KR101625580B1 - Mackerel muscle hydrolyzates production with biological functionalities using subcritical water - Google Patents

Abstract

The present invention relates to a method for hydrolyzing mackerel meat by using subcodes to recover and obtain a physiologically active ingredient. According to the present invention, a variety of physiologically active ingredients can be obtained by hydrolyzing mackerel meat using high temperature and high pressure ashing factors and subcritical carbon dioxide. Compared with conventional enzymatic treatment methods and chemical methods, according to the present invention, it is possible to safely recover physiologically active ingredients from mackerel meat through a simple process. Since the physiologically active ingredient thus obtained can be added as a functional ingredient such as medicine, food and / or cosmetic, it is expected that the physiologically active ingredient can be utilized in the medical industry, bio industry, cosmetics industry and food industry through the process of the present invention.

Description

MACKEREL MUSCLE HYDROLYZATES PRODUCTION WITH BIOLOGICAL FUNCTIONALITIES USING SUBCRITICAL WATER BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to a method for recovering a functional component from a living body, and more particularly, to a method for recovering and obtaining a physiologically active ingredient by hydrolyzing mackerel meat using an asymptotic coefficient.

Mackerel ( Scomber japonicus ) is a marine fish belonging to the mackerel family, and is widely distributed in the temperate and subtropical waters of the Pacific Ocean, Indian Ocean and Atlantic Ocean. As a seasonal breeding season, the mackerel living in the northern hemisphere migrates to the north in the summer when the water temperature rises and migrates to the south in winter.

Mackerel is one of the most important fishery resources for food, as it is very popular in the world as well as in Korea. Mackerel is mainly found in the upper layer of seawater, so it is tougher and more prone to decay than deep sea fish. Not only is mackerel so easy to decay, but the protein contained in mackerel has a high content of basic amino acid histidine.

Histidine turns into a harmful ingredient called histamine in the early days when mackerel decay begins when the lead falls. When histamine accumulates in the human body, it causes abnormalities in metabolism, causing various side effects and allergic phenomena, which can lead to food poisoning, abdominal pain and diarrhea if the mackerel is mistakenly ingested. Because of this, in Korea, mackerel is salted and stored in salt mackerel.

In particular, mackerel is rich in omega-3 fatty acids, an unsaturated fatty acid that serves as a blood coagulant, which cleans blood, and is therefore useful for the prevention of diseases related to blood vessels of modern people who consume high fat, high calorie foods, The intake of mackerel to prevent disease is also known to exert an excellent effect. Omega-3 fatty acids not only prevent vascular disease, but also help improve brain function to improve memory, as well as reduce the likelihood of mental illness such as depression, dementia and attention deficit disorder.

Therefore, it is necessary to utilize physiologically active ingredients contained in a large amount of fishery resources in addition to consumption of fishery resources such as mackerel, which is widely distributed in the coast of Korea, It is necessary to study how to do this. In particular, many mackerels are not commercially available as processed foods because they are scratched during the capture process. Taking this into consideration, the method of applying the fishery resources such as mackerel, which can not be utilized as food, to the high value added industry is a desirable choice in terms of efficient use of fishery resources.

To date, sugar components have been extracted and recovered by chemical methods such as hydrolysis methods using acids or acid decomposition methods using fish as raw materials. Hydrolysis using an enzymatic method specifically acts on the decomposition of macromolecules, but the enzymes used in the enzymatic methods are not only economical but also require a long time to complete the process because they are expensive .

On the other hand, the chemical decomposition method is economical, but it requires vigorous reaction and may cause serious environmental pollution when the reaction by-products are treated. Especially, the physiologically active ingredients that can be utilized as functional ingredients of drugs, foods, And the recovery rate is low. Therefore, there is a need in the related industry for a method that can safely recover the physiologically active ingredient contained in mackerel in an environmentally friendly manner and utilize it as a health functional food such as pharmaceuticals, foods, and cosmetics.

Korea Patent Publication No. 2009-0070629

DISCLOSURE Technical Problem The present invention has been proposed in order to overcome the problems of the prior art described above and it is an object of the present invention to provide a functional physiologically active ingredient contained in a large amount in mackerel meat such as antioxidant activity, A method for stably recovering and obtaining a functional physiologically active ingredient such as an amino acid and / or a reducing sugar exhibiting biological activity such as antimicrobial activity and anticoagulant activity in a short period of time.

Another object of the present invention is to provide a method capable of recovering a functional physiologically active ingredient such as an amino acid and / or a reducing sugar by an environmentally friendly method and recovering it at a low cost with high yield.

The present invention having the above-mentioned object is characterized by hydrolyzing mackerel meat by treating the mackerel meat obtained by the drying and deoiling treatment with a high temperature and high pressure index, thereby extracting, recovering and obtaining a physiologically active ingredient such as amino acid, peptide and reducing sugar ≪ / RTI >

More specifically, the present invention relates to a method for producing a mackerel muscle, comprising: drying a mackerel muscle; Crushing the dried mackerel meat; Removing oil from the crushed mackerel meat to obtain oat mackerel meat; And recovering the physiologically active ingredient by hydrolyzing the de-mackerel mackerel meat by reacting the oily mackerel meat with a sublimation factor having a temperature of 150 to 350 DEG C and a pressure of 4 to 400 bar, The step of obtaining the deacidized mackerel meat comprises treating the ground mackerel meat with supercritical carbon dioxide having a temperature of 40 to 60 DEG C and a pressure of 300 to 500 bar, and in the step of hydrolyzing the de- A catalyst selected from the group consisting of acetic acid, sodium hydroxide, sodium chloride, sodium bicarbonate, carbon dioxide gas, nitrogen gas and a combination thereof is used, and the step of recovering the physiologically active ingredient comprises: Provides a method for obtaining a physiologically active ingredient from mackerel meat mixed at a ratio of 1:10 to 1:30 w / v.

