WO2015182859A1 - Method for obtaining low-molecular weight collagen peptides from fish bones and shells 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. The hydrolysate obtained in the hydrolysis process contains collagen peptides (<3000 Da) with a low molecular weight, into which collagen proteins (116,000 - 126,000 Da) isolated from fish bones and shells are lysed. It was verified that the hydrolysate derived from fish collagen contains essential amino acids. The antioxidative activity of the fish collagen hydrolysate was higher than that of the initially isolated fish collagen. The fish collagen hydrolysate had a reduced smell that acts as a negative factor of products, but an increased smell that is in favor of sensory feeling. According to the present invention, the process is simple and eco-friendly, and the product safety is excellent, and thus the hardness enhancement is anticipated. Therefore, the fish collagen hydrolysate obtained in the present invention can be utilized as a functional material in food, medicinal, and cosmetic fields.

Description

Process for obtaining low molecular weight collagen peptides from bones and shells of fish using press hydrothermal hydrolysis

The present invention relates to a method for recovering functional components from fish. More specifically, collagen macromolecules (about 126 kDa) extracted from by-products such as shells, bones and intestines left after fish processing are hydrolyzed by pressurized hydrothermal reaction to convert into collagen peptides with low molecular weight (about 3 kDa). A method for obtaining collagen peptides.

Collagen is a protein with a variety of structures, generally found in animal bone, shell, and cartilage tissue. Collagen accounts for 25% of the total weight of the animal and contains 1-2% in muscle. Collagen is a major component of the cell matrix matrix of human and animal connective tissue. Collagen exists in various forms. The typical collagen type, collagen I, has a triple helical structure with three polypeptide chains twisted at about 126 kDa (Trofocollagen). There are three major amino acids in collagen, and the specific one is a repeating structure of "glycine-proline-X" or "glycine-X-hydroxyproline" (where X is any amino acid).

Many modern consumers are very interested in health foods or dietary supplements derived from natural products that enhance immunity. Collagen is a biomaterial for tissue regeneration due to its excellent bio-compatibility, bio-degrability and anti-aging properties, and is widely used in industries such as food, cosmetics, biopharmaceuticals, and pharmaceuticals. . In addition, collagen enhances immune function, promotes bone cell regeneration by adsorbing calcium in bone tissue, and strengthens joints. It also has excellent effects on skin beauty by activating skin metabolism and maintaining moisturizing power. It is known to exert, and is utilized as functional ingredients, such as a pharmaceutical and cosmetics. In particular, it is used as a component of the functional cosmetics to improve the wrinkles of the skin caused by aging or ultraviolet rays.

In extracting collagen from biological tissues, it is extracted with an organic solvent, treated with acid or alkali, and then collagen which is insoluble in water is obtained using appropriate enzymes such as trypsin and hyaluronidase. Most of the collagen in use today is isolated and purified from the bones and shells of land animals such as cattle, calves, pigs and poultry.

However, in the case of collagen isolated from terrestrial animals, recently, bovine spongiform encephalopathy (BSE), foot-and-mouth disease (FMD), avian influenza (AI), and transmissible spongiform encephalopathy (TSE) With the development of such diseases, safety issues are emerging. In addition, pigs are disgusted animals in the Islamic world, so collagen derived from pigs cannot be used in a society with a certain religion. Therefore, there is a need for the prevention of infectious diseases and the development of alternative collagen production technology for certain religious people.

Fish ( Scomber japonicus ) is an important food ingredient consumed around the world. It is consumed through various cooking methods and a large amount of fish is consumed every day. When fish are consumed as food, bones, shells and intestines are treated as by-products as non-edible parts or used as animal feed, but are also left unattended as environmental pollutants. However, given the high nutritional value and other industrial possibilities of the functional materials contained in these by-products, it is possible to solve the problems of environmental pollution caused by these materials that are currently being disposed of, and to obtain commercially useful materials if appropriate processing technology is introduced. Economic benefits can also be gained through recovery and use.

In addition, as described above, foot and mouth disease has recently been popularized locally and globally, and safety problems with collagen extracted from terrestrial animals have emerged. However, using collagen present in fish bones, shells, intestines, etc. can solve this problem. In particular, if the collagen hydrolyzate can be extracted from the shells, bones, intestines, etc. of fish that are currently discarded in large quantities in the domestic geographic environment surrounded by the sea on three sides, it may be preferable in view of efficient use of fishery resources.

Natural collagen obtained from fish has a relatively high molecular weight of about 126 kDa and thus has a problem in that it cannot be absorbed in vivo because it does not penetrate deep into the living body. Therefore, there is a need for the development of a technology capable of producing a relatively low molecular weight collagen peptide to efficiently penetrate collagen into the living body.

Conventionally, methods for decomposing high molecular substances derived from living bodies include chemical hydrolysis using an acid, an alkali or a catalyst, and enzymatic hydrolysis using an enzyme. However, chemical hydrolysis requires severe reaction conditions, causes severe environmental pollution, requires a process for recovering the injected chemicals, and may result in deterioration of the product. On the other hand, enzymatic hydrolysis is not economical because the reaction process is complicated and takes a long time to complete the production cycle.

[Preceding technical literature]

Patent Document 1: Japanese Unexamined Patent Publication No. H06-157233A (Published: June 3, 1994, "Odorless to low-odor component in shellfish peptides, preparation method thereof, and external preparation or solvent containing the component")

The present invention has been proposed to solve the problems of the prior art of producing low molecular weight collagen by chemical or enzymatic hydrolysis using acid, alkali or catalyst described above, namely by-product generation, environmental pollution, prolonged process conditions, etc. An object of the present invention is to provide a method for obtaining collagen peptide hydrolysates (low molecular weight collagen peptides) derived from fish from bones, shells, intestines and the like which are by-products after fish processing.

Another object of the present invention is to provide a method for obtaining collagen hydrolyzate derived from fish while being economical, environmentally friendly and safe through a simple process without using a catalyst.

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

The technical problem of the present invention described above is achieved by the following solving means.

1. A method of obtaining low molecular weight collagen derived from fish, comprising the step of hydrothermally hydrolyzing a high molecular weight collagen powder extracted from at least one fish processing by-product selected from the group consisting of fish bones, shells and intestines.

2. A method for producing low molecular weight collagen derived from fish, comprising hydrolyzing high molecular weight collagen powder extracted from at least one fish processing by-product selected from the group consisting of fish bones, shells and intestines with high temperature and high pressure water.

3. The method of 1 or 2 above, wherein the low molecular weight collagen has a molecular weight of 3,000 Da or less.

4. The method of 1 or 2 above, wherein water at a temperature of 150 to 370 ° C. and a pressure of 5 to 400 bar is used in the hydrothermal hydrolysis reaction.

5. Before the pressurized hydrothermal hydrolysis of the high molecular weight collagen powder, the method further comprises the step of extracting the high molecular weight collagen powder from the dried and ground fish processing by-product sample, wherein the high molecular weight collagen powder The method of 1 or 2, which is used as a starting material of the step of hydrothermal hydrolysis.

