / v) to the insoluble mackerel bones obtained by treatment with an alkali solution to degasify the calcined mackerel bones 1 to 5: 1 to 20: 1 (w / v) alcohol, and a step of adding pepsin to the degreased mackerel bone residue to hydrolyze the collagen hydrolyzate from fish .
3. The method of claim 1 or 2, wherein separating the collagen comprises: crushing the shell of the mackerel; and diluting the crushed shell of the mackerel at a ratio of 1:20 to 1:50 (w / v) A step of treating the insoluble mackerel bark obtained by treatment with an alkaline solution with an alcohol at a ratio of 1:20 to 1:50 (w / v) to degrease the treated mackerel bark, And adding pepsin to the collagen hydrolyzate to obtain a fish-derived collagen hydrolyzate.
The method according to claim 1 or 2, wherein the obtained collagen hydrolyzate has an antioxidative activity.

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