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At this time, the physiologically active component obtained by hydrolysis of the mackerel meat according to the present invention may contain an amino acid and a reducing sugar.

For example, the physiologically active ingredient obtained by the hydrolysis of the mackerel meat may have any one of the antioxidant activity, the inhibition of angiotensin converting enzyme (ACE) activity, and the antimicrobial activity effect.

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According to the present invention, it is possible to recover and obtain physiologically active components such as free amino acid and reducing sugar from mackerel meat by dehydrating and hydrolyzing mackerel meat using high temperature and high pressure index and subcritical carbon dioxide.

Compared with conventional chemical processes and enzymatic processes, the hydrolytic hydrolysis method according to the present invention is much simpler in process, which is not only cost effective and excellent in stability, but also can reduce the energy consumed in the recovery of bioactive components Environmentally friendly. It is possible to extract an antioxidant component having a characteristic of not emitting exhaust gas at all from dried mackerel meat.

In addition, the physiologically active ingredient recovered according to the present invention exhibits not only antioxidant activity, but also an antioxidant activity and an antioxidative activity of angiotensin converting enzyme (ACE). As described above, the physiologically active ingredient recovered and obtained from mackerel meat according to the present invention can treat and prevent chronic diseases of modern people, and can inhibit various physiological activities related to diseases or promote physiological activities related to inhibition of diseases.

Therefore, the physiologically active ingredient recovered from mackerel meat according to the present invention can be added as a functional ingredient of functional cosmetics, functional foods, and the like, as well as a raw material for medicines for the treatment and prevention of diseases. Thus, using the process according to the present invention, it is expected that a functional material having a high value-added can be obtained and widely used for pharmaceuticals, foods, and cosmetics. Ultimately, using the process according to the present invention, the domestic fishery resources, which are surrounded by the sea on three sides, can be utilized in the high-value-added industry, beyond the use of mackerel as a simple food.

Brief Description of the Drawings Fig. 1 is a diagram showing the process of recovering and obtaining a physiologically active ingredient such as free amino acid and reducing sugar from mackerel meat according to an exemplary embodiment of the present invention, analysis of the obtained physiologically active ingredient, , And a flow chart showing a process of measuring the flow rate.
2 is a schematic view showing a device used for hydrolyzing mackerel meat dehulled using a high temperature and high pressure subzero index to obtain a physiologically active ingredient from mackerel meat according to an exemplary embodiment of the present invention .
3A and 3B are graphs showing the total amino acid content of the hydrolyzate obtained from mackerel meat using different subcategories of temperature and pressure according to an exemplary embodiment of the present invention, , Non-dehydrated dried mackerel meat, and dehydrated mackerel meat, and FIG. 3B is a graph showing the total amino acid content of the hydrolyzate of dehydrated mackerel meat using different catalysts. Mean ± standard deviation (n = 3).
FIG. 4 is a graph showing the content of essential amino acids in the mackerel hydrolysates obtained using different catalysts at different temperatures and pressures as a percentage. Mean ± standard deviation (n = 3).
Figures 5A and 5B are graphs illustrating the content of reducing sugars in hydrolysates obtained from mackerel meat using different subcategories of temperature and pressure according to an exemplary embodiment of the present invention, , Non-dehydrated dried mackerel meat, and dehydrated mackerel meat, and FIG. 5B is a graph showing the content of reducing sugar in the hydrolyzate of dehydrated mackerel meat using different catalysts. Mean ± standard deviation (n = 3).
Figures 6a and 6b are graphs illustrating DPPH free radical scavenging activity for hydrolysates obtained from mackerel meat using different temperature and pressure subclaims according to an exemplary embodiment of the present invention, DPBH free radical scavenging activity on the hydrolysates of dried mackerel meat, non-dehydrated mackerel mackerel meat, and dehydrated mackerel meat, and FIG. 6b shows DPPH free radical scavenging activity on dehydrated mackerel meat hydrolyzate Curl scavenging activity. Mean ± standard deviation (n = 3).
Figures 7a and 7b are graphs of ABTS free radical scavenging activity for hydrolysates obtained from mackerel meat using different temperature and pressure subclaims according to an exemplary embodiment of the present invention, ABTS free radical scavenging activity for hydrolysates of dried mackerel meat, non-dehydrated mackerel mackerel meat, and dehydrated mackerel meat, FIG. 7b shows ABTS free radical scavenging activity for dehydrated mackerel meat hydrolyzate, Curl scavenging activity. Mean ± standard deviation (n = 3).
Figures 8a and 8b are graphs illustrating the reduction potentials for hydrolysates obtained from mackerel meat using different subcategories of temperature and pressure according to an exemplary embodiment of the present invention, FIG. 8B is a graph showing a reduction power of the hydrolyzate of dehydrated mackerel meat by using other catalysts, and FIG. Mean ± standard deviation (n = 3).
9A and 9B are graphs showing the ACE inhibitory activity of a hydrolyzate obtained from mackerel meat using different subcategories of temperature and pressure according to an exemplary embodiment of the present invention, FIG. 9B is a graph showing ACE inhibitory activity against hydrolysates of dehydrated mackerel meat using different catalysts, and FIG. 9B is a graph showing ACE inhibitory activity against hydrolysates of dehydrated and dried mackerel meat. Mean ± standard deviation (n = 3).