6. Extracting high molecular weight collagen powder comprises treating the ground mackerel bone with an alkaline solution diluted in a ratio of 1: 5-1:20 (w / v); Decalcifying by adding an acid in a ratio of 1: 5 to 1:20 (w / v) to the insoluble mackerel bone obtained by treatment of the alkaline solution; Degreasing by adding 1: 5-1:20 (w / v) alcohol to the decalcified mackerel bone; And hydrolyzing by adding pepsin to the degreased mackerel bone residue.

7. The step of extracting high molecular weight collagen powder comprises the steps of treating the ground mackerel with an alkaline solution diluted in a ratio of 1:20-1:50 (w / v); Degreasing by adding 1:20-1:50 (w / v) alcohol to the insoluble mackerel shell obtained by treatment of the alkaline solution; And hydrolyzing by adding pepsin to the degreased mackerel shell residue.

According to the present invention, a method of obtaining collagen peptide hydrolyzate having a low molecular weight by using hydrothermal hydrolysis using high-temperature and high-pressure water from by-products such as bones, shells and intestines of fishes is provided.

The present invention adopts a simple process that does not use substances such as enzymes or organic acids to ensure the efficiency and economic efficiency of the process, while ensuring environmental friendliness and safety, while being used as a functional ingredient such as food, medicine, cosmetics, etc. The advantage is that it can rapidly produce molecular weight collagen peptides.

In addition, the present invention can be used as a raw material collagen extracted from by-products such as bones, shells, intestines discarded after the processing of the fish, the fish collagen peptide produced in the present invention does not have the smell peculiar to the fish functionalities of the applied product , Stability and safety are also excellent.

Since the collagen peptide hydrolyzate finally recovered and obtained according to the present invention has a molecular weight of 3,000 Da or less, it is easy to penetrate and absorb into the human body, thereby making it a functional ingredient for enhancing immunity, preventing skin aging, and regenerating bone cells It can be added to pharmaceuticals, cosmetics, etc., can be utilized in these industries.

1 is a process diagram schematically illustrating a process for obtaining low molecular weight collagen peptide hydrolyzate from fish processing by-products and an analytical method for confirming the structure and function of collagen obtained in these processes according to an exemplary embodiment of the present invention. .

2 is a schematic of a pressurized hydrothermal hydrolysis process apparatus for obtaining low molecular weight collagen peptide hydrolyzate from fish processing by-products in accordance with an exemplary embodiment of the present invention.

3 is a photograph of Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for pepsin soluble collagen isolated from fish bones and shells in an embodiment of the present invention. In Figure 3, MMP represents molecular weight marker protein, CSC represents calf shell collagen, MSC represents fish shell collagen, MBC represents fish bone collagen, and BATC represents Achilles tendon collagen.

4 and 5 are FT-IR spectrum analysis results for pepsin soluble collagen isolated from fish bone and shell in the embodiment of the present invention.

Figure 6 is a heat denaturation measurement results for pepsin soluble collagen isolated from fish bone and shell in the embodiment of the present invention.

7 to 9 are MALDI-TOF mass spectrum analysis results for collagen hydrolysates obtained by pressurized hydrothermal hydrolysis of pepsin soluble collagen isolated from fish bones and shells in the examples of the present invention. 7 is a collagen hydrolyzate obtained by hydrothermal hydrolysis of pepsin soluble collagen isolated from fish bone at 200 ° C., 30 bar, and FIG. 8 is a hydrothermal hydrolysis of pepsin soluble collagen isolated from fish skin at 200 ° C. and 30 bar. The collagen hydrolyzate obtained by FIG. 9 is a collagen hydrolyzate obtained by hydrothermal hydrolysis of pepsin soluble collagen isolated from fish shells at 250 ℃, 70 bar.

10 to 13 is a measurement result of the antioxidant activity of the collagen hydrolyzate obtained by the hydrothermal hydrolysis of pepsin soluble collagen isolated from the bone and shell of fish in the embodiment of the present invention. 10 shows DPPH free radical scavenging activity, FIG. 11 shows ABTS free radical scavenging activity, FIG. 12 shows ferric reducing power analysis, and FIG. 13 shows Fe ²⁺ chelate activity measurement results.

[Description of the code]

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

In order to solve the problems of the prior art, the present inventors obtained a low molecular weight collagen peptide by pressurized hydrothermal hydrolysis using high-temperature, high-pressure water to collagen derived from fish. Collagen poptide has completed the present invention with the focus on the fact that it can be used in a variety of industries, such as pharmaceuticals, health foods, cosmetics.

The present invention provides a method for obtaining low molecular weight collagen derived from fish, comprising the step of hydrothermally hydrolyzing a high molecular weight collagen powder extracted from at least one fish processing by-product selected from the group consisting of fish bones, shells and intestines. It is about.

The present invention also provides a low molecular weight collagen derived from fish, comprising hydrolyzing high molecular weight collagen powder extracted from at least one fish processing by-product selected from the group consisting of fish bones, shells and intestines with high temperature and high pressure water. It is about a method.

In the present invention, the term 'fish processing by-product' refers to by-products remaining as non-edible parts after being processed into food materials, for example bone, shells, intestines and the like. Fish includes, for example, mackerel, tuna, and the like, but is not limited thereto, and may be any kind having a high content of collagen in processed by-products.

In the present invention, the high molecular weight collagen extracted from fish is used as a starting material of the pressurized hydrothermal reaction. In this case, 'high molecular weight collagen' is a natural collagen extracted from a fish by a known method means that the average molecular weight is about 126 kDa.

In the present invention, low molecular weight collagen, specifically, collagen having a molecular weight of 3,000 Da or less is obtained by the hydrothermal hydrolysis of high molecular weight collagen as described above.

Hydrolysis using subcritical water hydrolysis (also called 'SWH') generated by high pressure and high temperature is an environmentally friendly and fast hydrolysis method. The boiling point of water is 0.1 MPa, 100 ° C., the critical points are 22 MPa, 374 ° C. and the subcritical water is near the critical point of water. Subcritical water exhibits different properties from those of water at room temperature, atmospheric pressure, or near boiling point, that is, low dielectric constant and high ionicity. Low dielectric constants have high solubility in organics, and high ionicity can act as an acid or basic catalyst to promote hydrolysis. Subcritical water hydrolysis processes are environmentally friendly because they do not generate environmental pollution and by-products.

Accordingly, in the present invention, in order to obtain high molecular weight collagen by hydrolyzing high molecular weight collagen, a pressurized hydrothermal hydrolysis 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 area, but the experimental range for temperature change without changing the pressure is limited. However, the critical point of water is at a temperature of 374 ° C. and a pressure of 22 MPa, below which water maintains a subcritical state, in which water maintains a liquid phase due to high pressure. That is, in the subcritical state, water is not divided into gas and liquid regions, and different process conditions may be formed according to various temperature and pressure changes, and the subcritical water in various regions may have different physical properties. Exists as.