The inventors of the present invention have developed a method for hydrolyzing mackerel meat using high temperature and high pressure asymmetric coefficients in order to extract, recover and obtain physiologically active components such as peptides, free amino acids and reducing sugars from mackerel muscle. The hydrolyzate derived from the mackerel meat obtained in accordance with the present invention contains a large amount of physiologically active components. These physiologically active components, amino acids and reducing sugars, have antioxidant activity, antihypertensive activity, antimicrobial activity, anticoaglutant ) Activity. Hereinafter, the present invention will be described in detail with reference to the accompanying drawings when necessary.

Brief Description of the Drawings Fig. 1 is a diagram showing the process of recovering and obtaining a physiologically active ingredient such as free amino acid and reducing sugar from mackerel meat according to an exemplary embodiment of the present invention, analysis of the obtained physiologically active ingredient, , And a flow chart showing a process of measuring the flow rate.

As shown in Fig. 1, non-dried raw mackerel meat is first prepared. Next, the prepared non-dried raw mackerel meat is cleanly washed with water, and then non-dried raw mackerel meat is dried to obtain non-deoiled dry mackerel meat. As a method for drying the washed non-dried raw mackerel meat, a method of naturally drying in a shade in the wind can be used, but a method of freeze-drying raw mackerel meat at low temperature is adopted can do.

 When the freeze-drying method is adopted, a large amount of physiologically active ingredients contained in raw mackerel meat can be particularly useful because it is not deformed by heat. When the raw mackerel meat is 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 may be 48 to 96 hours. When freeze-dried under these conditions, dried mackerel meat is completely dried and dried to obtain dried mackerel meat. However, the freeze-drying temperature and the drying time may be changed depending on the prepared mackerel meat condition.

Then, the dried mackerel meat is cut and pulverized to an 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. A deoiled process may be performed to remove the fat content contained in the mackerel meat prior to processing the milled mackerel meat with the ash factor.

In an exemplary embodiment, a supercritical extraction method using high temperature, high pressure supercritical carbon dioxide (CO ₂ ) may be utilized for the deoiling process to remove the fat component from mackerel meat. The supercritical fluid is supercritical fluid which is in a state of exceeding the critical temperature and the critical pressure. The supercritical fluid used in accordance with the present invention is not only low in density but high in density, Therefore, it can be used to efficiently extract and remove the preservative ingredient from dried mackerel meat.

For example, the supercritical carbon dioxide used in the deoiling process to remove the maintenance component from dried mackerel meat may be supercritical carbon dioxide at a temperature of 40-60 < 0 > C and a pressure of 300-500 bar, Can be treated for about 1 to 3 hours. In an exemplary embodiment according to the present invention, supercritical carbon dioxide at a temperature of 55 ° C and a pressure of 400 bar was treated with dried mackerel meat for 2 hours to remove the fat component from the mackerel meat.

Subcritical water hydrolysis (SWH) processes are then carried out on dehydrated mackerel meats obtained by treatment with supercritical carbon dioxide, with high temperature and high pressure subcritical water. The reaction using the asymptotic coefficients is attracting attention as a new environmentally friendly conversion method.

That is, the hydrolysis step of the hydrolysis step requires no pre-treatment, has a relatively short reaction time, produces less corrosion or residue by the reaction, does not require the use of a toxic solvent, It is a method for the hydrolysis of environmentally friendly and rapid biomass which can be used as an alternative to chemical methods or catalytic methods using existing acids or bases because the formation of the product has a small advantage.

The boiling point of water at atmospheric pressure is 100 ° C, but it maintains the liquid phase even at elevated temperatures at high pressures. For example, water maintains a subcritical state at a boiling point of 100 ° C of 0.1 MPa and a critical point of 374 ° C and 22 MPa of SWH, while in a subcritical state, water retains its liquid state due to high pressure. Compared with liquid water at atmospheric pressure, water in the subcritical state exhibits unique physical and chemical properties, especially high frequency of hydrogen bonding, low dielectric constant and high ion product content.

Due to the physical and chemical properties of the subcritical condition, that is, the critical temperature and subcritical condition, the subcritical coefficient can function as a new reaction medium in many chemical reactions. For example, due to the low dielectric constant of the water in the subcritical state, it can function as a solvent suitable for dissolving these organic compounds by reacting with a wide range of organic compounds, and the high ionic product is such that the sub- Thus giving the catalyst the potential to function. In addition, the hydrolysis reaction using an asymptotic coefficient is environmentally friendly since it does not generate contaminants, so it is important from an economical point of view as well as from an ecological point of view.

In the present invention, the mackerel meat is treated using the subclaims to hydrolyze the mackerel meat to produce many physiologically active functional substances such as peptides, amino acids and reducing sugars contained in the mackerel meat. Most biomass is readily hydrolyzed at a structurally different submixture from conventional liquid water. In this process, the hydrolysis of the asymptotic factors is environmentally friendly, and the hydrolysis of polymeric substances such as proteins can be performed at a relatively low cost from an economical point of view, .

Illustratively, the submerged factor used to obtain the hydrolyzate derived from dehydrated mackerel meat according to the present invention is a temperature of 200 to 300 ° C, such as a temperature of 150 to 350 ° C, preferably 220 to 260 ° C, Sub-critical conditions such as 20 to 100 bar pressure, such as a pressure of 400 bar, preferably 30 to 70 bar, can be used.

For example, the deaeration process using supercritical carbon dioxide and / or the hydrolysis process using the asymptotic coefficients described above according to the present invention can be performed using the hydrolysis equipment exemplarily shown in FIG.

In FIG. 2, reference numeral 1 denotes a gas cylinder, 2 denotes a gas regulator, 3 denotes an on / off valve, 4 denotes an impeller / stirrer, 5 denotes an electric heater, 6 is a safety valve, 7 is a pressure gauge, 8 is a reactor such as a high-pressure reactor, 9 is a temperature controller, and 10 is a sample collector.

For example, the mackerel meat treated by the drying process and the deoiling process is transferred from the sample collector 10 to the reactor 8, and the reactor is filled with water such as distilled water. At this time, the water that can be converted into the subcritical state for the dehydrated dried mackerel meat sample can be mixed and reacted in the reactor 8 at a ratio of 1:10 to 1:30 w / v.

Subsequently, the inside of the reactor 8 is operated to reach the temperature and pressure of the subcritical condition by using the temperature controller 9 and the pressure gauge 7 for the hydrolysis of the mackerel meat. If necessary, a gas such as nitrogen, carbon dioxide, or air is supplied to the reactor 8 through a gas cylinder 1 to change the temperature and pressure inside the reactor 8, Can be enlarged. At the same time, by using the stirrer 4 attached to the inside of the reactor 8, the uniformity of the mackerel oil and the asymptotic mixture injected into the reactor 8 is maintained, while the predetermined pressure and heat are kept constant in the mackerel meat .

Particularly, according to the present invention, it is preferable to use a suitable catalyst when hydrolyzing mackerel meat. Catalysts that may be used in addition to the subcooling according to exemplary embodiments include organic acid solutions such as formic acid, acetic acid, inorganic base solutions such as sodium hydroxide, sodium bicarbonate, salt solutions such as sodium chloride, and gases such as carbon dioxide and nitrogen . Sodium hydroxide and / or sodium hydrogencarbonate, and particularly preferably sodium hydrogencarbonate. When these catalysts are used together with the subliming factor, the conversion yield to the hydrolyzate is improved and the content of the biologically active component in the hydrolyzate is increased, thereby improving the overall effect of physiological activity.

The protein or polysaccharide constituting the mackerel meat is insoluble in water under ordinary conditions. However, in such a process and condition, the mackerel meat put in the reactor (8) is decomposed and converted under the set hydrolysis conditions to produce a new physiologically active functional substance . Specifically, the insoluble polymer substance present in the high-temperature, high-pressure water of the mackerel which has been set in an appropriate range according to the present invention to have a unique physical property is hydrolyzed into physiologically active components such as peptides, amino acids and reducing sugars, ). ≪ / RTI > If necessary, a step of cooling the obtained hydrolyzate to room temperature may be carried out before confirming the composition of the physiologically active functional substance contained in the hydrolyzate obtained by treating the sublimation factor at high temperature and high pressure.

The general composition of the hydrolyzate derived from mackerel meat, the composition of the active ingredient, and the physiological activity effect recovered through such a process can be analyzed by a known method. Analysis of the histamine content can be performed using a p-phenyldiazonium reagent. For example, amino acid, which is one of the physiologically active components remaining in the hydrolyzate derived from mackerel meat, can be analyzed using an amino acid analyzer. The total content as well as the content of each amino acid can be confirmed. The content of reducing sugars in the hydrolysates derived from mackerel meat can be measured by 3,5-dinitrosalicylic acid (DNS) method. The effect of the physiologically active ingredient contained in the hydrolysates derived from these mackerel beef can also be confirmed by known methods.

Exemplary antioxidant activity effects include the free radical scavenging assay of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), ABTS free radical scavenging assay and reducing power assay. In addition, the inhibitory effect of angiotensin converting enzyme (ACE) activity can be confirmed by o-phthalaldehyde (OPA) assay, and the antimicrobial effect can be measured by a paper disk diffusion assay.

Histidine, a basic amino acid contained in mackerel, is converted into histamine by decomposing mackerel. Histamine promotes abnormalities of metabolism such as allergy in human body. According to the present invention, it was confirmed that when the mackerel meat is treated with the subcritical water, the amount of histamine which adversely affects the human body is reduced (Example 3). Therefore, according to the present invention, the mackerel meat that has undergone the sub-counting treatment can be stably taken as a food.