When hydrolyzing the polymer material using unique physical properties such as high solubility of subcritical water, low dielectric constant, small interfacial tension, fast permeability, and low viscosity, the hydrolysis reaction can be efficiently proceeded. For example, hydrothermal reactions with sub-critical water at various conditions have much more ions than at ambient atmospheric pressure, and these ions catalyze as acids or bases. In addition, subcritical water, for example, can function as a solvent suitable for the dissolution of a wide range of organic compounds due to its low dielectric constant. As such, subcritical water below supercritical conditions, i.e. below critical temperature and critical pressure, may function as a new reaction medium in a variety of chemical reactions due to its unique physical and chemical properties.

In particular, the hydrothermal hydrolysis process in the subcritical state is recognized as an alternative to the conventional hydrolysis process due to the short reaction time, high hydrolysis rate, the safety of the hydrolyzate, the simplicity of the process, and the environmentally friendly process. . Therefore, in the present invention, by using hydrothermal hydrolysis using high-temperature and high-pressure water in a subcritical state, when the subcritical water is out of the saturated gas region and becomes a liquid state, the polymeric material is not carbonized and the liquid hydrothermal hydrolysis is performed. (pressurized hydrothermal hydrolysis) is characterized in that the reaction proceeds to obtain a product having functional properties. Subcritical water hydrolysis process requires no pre-treatment, relatively short reaction time, less corrosion or residue generation by reaction, no need for toxic solvents, and formation of decomposition products This small advantage is an environmentally friendly and rapid biomass hydrolysis method that can be used as an alternative to chemical or catalytic methods using existing acids or bases.

Illustratively, the high temperature, high pressure water used to obtain the hydrolyzate of fish-derived high molecular weight collagen, eg pepsin soluble collagen, ie low molecular weight collagen, according to the present invention has a temperature of 150-350 ° C. and 4 A subcritical condition with a pressure of 400 bar, preferably at a temperature of 200-300 ° C. and a pressure of 20-100 bar, more preferably at a temperature of 220-260 ° C. and a pressure range of 30-70 bar. At temperatures above 350 ° C. or pressures above 400 bar, the negative organoleptic properties of the hydrolyzate recovered are induced and are undesirable because they are not competitive in terms of energy and economy.

In the method for obtaining low molecular weight collagen or the method for producing low molecular weight collagen according to the present invention, the high molecular weight collagen powder is extracted from dried and ground fish processing by-product samples before the step of hydrothermal hydrolysis of high molecular weight collagen powder. It further comprises a step, and may be used as a starting material of the step of hydrothermal hydrolysis of the high molecular weight collagen powder obtained here.

In one exemplary embodiment of the present invention, the step of extracting the high molecular weight collagen powder comprises the steps of treating the ground fish bone with an alkaline solution diluted at a ratio of 1: 5-1:20 (w / v). And demineralizing the fish bone by adding a demineralizing agent in a ratio of 1: 5-1:20 (w / v) to the insoluble fish bone obtained by the treatment of the alkaline solution, and adding the demineralized fish bone to the 1: 5-1:20 (w / v) of alcohol to degreasing and adding pepsin to the degreased fish bone residue may comprise hydrolysis.

In another exemplary embodiment of the present invention, the extracting of the high molecular weight collagen powder is performed by treating the ground fish skin with an alkaline solution diluted at a ratio of 1:20-1:50 (w / v). And degreasing by adding 1:20-1:50 (w / v) alcohol to the insoluble fish shell obtained by treating the alkaline solution, and hydrolyzing by adding pepsin to the degreased fish shell residue. It may include.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated with reference to an accompanying drawing.

1 is a process diagram schematically illustrating a method for recovering, obtaining and analyzing collagen hydrolysates derived from fish in accordance with one exemplary embodiment of the present invention. When directly extracting high molecular weight collagen from fish processing by-product and using it as a hydrolysis reaction raw material, as shown in FIG. 1, at least one fish processing by-product sample such as bones, skin and intestines of fish is prepared. Wash it thoroughly with water and dry it. It can be dried naturally in a windy shade or freeze-dried at low temperatures. The freeze-drying method is particularly useful because the collagen contained in fish bones and shells is not modified by heat. If the bone and skin of the fish as a fish sample are to be dried using a freeze drying method, the freeze drying temperature is approximately -20 to 10 ° C, preferably -5 to 10 ° C, and the drying time is in the range of 48-96 hours. Can be. Lyophilization under these conditions yields a completely dried sample. The freeze drying temperature and drying time may be chosen differently depending on the state of the prepared fish processing by-product.

The dried sample is then cut and ground to the appropriate size. In an exemplary embodiment, the dried sample may be cut to a size of approximately 0.1-3 cm using a mechanical blend and then ground into powder form. Alternatively, the dried sample may be ground into a powder form using a homogenizer.

The collagen is then separated from the milled sample. Exemplary proteases, such as pepsin, may be used as proteolytic enzymes other than collagenase that completely degrades collagen, for example in milled samples. As an example, pepsin can be used to isolate pepsin-solubilized collagen (PSC) from fish samples and recover it in powder form.

In one embodiment of the present invention, when the fish bone is used as a starting material, the ground fish bone is mixed with an alkaline solution of 1: 5-1:20 (w / v) to remove the collagen free portion. To insoluble fish bones obtained by treatment with an alkaline solution, demineralized by adding a deliming agent at a ratio of 1: 5-1:20 (w / v), and then 1: 5 to decalcified fish bones. -Degreasing by adding alcohol at a ratio of 1:20 (w / v) and hydrolyzing by adding pepsin to the degreased fish bone residue. Examples of alkaline solutions may be sodium hydroxide or calcium hydroxide solutions in concentrations of 0.01-1.0 M. Examples of deliming agents include formic acid, acetic acid, picric acid, and acid solutions such as trichloroacetic acid, buffers such as citric acid and citric acid buffer, and metals that form chelates. For example, histochemical deliming methods such as using a complex salt of ethylene diamine tetra acetic acid (EDTA) at a concentration of 0.1-1.0 M may be used. In the degreasing treatment step, alcohols having 1 to 4 carbon atoms, for example butyl alcohol, can be used, and pepsin can be used, for example, in a form diluted in an acid such as acetic acid.

If necessary, after treating with alkaline solution, washing or neutralizing with distilled water or the like to remove alkali, washing or neutralizing with distilled water, etc. to remove the deliming agent after decalcification, or degreasing It may further comprise any one of the steps of washing with distilled water in order to remove.

In addition, for example, in the case of using pepsin diluted in an acid such as acetic acid, thereafter, adding a salt such as sodium chloride to adjust the concentration of the solution, centrifuging the precipitate and dissolving it with an acid such as acetic acid, Dialysis using acetic acid / distilled water, and freeze-drying (eg, -20 to 10 ° C) the sample obtained by dialysis.