In addition, according to the results of the analysis in the exemplary embodiment of the present invention, the hydrolyzate obtained by treating the mildew meat factor with mildew has an important role in physiological functions such as osmotic pressure control and buffering capacity in aquatic animal tissues (Figs. 3A, 3B and 4; Tables 6 to 10) and reducing sugars (Figs. 5A and 5B) involved in the physiological activity of living organisms. Especially, when the appropriate catalyst such as sodium hydrogencarbonate is used, the content of amino acid and the content of reducing sugar are increased in comparison with the case of using only the sublimation factor alone.

Proteins in food have long been recognized in terms of nutrition and function. The biological activity of proteins, including peptides, is widely recognized as associated with the composition of the amino acids that make up the protein, combined with the physiological utilization of certain amino acids during digestion and absorption. In particular, peptides are one of the valuable biologically active ingredients because they can be used to form functional foods or nutraceuticals because they prevent damage caused by oxidative stress and are effective in preventing human diseases.

Recently, in order to utilize peptides and amino acids as functional ingredients of foods for maintenance of health, many studies have been conducted to extract peptides and amino acids which are bioactive components in food proteins. Life-size peptides and amino acids are absorbed into the human body through the intestines and enter the circulation system from the intestines to exert diverse physiological effects or induce local effects in the digestive organs. Food-derived peptides and amino acids are known to exhibit various categories of physiological functions including antihypertensive, antioxidant, antimicrobial and hypocholesterolemic effects.

Amino acids are involved in strength, repair and rebuilding in the human body. Amino acids are sweet, but contribute to the flavor of the food. Thus, hydrolysates derived from proteins and / or peptides, such as amino acids, are useful as additives in the food industry. On the other hand, since humans can not synthesize essential amino acids, they must take appropriate food containing essential amino acids. According to the present invention, it is confirmed that the content of essential amino acids is also rich in the hydrolyzate derived from mackerel meat, thus showing that the present invention can be applied to the food industry.

On the other hand, many carbohydrates are very good scavengers for metal ions. For example, reducing sugars such as glucose and fructose have the ability to block the reaction sites of metal ions such as copper, iron and less cobalt. These characteristics of the monosaccharide can slow the oxidation reaction by the catalyst, and these reducing sugars can help preserve the food. It is also known that reducing sugars in food systems have antioxidant properties. Because of this, reducing sugar is being used in the food industry as a food additive for biscuits, cookies or sausages.

In the exemplary embodiment, the DPPH free radical scavenging activity of all the hydrolysates derived from the mackerel meat obtained by the submerged counting process according to the present invention was good, and the scavenging activity using the sodium hydrogencarbonate catalyst was the most excellent in the submerged count (See Figs. 6A and 6B). The ABTS free radical scavenging activity exhibited a considerably good effect even when the sublimation coefficient was used alone or in combination with most of the catalysts (see FIGS. 7A and 7B), and the reducing power was particularly excellent when the sodium hydrogencarbonate catalyst was used And 8b).

While synthetic antioxidants may exhibit toxic side effects when used, natural antioxidants are particularly desirable in the human body because they can be used at much higher concentrations than synthetic antioxidants without these side effects. The hydrolyzate obtained from the biomaterial of mackerel meat according to the present invention is free from the side effects of the synthetic antioxidant and therefore the hydrolyzate derived from the mackerel meat obtained according to the present invention can be utilized as a drug or a component of health functional food .

On the other hand, the renin-angiotensin-aldosterone system (RAAS) plays an important role in regulating body water and electrolytes as well as controlling blood pressure in cardiovascular and kidneys. For example, when the blood pressure of the kidney artery is lowered, angiotensinogen is degraded by renin secreted from the kidney, and first, angiotensin I is converted to angiotensin converting enzyme (ACE ) Is converted to angiotensin II.

Angiotensin II is a peptide that plays a major role in RAAS in association with the angiotensin II type 1 receptor (AT1). When this peptide acts on RAAS, it mainly causes vasoconstriction, secretion of aldosterone and vasopressin, retention of salt and water, Neuronal activity, etc., resulting in changes in kidney blood vessels, hypertension in the glomerulus occurs and may progress to glomerulosclerosis.

That is, it is known that ACE causes hypertension in the human body and further contributes to hemodynamic changes of congestive heart failure. In addition, cardiovascular diseases such as atherosclerosis and atherosclerosis may be caused by platelet aggregation due to hypertension. Therefore, when the action of angiotensin converting enzyme is inhibited, it can induce blood pressure lowering and can treat and prevent cardiovascular diseases such as hypertension, congestive heart failure and atherosclerosis through anticoagulant effect.

The most potent ACE inhibitors contain hydrophobic amino acid residues at each of the three C-terminal positions that interact with S1, S19, S29 at the ACE active site. In many studies, peptides with high ACE-inhibiting activity have tryptophan, phenylalanine, tyrosine or proline at the C-terminus, aliphatic amino acids at the N-terminus and side chain aliphatic amino acids, and ACE is a C- It is known that there is little affinity for inhibitors having an amino acid (e.g., glutamic acid) having a carboxylic acid.

According to an exemplary embodiment of the present invention, the hydrolyzate derived from mackerel meat obtained by treating with a sub-coefficient inhibited the activity of ACE (see Fig. 9a 9b). Therefore, according to the present invention, the hydrolyzate derived from mackerel meat obtained by treating with the sublimation factor is useful as a medicament for treating and / or preventing cardiovascular diseases such as hypertension, congestive heart failure and arteriosclerosis and / It is expected to be used as an active ingredient in foods.

Recently, many microorganisms are known to acquire resistance to synthetic antibiotics. So scientists are trying to find natural antimicrobials. The antimicrobial peptide acts directly on the microorganism through a mechanism associated with membrane disruption and pore formation, for example, which allows for the supply of essential ions and nutrients. The molecular mechanism of antimicrobial peptides is not fully understood. Recently, it has been found that transmembrane pore formation is not the only mechanism to remove microorganisms, and that antimicrobial peptides can affect microbial cells in other ways. For example, inhibition of cell wall and / or nucleic acid synthesis of microorganisms, activation of the autolytic enzyme system, or synergy with other host immune substances.

According to the exemplary embodiment of the present invention, the hydrolyzate derived from the mackerel meat obtained by treating with the subliming factor showed antimicrobial activity against various pathogenic microorganisms when sodium hydroxide and sodium hydrogencarbonate were used as catalysts (see Example 8 and Table 11).

As a result, the present invention relates to a method for extracting, recovering and obtaining a physiologically active ingredient from mackerel muscle. The mackerel meat obtained by the drying and deoiling process is subjected to high temperature and high pressure ashing and, if necessary, Amino acids and reducing sugars having a variety of physiologically active functions can be obtained from proteins and polysaccharides which are a poorly soluble macromolecular substance contained in a large amount in mackerel meat. These physiologically active ingredients can be used in medicines, foods, cosmetics As a functional material.

Hereinafter, the present invention will be described in detail with reference to exemplary embodiments, but the present invention is by no means limited to the invention described in the following embodiments.

<Materials and Statistics Processing>

The washed mackerel meat samples used in the present invention were collected from F & F (Pusan, Korea) and sent to the laboratory under freezing conditions. (ACE), hippuryl-L-histidyl-L-leucine (HHL), ophthalaldehyde (OPA), captopril, Trolox and ascorbic acid were all available from Sigma Aldrich Chemical Co. (St. Louis, MO, USA).

All other reagents and reagents used in the present invention were of analytical grade or high performance liquid chromatography (HPLC) grade. Each experiment was performed in triplicate in the examples and the data were expressed as mean ± standard deviation (P <0.05). SPSS statistical program (SPSS version 15.0 for Windows, SPSS Inc., Chicago, IL, USA) was used for data analysis.

Example 1: Production of hydrolyzed mackerel hydrolyzate (physiologically active ingredient)

The raw mackerel muscle samples that had not been dried were lyophilized for 72 hours using a freeze dryer (EYELA FDV-2100, Rikakikai Co. Ltd., Tokyo, Japan). A completely dried mackerel meat sample was ground using a mechanical blender. Subsequently, supercritical carbon dioxide (SC-CO ₂ ) was added to the dried mackerel meats at 55 ° C and 400 bar pressure for 2 hours to obtain de-oiled mackerel meat with oil removed. The samples of oyster mackerel meat were stored at -20 ° C prior to hydrolysis using the asymptotic counting method.

General compositions such as moisture, ash, lipid and protein for non-dried fresh mackerel meat, non-dehydrated dried mackerel meat and dehydrated dried mackerel meat, (proximate composition) was analyzed according to the standard AOAC method. The results of analysis for the general composition are shown in Table 1 below. The protein contents of mackerel meat were different in each process. Especially, the protein content of mackerel meat was 74.13%.

Raw mackerel meat, dried mackerel meat and de-oiled mackerel

After the general composition analysis, all processed mackerel meat was treated with the hydrolysis hydrolysis process. The subcritical water hydrolysis (SWH) was carried out in 200 mL of a batch reactor made of 276 Hastelloy equipped with a temperature controller, shown in FIG. The mackerel meat residue and distilled water obtained according to each process were put into the reactor and the reactor was closed. As a control, deodorized mackerel meat residue 10 g / 160 mL of deionized mackerel meat residue was added to distilled water (the ratio of sample deinked mackerel meat to distilled water was 1:25 w / v).

A solution of formic acid, acetic acid, sodium chloride and sodium bicarbonate as a catalyst was prepared at a concentration of 0.6 M and 0.013 M sodium hydroxide solution was added thereto so as to prevent the hydrolysis from occurring before the hydrolysis by the sub- Respectively. 1.52 g and 1.04 g of carbon dioxide gas and nitrogen gas were fed into the reactor, respectively. The interior of the reactor was heated using an electric heater previously heated to the desired temperature (220-260 DEG C) and treated with pressure (30-70 bar). In each experiment, the temperature and pressure inside the reactor were measured using a temperature controller and a pressure gauge.

Each sample was magnetically stirred at 150 rpm. The submixing reaction time for each sample was 3 minutes. After rapid cooling to room temperature, the hydrolyzate was collected in the reactor, filtered using filter paper (Advantec No. 5A) and stored at -4 ° C for later analysis. The liquid portion, referred to as the hydrolyzate, was used for further histamine content analysis, amino acid and reducing sugar analysis, antioxidant activity, ACE-inhibiting activity and antimicrobial activity.