On the other hand, when using the shell of the fish as a starting material, the step of separating the collagen, the shell of the pulverized fish with an alkaline solution such as sodium hydroxide or calcium hydroxide solution of 0.01-1.0 M concentration 1:20-1:50 ( mixing and treating at a ratio of w / v), insoluble fish shell obtained by treatment of an alkaline solution, such as for example butyl alcohol, having 1 to 4 carbon atoms such as butyl alcohol, 1:20 to 1:50 (w / and degreasing by adding in a proportion of v), and hydrolyzing by adding pepsin to the degreased fish shell residue. Like fish bones, pepsin can be used, for example, in diluted form with an acid such as acetic acid.

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

Collagen including collagen type I has three polypeptide chains bound together to form a helix structure, and has a telopeptide structure having no helix structure at both ends of the molecule. In the collagen molecular structure, telopeptide is the part where intra-molecule and inter-molecule crosslinking takes place. That is, in vivo, collagen is insoluble by crosslinking of molecules in the telopeptide moiety. When this insoluble collagen is treated with a protease such as pepsin, the telopeptide of the crosslinked moiety can be removed and digested to obtain a solubilized atherocollagen.

Subsequently, collagen hydrolyzate is recovered and obtained by a pressurized hydrothermal hydrolysis reaction in which high-temperature, high-pressure water is added to pepsin soluble collagen derived from fish.

For example, the pressurized hydrothermal hydrolysis process using high temperature and high pressure water according to the present invention may be performed using the hydrolysis equipment shown in FIG. 2. For example, collagen samples obtained from fish-derived bones and / or shells, such as fish, are transferred from sample collector 8 to reactor 5 and filled with water, such as distilled water. In one exemplary embodiment of the present invention, the collagen sample and water that can be converted into a subcritical state can be reacted by mixing in the reactor 5 at a ratio of 1: 100-1: 300 w / v. Then, the temperature controller 7 and the pressure gauge 2 are operated to hydrolyze the collagen by the pressurized hydrothermal reaction to adjust the inside of the reactor 5 to a predetermined temperature and pressure. At the same time, using a stirrer 6 attached inside the reactor 5 to maintain uniformity of the collagen and subcritical water mixture injected into the reactor 5 while inducing a uniform pressure and heat distribution throughout the sample. can do.

SDS-PAGE analysis of pepsin soluble collagen isolated from fish processing by-products such as bone, shell and / or intestine of fish through the process described above according to an exemplary embodiment of the present invention resulted in three chains (two α-chains). And 1 β-chain) to which collagen type I was bound (see FIGS. 3 to 5), and the heat denaturation characteristics and viscosity were also shown to be good (see Example 5). Thermal denaturation properties may be related to the content of proline and hydroxyproline, which are the major amino acids of collagen, as well as the imino acids involved in the thermal stability of collagen.

In the present invention, the collagen hydrolyzate derived from the bone and / or shell of the fish recovered through the above-described process was measured by molecular weight measurement, amino acid composition analysis and antioxidant activity by MALDI-TOF-MS analysis. According to the embodiment of the present invention, all of the molecular weight of the collagen hydrolyzate obtained by the hydrothermal hydrolysis reaction was 2000 Da or less, it was confirmed that significantly reduced compared to the molecular weight of several hundred kDa of the general collagen (Figs. 7 to 9 and Examples) 7). This analysis means that collagen hydrolysates derived from fish bones and / or shells finally recovered and obtained according to the present invention can be rapidly introduced into the human body.

In addition, the collagen hydrolyzate obtained according to an exemplary embodiment of the present invention has a relatively high content of lysine, an amino acid that helps people suffering from anorexia and anemia, and also has an important function in forming connective tissue of the body. It contains a lot of glycine, proline and hydroxyproline to be performed (see Table 7 in Example 8). 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 FIGS. 10 to 13).

In the embodiment of the present invention, the high molecular weight collagen was isolated using the bone or shell of the fish as a starting material, and collagen hydrolyzate, that is, low molecular weight collagen, was obtained by the hydrothermal hydrolysis. However, it is also possible to obtain low molecular weight collagen by press hydrothermal hydrolysis using commercially available high molecular weight collagen products extracted from fish processing by-products including fish bones, shells, guts and the like as starting materials.

As described above, according to the present invention, collagen may be separated from fish processing by-products, and hydrolysates containing collagen peptides and amino acids may be obtained through pressure hydrothermal hydrolysis. Since the collagen hydrolyzate recovered according to the present invention not only has an antioxidant activity but can be relatively easily administered to the human body, it can be added as a functional ingredient of cosmetics, drugs and / or foods, and thus can be utilized in various industries. It is expected to be.

Example

In the following, the present invention will be described in more detail by way of exemplary examples, but the present invention is in no way limited to the invention described in the examples described below.

material

The mackerel (Scomber japonicus ) processing by-product was provided by a seafood processing company in Dongwonhae Sarang Co., Ltd. in Busan. Reagents used (pepsin, protein marker, terrestrial collagen, 2,2-diphenyl-1-picrylhydrzyl (DPPH), 2,2-azino-bis (3-ethylbeznothiazoline-6-sulfonic acd) diammonium salt (ABTS), potassisum ferricyanide, 3- (2-pyridyl) -5,6-bis (4-phenyl-sulfonic acid) -1,2,4-triazine (ferrozine), 6-hydroxy-2,5,7,8-tetramethyl chroman- 2-carboxylic acid (trolox) was purchased from Sigma Aldrich (St. Louis, MO., USA), and other reagents and solvents were used for analytical or HPLC grade.

Example 1 Component Analysis of Mackerel Bones and Shells

The bones and shells of the mackerel were washed in cold water and the samples were freeze dried for 72 hours. The dried sample was ground in a homogenizer and stored at -20 ° C. In order to analyze the general composition of mackerel bone and skin, water, ash, crude fat and protein content were measured according to the Association of Official Analytical Chemists (AOAC) method. Non-protein content was calculated by subtracting the total content of moisture, ash, fat and protein. In this example, freeze-dried mackerel bone and skin samples were used. Table 1 is a result of the analysis of the content of the major components in the dried mackerel bone and shell, it can be seen that the dried mackerel shell is relatively higher in the content of protein and lipid than the dried mackerel bone.

Table 1 Main Components of Dried Mackerel Bones and Shells

sample	moisture (%)	Ash content (%)	Crude fat (%)	Crude Protein (%)	Non-protein (%)
Dry mackerel bone	6.68 ± 0.07	6.89 ± 0.06	17.59 ± 1.10	47.25 ± 1.25	21.59 ± 1.28
Dry mackerel shells	4.60 ± 0.06	4.50 ± 0.05	29.47 ± 1.26	49.49 ± 1.52	11.94 ± 0.76

Example 2: Pepsin Soluble Collagen Isolation from Mackerel Bones and Shells

Pepsin-solubilized collagen (PSC) in the mackerel bones and shells was isolated at -4 ° C as follows.