Example 2: Yield analysis of meat hydrolyzate of mackerel by submerging treatment

According to Example 1, non-dried fresh mackerel meat, non-dehydrated dried mackerel meat and other dehydrated dried mackerel meats using different catalysts at 220-260 &lt; 0 &gt; C temperature and 30-70 bar pressure, The hydrolysis yield and pH were analyzed. The hydrolytic conversion yield (X) of the mackerel meat was determined from the following equation (where W ₀ is the total amount of the whole of the mackerel introduced into the reactor The amount of mackerel meat, and W is the amount of mackerel meat recovered after SWH)

Meanwhile, the pH of the hydrolyzate was measured using a Mettler toledo pH meter. The results of hydrolysis yield and pH measurement according to this example are shown in Tables 2 and 3 below. Among mackerel meats, the yield of hydrolysis was higher in non - dried mackerel meat. The yield of hydrolysis of deaerated mackerel meat using catalyst was increased as compared with that using only asimens. Among them, the hydrolysis yield of the dehydrated mackerel meat using sodium hydrogencarbonate was the highest at 99.50 ± 0.60% when 260 ℃ and 70 bar pressure was used.

Comparison of yield and pH of hydrolyzate obtained from mackerel meat by subc

Comparison of the yield and pH of the hydrolyzate obtained from deaeration mackerel meat using a catalyst

Of all the catalysts used, sodium hydroxide and sodium bicarbonate in particular influenced the hydrolysis efficiency. There was a positive interaction with the catalyst, temperature and pressure. Increasing the temperature and pressure increased the hydrolysis yield. The pH of the hydrolyzate increased with increasing temperature and pressure when using each catalyst and water. Similar results were observed after the addition of the catalyst to the hydrolysis of subcycle when the proteins were separated.

Example 3: Analysis of histamine content of mackerel hydrolyzate

The histamine content of the mackerel meat obtained according to Example 1 before and after hydrolysis was analyzed. The reagent p-phenyldiazonium was prepared according to the method of Patange et al. (2005). 1.5 mL of chilled 0.9% (w / v) sulfanilic acid in 4% hydrochloric acid and 1.5 mL of 5% (w / v) sodium nitrite were mixed in a 50 mL standard flask and placed in an ice bath for 5 minutes. 6 mL or more of a 5% sodium nitrite solution was added, and after 5 minutes, the mixture was cooled with distilled water.

After 15 minutes of dilution with distilled water, the reagent stored in the ice bath was used and stable for 12 hours. 2.5 g each of non-dried mackerel meat, non-mackerel dried mackerel meat and dehydrated mackerel meat were taken in a centrifuge tube and homogenized using 30 mL of 0.85% NaCl solution for 5 minutes using a high-speed blender , And centrifuged at 10,000 rpm for 10 minutes at 4 占 폚. The supernatant was stored at -20 ° C for further analysis.

In a glass stoppered test tube, dilute 1 mL of mackerel (before and after hydrolysis) with 2 mL of physiological saline, add 6.25 g of anhydrous sodium sulfate to 1 g of trisodium phosphate monohydrate Was added. The tube of the test tube was closed and completely shaken.

Subsequently, 2 mL of n-butanol was added and the tube was shaken vigorously for 1 minute, allowed to stand for 2 minutes, and shaken again to break the protein gel. The tube was vigorously shaken again for a few seconds and then centrifuged at 3,000 rpm for 10 minutes. The upper butanol layer (only 1 mL) was transferred to a cleanly-dried test tube and then evaporated to dryness under nitrogen reflux. The residues were dissolved in 1 mL of distilled water and reacted with the reagents according to the procedure described below.

Take 5 mL of 1.1% sodium carbonate solution in a clean test tube and add 2 mL of the cooled reagent slowly and mix. And then added to a test tube containing 1 mL of the mackerel meat residue collected in the extraction process. The absorbance of the color immediately produced after 5 minutes at a wavelength of 496 nm was measured using distilled water as a reference. 1 ml of a standard histamine solution fraction containing 0-75 占 퐂 / ml dissolved in distilled water was reacted in the same manner to obtain a reference color standard and a standard curve of absorbance against histamine concentration. An ultraviolet-visible spectrometer equipped with glass cuvettes was used for this purpose.

The histamine concentration in the sample was determined from a standard curve for the corresponding absorbance measured at 496 nm by regression analysis. The histamine concentration in the sample was evaluated using the following equation.

The histamine content in the mackerel meat before and after the hydrolysis is shown in Table 4. The histamine contents were 149.70, 179.25 and 183.83 mg / 1000g, respectively, before hydrolysis of non-dried mackerel meat, non-mackerel dried mackerel meat, and dehydrated mackerel meat. After hydrolysis with subclaims, the histamine content in these mackerel meats decreased.

Measurement of histamine content of mackerel meat before and after hydrolysis

Of all the hydrolysates produced, the histamine content was low (51.42 mg / 1,000 g) in non-dried raw mackerel hydrolyzate using the sublimation factor alone at 260 ° C and 70 bar compared to other hydrolysates. Increasing temperature and pressure resulted in a slight decrease in histamine concentration. Although the exact mechanism is uncertain, it is predicted that the histamine structure has collapsed due to temperature and pressure.

Example 4 Analysis of Amino Acids of Mackerel Hydrolyzate

(1) Analysis of total amino acid composition

Amino acid composition analysis for the hydrolyzate was carried out according to Blackburn's method. All the hydrolyzate obtained from the degumming mackerel meat was filtered by hydrolysis of the hydrolyzate, and then loaded into the amino acid automatic analyzer S430 (SYKAM) for amino acid analysis. A cation separation column LCA K07 / Li (4.6 x 150 mm) was used for amino acid analysis.