To remove the collagen free portion, the ground mackerel bone was mixed with 0.1 M NaOH in a 1:10 (w / v) ratio. The mixture was continuously stirred at 250 rpm for 24 hours with a magnetic stirrer. At this time, the alkaline solution was exchanged every 6 hours. The sample was then washed with cold distilled water and lyophilized until the pH of the sample reached neutrality (EYELA FDV-2100, Rikakikai Co. Ltd., Tokyo, Japan). Thereafter, 0.5 M ethylenediaminetetraacetic acid (EDTA, pH 7.5) was added to the sample in a 1:10 (w / v) ratio and decalcified for 4 days. EDTA solution was changed daily. The residue was washed with cold distilled water and the recovered residue was mixed with 10% (v / v) butyl alcohol aqueous solution at a ratio of 1:10 (w / v) and treated for 24 hours. After washing again with distilled water, the residue was mixed with 0.5% M acetic acid solution containing 0.1% (w / v) pepsin at 1:10 (w / v) for 3 days and treated at 12,000 rpm for 50 minutes. Centrifugation was performed. The residue was extracted again with the same solution for 3 days and centrifuged under the same conditions. NaCl was added to the viscous solution to finally adjust the concentration to 2.0M. The solution was incubated for 24 hours and the resulting precipitate was centrifuged at 12,000 rpm for 20 minutes and then dissolved in 0.57M acetic acid. The solution was dialyzed with 0.1 M acetic acid and distilled water, placed in a dialysis bag and stored for 2 hours, and then lyophilized to obtain pepsin soluble collagen powder.

Collagen was isolated from the mackerel shell as follows. 0.1M NaOH was mixed at a ratio of 1:35 (w / v) in the ground mackerel shell, and then treated with a magnetic stirrer for 24 hours while stirring. The alkaline solution was then replaced four times a day. And washed with cold distilled water, and then lyophilized. The freeze-dried sample was mixed with 10% (w / v) butyl alcohol aqueous solution at a ratio of 1:35 (w / v) and degreased for 24 hours. The degreased residue was washed with cold distilled water, and the residue was then treated for 3 days by mixing 1:35 (w / v) in 0.57M acetic acid solution containing 0.1% (w / v) pepsin. Then, the same procedure as when separating collagen from mackerel bone was performed to obtain pepsin soluble collagen powder.

The collagen content in pepsin soluble collagen powder obtained from mackerel bone and skin was determined by hydroxyproline according to the method described in the literature (DE Goll et al., 1963 J. Food Science , 28 (5): 503-509), Obtained by multiplying the conversion factor. The content of hydroxyproline was quantified by slightly modifying the method described in the literature (I. Bergman et al., 1963, Analytical Chemistry , 35 (12): 1961-65).

Table 2 below shows the results of collagen (protein) and general ingredient contents in pepsin soluble collagen powder obtained from mackerel bone and skin, respectively. The skin content of pepsin soluble collagen was higher than that of mackerel bone. Mackerel bone and skin protein contents were 90.05 ± 2.34 and 86.89 ± 2.48%, respectively. The mackerel bone and skin moisture content was 6.28 ± 0.12 and 7.48 ± 0.10%, the ash content was 3.48 ± 0.09 and 5.37 ± 0.06%, and the fat content was 0.19 ± 0.02 and 0.26 ± 0.03%.

TABLE 2 Protein (collagen) and general ingredient content in pepsin soluble collagen powder obtained from dried mackerel bone and skin

Furtherance(%)	Pepsin Soluble Collagen Powder
	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
Geology	0.19 ± 0.02	0.26 ± 0.03

Example 3: Determination of Molecular Weight of Pepsin Soluble Collagen

The molecular weight of the pepsin soluble collagen powder obtained in Example 2 was measured using SDS-PAGE. Proteins were analyzed by Laemmli's modified method using 3.0% stacking gel and 5.0% resolving gel. After dissolving 2 g of the pepsin soluble collagen (PSC) powder sample obtained in Example 2 in 1.0 mL of 0.02 M sodium phosphate buffer (pH 7.2), the dissolved sample was buffered (1M Tris-HCl, pH 6.8, 10% SDS, 25% glycerol, 2% bromophenol blue, 5% 2-mercaptoethanol) and 1: 1 (v / v) ratio was mixed. 20 μL of the mixed sample prepared above was attached to polyacrylamide gels and separated by continuous electrophoresis at 30 mA current. In the same manner, collagen isolated from calf crust and bovine tendon was used as a standard, and compared with pepsin soluble collagen isolated from bone and skin of mackerel in Example 2. In addition, markers with high molecular weight (MW) were used to specify the molecular weight of the protein.

SDS-PAGE pattern results for the molecular weight size of pepsin soluble collagen (PSC) isolated from mackerel bone and skin are shown in FIG. 3. The two α-chain (α ₁ and α ₂ ) collagens had molecular weights of 116 kDa (α ₁ ) and 126 kDa (α ₂ ), respectively, and showed similar values to those of standard collagen. The β-chain collagen isolated from the bones and shells of the mackerel had a molecular weight of 205 kDa. These results indicate that the collagen isolated from the bones and shells of the mackerel has a molecular weight similar to that of animal-derived collagen.

Example 4 FT-IR Analysis of Pepsin Soluble Collagen

FT-IR analysis was performed on pepsin soluble collagen (PSC) powders obtained from bone and skin of mackerel respectively in Example 2. PSCs FT-IR spectra of freeze-dried mackerel bone and skin were obtained using Perkin Elmer (USA), Spectrum X, and the spectra were measured at resolutions of 4000-650 cm ^-1 and 4 cm ^-1 . FT-IR spectra of collagen isolated from mackerel bone and skin are shown in FIGS. 4 and 5, respectively. As can be seen in these figures, the band of collagen isolated from the mackerel bone and skin was 3283 cm ^-1 and It appeared at 3285 cm ^-1 . The wavelengths for 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 (1600-1660 cm ^-1 ) is derived from the carbonyl groups (C = O) in the polypeptide. It is related to vibration. Amide II bands (~ 1550 cm ^-1 ) are associated with NH bending and CN stretching vibrations, while amide III bands (1220-1320 cm ^-1 ) are associated with CN stretching vibrations and NH deformation. From the results of the FT-IR spectrum analysis, it was confirmed that the material obtained in Example 2 was collagen.

Example 5: Determination 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 viscosity of the bone and skin of the mackerel was measured using a Brookfield DVII + Pro viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA 02346 USA) as follows. 8 mL of 0.1M acetic acid containing 0.1% (w / v) PSC was incubated at 10 ° C. for 20 minutes and then placed in a container. Then, the viscosity of the sample was measured by rotating at 150 rpm using a viscosity measuring device component SC4-18, and the unit of viscosity was expressed in centipose (cP). Collagen has a high viscosity is an important feature, the viscosity of collagen (PSC) separated from the mackerel bone and shell according to the present embodiment is shown in Table 3. The viscosity ranges were 18.34 ± 0.25 and 20.26 ± 0.21 cP.

In addition, denaturation temperature (Td) was measured as follows. Samples were taken at 3 ° C intervals while heating 8 mL of 0.1M acetic acid containing 0.1% (w / v) PSC from 10 ° C to 40 ° C. Viscosity was measured after the collected solution was stored at the collected temperature for 20 minutes. The heat denaturation curve of the PSC showed a slight change in viscosity with temperature (see FIG. 6). The fractional viscosity was calculated by dividing the maximum viscosity by the viscosity measured at each temperature, and the temperature at which the fractional viscosity approached 0.5 was determined as the denaturation temperature. Table 3 below shows the viscosity and denaturation temperature of the collagen isolated from the bone and shell of the mackerel.

TABLE 3 Viscosity and Denaturation Temperature of Collagen Isolated from Mackerel Bones and Mackerel Shells

Properties	Collagen
	bone	skin
Viscosity (cP)	18.34 ± 0.25	20.26 ± 0.21
Denaturation temperature (℃)	27	30

Example 6: Obtaining Collagen Hydrolyzate Using Pressurized Hydrothermal Hydrolysis

The pepsin soluble collagen obtained in Example 2 was hydrolyzed using a high temperature, high pressure batch reactor such as that shown in FIG. 2. As a hydrolysis reactor, a hydrolysis reaction was carried out using a high-pressure reactor (material: Hastelloy 276, Ilshin Autoclave Co., Ltd.) equipped with a temperature controller and a pressure gauge as described below in a batch reaction of 200 mL.

Collagen (0.5 g) and water obtained in Example 2 were mixed at a ratio of 1: 200 (w / v) and injected into the reactor. The heater was heated to a temperature of 200-250 ° C. and a pressure range of 30-70 bar using an electric heater, and the temperature and pressure of the reactor were measured using a thermostat and a pressure gauge during the experiment. Stirred using a magnetic stirrer at 150 rpm. The desired temperature was reached after 26-54 minutes heating and held at this temperature for 3 minutes. After cooling by connecting a cooling circulation jacket to the reactor, the hydrolyzate was recovered from the reactor and filtered using filter paper.

Example 7: MALDI-TOF Mass Spectrometry on Collagen Hydrolysates

In order to determine the molecular weight of the collagen hydrolyzate obtained in Example 6, Matrix Assisted Laser Desorption / Ionization-Time Of Flight-Mass Spectrometry (MALDI-TOF-MS) analysis was performed on the recovered collagen hydrolyzate. In Example 6 1 μL of a sample of pepsin soluble collagen (PSC) hydrolyzate isolated from the bone and shell of the mackerel was attached to a polished steel 384 target plate. 1 μL of 2,5-dihydroxybenzoic acid (DHB) and 1% THF were 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 of 377 nm. Data were obtained by accumulating data from 200 laser shots in cationization and reflection mode at 700-6000 m / z.

The analysis results of Example 7 are shown in FIGS. 7 to 9 and Tables 4 to 6 below. As shown in these figures and tables, when the collagen isolated from the mackerel bone was hydrolyzed at 200 ° C. and 30 bar, the molecular weight range of the peptide was about 759-988 Da, and the collagen singer of the shell of the mackerel treated under the same conditions Peptide molecular weights of the digests ranged from 789-1632 Da. Hydrolysis reaction conditions The molecular weight of the peptide present in the collagen hydrolyzate of the mackerel shell treated at 250 ° C. and 70 bar showed a range of 952-1638 Da. From these results, it can be seen that the molecular weight of the collagen hydrolyzate is significantly reduced compared to the molecular weight range 116-126 kDa of the non-hydrolyzed collagen. Peptides present in the hydrolyzate were identified using the Mascot Database, and the expected peptides are shown in Tables 4-6.

Table 4 shows the results of MALDI-TOF mass spectrometry (200 ° C., 30 bar) of mackerel bone collagen hydrolyzate, and Table 5 shows the results of MALDI-TOF mass analysis (200 ° C., 30 bar) for mackerel skin collagen hydrolyzate. 6 shows the results of MALDI-TOF mass spectrometry (250 ° C., 70 bar) on mackerel bone collagen hydrolysates, respectively. (Abbreviations shown in Tables 4-6: A-alanine, C-cysteine, D-aspartic acid, E-glutamic acid, F-phenylalanine, G-glycine, H-histidine, I-isoleucine, K-lysine, M-methionine, N-asparagine, P-proline, Q-glutamine, R-arginine, S-serine, T-threonine, V-valine, W-tryptophan, Y-tyrosine; ^* Predicted peptides are mascot databases (http: // www. available at matrixscience.comd)

Table 4 MALDI-TOF Mass Spectrometry of Mackerel Bone Collagen Hydrolysates (200 ° C, 30 bar)

m / z	Molecular weight (dalton)	Predicted peptide *	Intensity
760.13	759.12	K.TKATLAR.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

Table 5 MALDI-TOF Mass Spectrometry of Mackerel Shell Collagen Hydrolysates (200 ° C, 30 bar)

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

Table 6 MALDI-TOF Mass Spectrometry of 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

Example 8: Amino Acid Composition Analysis for Collagen Hydrolysate

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. S430 (SYKAM) amino acid automated analyzer was used for amino acid content analysis. The cation separation column LCA K07 / Li (4.6 × 150 mm) had a temperature of 37-74 ° C. and a buffer pH range of 2.90-7.95. The mobile phase was 1, was used a flow rate 5 mM p-toluenesulphonic acid solution per minute at 0.45 mL, 5 mM p -toluenesulphonic acid and, 20 mM of Bis-Tris, post-column reagent a mixture of EDTA 100 m at a flow rate of 0.25 mL per minute Used as. The emission wavelength was 440 and 570 nm. The composition and content of amino acids in the hydrolyzate of collagen isolated from mackerel bone and skin are shown in Table 7. In view of the change in amino acid content depending on the applied temperature and pressure conditions, it can be seen that the temperature and pressure of the hydrothermal reaction are important variables in the hydrolysis reaction. The glycine content was high in the amino acids present in the hydrolyzate, about 27.90-29.44% for the hydrolyzate of mackerel and about 32.38-35.25% for the hydrolyzate of the mackerel shell.

TABLE 7 Amino Acid Composition of Collagen Hydrolyzate

	(mg / g)
Amino acid composition	Bone collagen hydrolyzate		Skin collagen hydrolyzate
	Temperature (℃) / 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
α-aminobutyric acid	1.09 ± 0.04 a	1.15 ± 0.03 a	0.59 ± 0.03 b	0.31 ± 0.02 b
Valine	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
Cystation	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
β-alanine	0.01 ± 0.01	0.04 ± 0.01	0.02 ± 0.01	0.01 ± 0.01
β-aminobutyric acid	0.18 ± 0.02	0.12 ± 0.02	0.10 ± 0.01	0.11 ± 0.01
γ-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

Example 9 Analysis of Antioxidant Activity of Collagen Hydrolysates

The antioxidant activity of the collagen hydrolyzate obtained in Example 6 was confirmed by DPPH free radical scavenging ability, ABTS free radical scavenging ability, Ferric reducing power activity, Fe ²⁺ chelating activity method.

DPPH Free Radical Scavenging Assay

For DPPH free radical scavenging analysis, 3.95 mL of 0.1 mM DPPH methanol solution was added to Test tubes containing 0.1 mL of collagen samples at various concentrations before and after hydrolysis. Mix the mixtures for 10 seconds each, in the dark Incubated at room temperature for 30 minutes. Absorbance of all samples was measured at 517 nm. DPPH free radical scavenging ability was calculated by the following equation.

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

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

Experimental and measured experiments (variable concentrations of trolox in methanol) were analyzed using the same procedure described above.

ABTS + Free Radical Removal Assay

The ABTS ⁺ free-radical-removing method was performed as follows. A 7 mM ABTS aqueous solution and a 2.45 mM potassium sulfate aqueous solution were mixed at a volume ratio of 1: 1, and reacted at room temperature in a dark room to prepare an ABTS ⁺ solution, and stored for 12-16 hours before use.

ABTS ⁺ solution was diluted to 80% methanol such that the absorbance 0.70 ± 0.02 at 734nm, diluted with ABTS ⁺ solution 3.95 mL to PSC and its hydrolyzate samples in a respective addition of 0.05 mL, and then, the mixture was a dark environment 6 minutes It was kept at room temperature. Absorbance of all samples was measured at 734 nm. The free radical scavenging activity ABTS ⁺ percent was calculated by the following formula.

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

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

The blank and measured experiments (variable concentrations of trolox in 80% methanol) were analyzed as described above.

Ferric reducing power assay

The Fe ³⁺ reducing power of the PSC sample and the hydrolyzate was determined in the following manner, respectively. Before and after hydrolysis, 0.125 mL of PSC samples were mixed with 0.625 mL of 0.2M phosphate buffer, pH 6.6, 0.625 mL of 1% potassium ferricyanide at various concentrations. This mixture was incubated at 50 ° C. for 20 minutes. After centrifugation at 3,000 rpm for 10 minutes, 0.625 mL of 10% trichloroacetic acid was added. 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. And absorbance was read at 700 nm. The increase in absorbance was found to be associated with an increase in reducing power.

Fe ²⁺ Chelation Assay

The Fe ²⁺ chelate capacity of the samples was determined in the following manner. PSC samples (0.1 mL) were mixed with 3.0 mL of distilled water at various concentrations before and after hydrolysis. In addition, 50 μL of 2 mM FeCl ₂ and 0.1 mL of 5 mM ferrozine were added to PSC samples before and after hydrolysis, and 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. Chelating activity (%) was calculated using the following formula.

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

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

The analysis results of this example are shown in FIGS. 10 to 13 and Table 8 below. Figure 10 shows the DPPH radical scavenging ability of the hydrolyzate of collagen separated from the mackerel bone and skin by hydrothermal reaction, showing a relatively high antioxidant activity at a concentration of 10 mg / mL. 11 is a comparison of the ABTS scavenging ability of the collagen (PCS) and the hydrolyzate isolated from the mackerel bone and skin, the antioxidant properties of the hydrolyzate was higher than the collagen before hydrolysis. 12 and 13 show the results of Fe ³⁺ reducing power and Fe ²⁺ chelate activity of the collagen before hydrolysis and the collagen peptide-containing hydrolysate after hydrolysis, and showed higher activity in hydrolyzate after hydrothermal reaction than before hydrolysis of fish collagen. It is showing. The hydrolyzate of collagen isolated from the mackerel shell obtained at 250 ° C and 70 bar showed higher Fe ³⁺ reducing power than the collagen hydrolyzate from mackerel bone at various concentrations. The collagen hydrolyzate of mackerel bone and skin shows a similar Fe ²⁺ chelating effect, indicating that the hydrolyzate of collagen isolated from mackerel bone and skin can function as a Fe ²⁺ chelator. PSCs IC ₅₀ values in mackerel bone were 8.21 ± 0.15 mg / mL in samples hydrolyzed at 200 ° C and 30 bar, and 7.27 ± 0.14 mg / mL in samples hydrolyzed at 250 ° C and 70 bar. In the case of the sample hydrolyzed at 200 ℃, 30 bar condition is 7.91 ± 0.11 mg / mL, the sample hydrolyzed at 250 ℃, 70 bar condition shows an IC ₅₀ value of 7.01 ± 0.12 mg / mL. Table 8 below shows the DPPH, ABTS radical scavenging activity and Fe ²⁺ chelating activity (IC ₅₀ ) of the collagen hydrolyzate, and these values are higher than the reference value.

Table 8 DPPH, ABTS radical scavenging activity and Fe <sup> 2 + </ sup> chelating activity of collagen hydrolysates (IC <sub> 50 </ sub>)

Activity	Collagen / Reference	IC 50 (mg / mL)
DPPH free radical scavenging activity	Collagen hydrolyzate of bone (200 ℃, 30 bar)	8.71 ± 0.14 c
	Collagen hydrolyzate of bone (250 ℃, 70bar)	8.38 ± 0.13 c
	Collagen hydrolyzate of skin (200 ℃, 30 bar)	9.57 ± 0.12 d
	Collagen Hydrolyzate of Skin (250 ℃, 70bar)	7.58 ± 0.10 b
	Trolox (reference)	0.35 ± 0.05 a
ABTS free radicalscavenging	Collagen Isolated From Bone	10.77 ± 0.11 d
	Collagen hydrolyzate of bone (200 ℃, 30bar)	4.18 ± 0.07 c
	Collagen hydrolyzate of bone (250 ℃, 70bar)	2.61 ± 0.05 b
	Collagen Isolated from Peel	12.12 ± 0.13 d
	Collagen hydrolyzate of skin (200 ℃, 30bar)	4.05 ± 0.08 c
	Collagen Hydrolyzate of Skin (250 ℃, 70bar)	2.50 ± 0.05 b
	Trolox (reference)	0.25 ± 0.03 a
Fe 2+ chelating	Collagen hydrolyzate of bone (200 ℃, 30bar)	8.21 ± 0.15 c
	Collagen hydrolyzate of bone (250 ℃, 70bar)	7.27 ± 0.14 b
	Collagen Hydrolyzate of Skin (200 ℃, 30bar)	7.91 ± 0.11 c
	Collagen hydrolyzate of skin (250 ℃, 70 bar)	7.01 ± 0.12 b
	EDTA (reference)	0.34 ± 0.06 a

Example 10 Analysis of Volatile Materials (VOCs)

Various volatile organics resulting from collagen recovered from mackerel bone and skin are shown in Tables 9 and 10. The hydrolyzate of mackerel bone and skin produced by subcritical water pressurized hydrolysis has either lost some volatile organics or produced new volatile organics. Among the volatile organic compounds identified in the collagen peptides and amino acid-containing hydrolysates produced in the mackerel bones and shells, methylene chloride, methyl ethyl ketone, 3-methylbutanal and 2-methylbutanal are the components that have a good effect on quality. This volatile organic substance was superior to the odor sensor than the volatile organic substance generated from the collagen of mackerel bone and skin before hydrolysis by subcritical water. Table 9 shows the volatiles (VOCs) composition of mackerel bone collagen and hydrolyzate, respectively.

Table 9 Composition of Volatile Compounds (VOCs) for Mackerel Bone Collagen and Hydrolysates

Volatile organic compounds	Area (%)
	Mackerel bone	Mackerel bone collagen	Bone collagen hydrolyzate (200 ℃, 30bar)	Bone collagen hydrolyzate (250 ℃, 70bar)
Ethanal	3.50	ND	ND	ND
Parapropionaldehyde	15.57	7.53	ND	ND
Furan	2.21	ND	ND	ND
Methylene chloride	ND	1.89	3.04	2.11
Isobutanal	ND	ND	14.42	14.90
Propanal, 2-methyl-	3.14	ND	ND	ND
Propanol	2.52	ND	ND	ND
Butanal	9.85	3.58	2.50	2.16
Methyl ethyl ketone	2.75	ND	5.50	6.76
Furan, 2-methyl-	1.50	ND	ND	ND
2-Butenal	1.60	ND	ND	ND
3-Methylbutanal	5.67	ND	26.61	23.11
2-Methylbutanal	2.89	ND	17.01	16.03
1-Penten-3-one	3.40	ND	ND	ND
2-pentanone	ND	ND	1.18	1.44
2-Ethylfuran	4.01	ND	ND	ND
Furan, 2-ethyl-	9.44	ND	ND	ND
Heptane	2.17	ND	ND	ND
2-Pentenal, (E)-	1.69	ND	ND	ND
1-Pentanol	2.51	ND	ND	ND
n-Hexanal	9.65	5.03	1.26	ND
Heptane, 2,4-dimethyl-	2.09	ND	ND	ND
Heptanal	3.93	2.02	ND	ND
Octanal	2.70	2.07	ND	ND

Table 10 below shows the composition of volatiles (VOCs) of mackerel shell collagen and hydrolyzate, respectively.

Table 10 Composition of Volatile Compounds (VOCs) for Mackerel Shell Collagen and Hydrolysates

Volatile organic compounds (VOCs)	Area (%)
	Mackerel skin	Mackerel skin collagen	Skin collagen hydrolyzate (200 ℃, 30bar)	Skin collagen hydrolyzate (250 ℃, 70bar)
Diethoxyethane	16.66	ND	ND	ND
Propionaldehyde	22.37	8.13	ND	ND
Ethyl formate	4.48	ND	ND	ND
Methylene chloride	ND	ND	5.15	3.09
Isobutanal	1.48	ND	14.13	14.09
Butanal	7.78	2.64	1.85	2.36
2-Butanone	2.22	ND	ND	ND
Methyl ethyl ketone	ND	ND	6.03	6.61
Chlororoform	1.91	ND	ND	ND
2-Butenal	2.58	ND	ND	ND
3-Methylbutanal	2.08	ND	19.18	20.05
2-Methylbutanal	ND	ND	12.49	14.48
1-Penten-3-one	3.11	ND	ND	ND
Hydroxycyclopentane	8.90	ND	ND	ND
Furan, 2-ethyl-	2.58	1.32	ND	ND
n-Heptane	1.58	ND	ND	ND
n-Hexanal	6.92	5.27	ND	ND
1,2-Butylene glycol	1.38	ND	ND	ND
Heptane, 2,4-dimethyl-	2.01	ND	ND	ND
Cyclotrisiloxane, hexamethyl-	ND	1.34	ND	ND
Allyl Isothiocyanate	ND	1.29	ND	ND
Heptanal	2.44	4.72	ND	ND
Octanal	1.31	3.90	ND	ND
Nonane, 3-methyl-5-propyl-	1.53	ND	ND	ND

As mentioned above, this invention was demonstrated based on the preferable Example of this invention. The above embodiments are merely examples of the present invention, and the scope of the present invention is not limited to the above-described embodiments. Rather, those skilled in the art to which the present invention pertains will be able to easily make various modifications and changes based on the above-described embodiments. However, it will be more apparent from the appended claims that such variations and modifications are all within the scope of the present invention.

According to the present invention, the low molecular weight collagen peptide hydrolyzate produced by using a pressurized hydrothermal reaction from fish bones, shells, intestines, etc. generated by the processing of fish can be absorbed into the living body due to its molecular weight of 3 kDa or less. It contains glycine and proline, which are important materials for tissue regeneration, and exhibits antioxidant activity, so it can be usefully used in various industrial fields, including medicines, dietary supplements, and cosmetics.

Claims (8)

1. A method of obtaining low molecular weight collagen derived from fish, comprising the step of hydrothermally hydrolyzing a high molecular weight collagen powder extracted from at least one fish processing by-product selected from the group consisting of fish bones, shells and intestines.
2. The method of claim 1,

   The low molecular weight collagen has a molecular weight of less than 3,000 Da, a method for obtaining a low molecular weight collagen derived from fish.
3. The method of claim 1,

   The method for obtaining low molecular weight collagen derived from fish, characterized in that the pressurized hydrothermal hydrolysis reaction is used water at a temperature of 150-370 ℃ and 5-400 bar pressure.
4. The method of claim 1,

   Before the step of hydrothermal hydrolysis of the high molecular weight collagen powder, further comprising the step of extracting a high molecular weight collagen powder from the dried and ground fish processing by-product sample, wherein the high molecular weight collagen powder A method for obtaining low molecular weight collagen derived from fish, which is used as a starting material for the decomposition reaction step.
5. The method of claim 1,

   Extracting high molecular weight collagen powder may include treating the ground mackerel bone with an alkaline solution diluted in a ratio of 1: 5-1:20 (w / v); Decalcification by mixing acid in an insoluble mackerel bone obtained by treatment of an alkaline solution in a ratio of 1: 5-1:20 (w / v); Degreasing treatment by adding alcohol to the decalcified mackerel bone in a ratio of 1: 5-1:20 (w / v); And hydrolyzing by adding pepsin to the degreased mackerel bone residue.
6. The method of claim 1,

   Extracting the high molecular weight collagen powder may include treating the ground mackerel with an alkaline solution diluted in a ratio of 1:20-1:50 (w / v); Degreasing treatment by mixing alcohol in an insoluble mackerel shell obtained by treatment of an alkaline solution in a ratio of 1:20-1:50 (w / v); And hydrolyzing by adding pepsin to the degreased mackerel shell residue.
7. A method for producing low molecular weight collagen derived from fish, comprising hydrolyzing high molecular weight collagen powder extracted from at least one fish processing by-product selected from the group consisting of fish bones, shells and intestines with high temperature and high pressure water.
8. The method of claim 7, wherein

   The low molecular weight collagen has a molecular weight of less than 3,000 Da, a method for producing low molecular weight collagen derived from fish.

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