The mobile phase was a 5 mM p-toluenesulfonic acid solution with a flow rate of 0.45 ml / min. A mixture of 5 mM p-toluenesulfonic acid, 20 mM Bis-Tris and 100 mM EDTA was used as a post column reagent and the flow rate was 0.25 ml / min. Under two operating conditions, the excitation and emission wavelengths were 440 ㎚ and 570 ㎚, respectively. The operating conditions for amino acid analysis are shown in Table 5 below.

Operating conditions of amino acid analyzer for amino acid measurement

The results of the analysis of the total amino acid content of the hydrolyzate of mackerel meat obtained by another process according to this Example are shown in FIG. The highest amino acid content was observed in hydrolyzate of dehydrated dried mackerel meat. On the other hand, the results of analysis of total amino acid content in the hydrolyzate derived from dehydrated mackerel meat using different catalysts at 220-260 ° C and 30-70 bar pressure are shown in FIG. 3b.

The total amino acid content of the hydrolyzate using sodium hydrogencarbonate as a catalyst was the highest, and the total amino acid content in the dried residue was 328.63 ± 4.58 mg / g when 220 ° C temperature and 30 bar pressure was used, The total amino acid content in the dry residue was 400.36 ± 4.62 mg / g when the temperature and an asymptotic factor of 70 bar pressure were used. In the case of using sodium hydrogencarbonate as a catalyst, the production of amino acid was increased about 4 times as compared with the case of using only the asymptotic coefficient. In the case of the other catalysts, a positive effect on the amino acid production was observed as compared with the case of using only the asymmetry coefficient.

(2) Analysis of essential amino acid content

Subsequently, the content of essential amino acids in the hydrolyzate of mackerel was analyzed according to this Example. The analysis of the essential amino acids for the hydrolyzate obtained by treating the dehydrated mackerel meat with different catalysts at a temperature of 220-260 ° C and a pressure of 30-70 bar is shown in FIG. As shown in FIG. 4, the hydrolyzate of the dehydrated mackerel meat obtained by treating with the catalyst having the subliming factor added thereto has a high content of essential amino acids, so that it can be utilized as a nutritionally valuable food .

The hydrolyzate with sodium bicarbonate at 260 ° C and 70 bar pressure had almost the same amino acid content as almost 44% of the total amino acids, and the content of essential amino acids was higher than that of other catalysts. Essential amino acid profiles in hydrolysates using sodium bicarbonate at subcritical temperatures of 260 ° C and 70 bar pressure, except for histidine, threonine and methionine, are required by FAO / WHO for adults .

(3) Analysis of composition of individual amino acids

The individual amino acid contents in the hydrolyzate obtained by treating the subclaims of 220 ° C and 30 bar pressure, 260 ° C and 30-70 bar pressure on non-dried fresh mackerel meat, non-dehydrated dried mackerel meat and dehydrated mackerel meat respectively Are shown in Tables 6 and 7. Amino acids recovered from hydrolysates from three processed mackerel meats mainly consist of glycine, alanine, histidine and valine. As analyzed in Table 1 of Example 1, due to the high protein content of dehydrated dried mackerel meat, the content of individual amino acids increased after the hydrolysis treatment by the asymmetric coefficient.

Amino acid composition of hydrolysates of mackerel by 220 ° C, 30 bar asymmetric treatment

Amino acid composition of hydrolyzate of mackerel by 260 ° C, 70 bar subcritical water treatment

In addition, the individual amino acid contents in the hydrolyzate obtained by treating the dehydrated mackerel meat with the catalyst at the 220 DEG C and 30 bar pressure, 260 DEG C and 30-70 bar pressure sub-catalysts are shown in Tables 8 and 9 have. Especially at the same temperature and pressure, as compared with the case where only the subcycle was used alone or in the presence of other catalysts, especially in the sodium bicarbonate and in the high temperature and high pressure subcycle.

During the application of a fixed concentration of sodium hydrogencarbonate, the amino acid production varied with temperature and pressure. Serine, glutamic acid, sarcocine, proline, methionine, histidine (aspartic acid), glutamic acid, glutamic acid, And ornithine, respectively. The concentration of these amino acids decreased with increasing temperature and pressure.

On the other hand, it is also known that threonine, glycine, alanine, valine, cysteine, cystathionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan and lysine ) Was the highest at 260 ℃ and 70 bar pressure.

Amino acid content of hydrolysates of mackerel hydrolyzate by catalytic treatment at 220 ℃, 30 bar

Amino acid content of mackerel hydrolysates by 260 ° C, 70 bar subcritical rate and catalytic treatment

Example 5: Analysis of reducing sugar content of mackerel hydrolyzate

The reducing sugar content of the hydrolyzate was analyzed by slightly modifying the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). D-glucose was used as a standard.

1.0 mL of the hydrolyzate derived from each mackerel meat obtained in Example 1 was diluted with 3 mL of HPLC grade water, and 3 mL of the DNS reagent was mixed with each mackerel sample. The mixture was heated in boiling water for 5 minutes. After cooling to room temperature, 1 ml of a 40% solution of potassium sodium tartarate (Rochelle salt) was added to stabilize the color. After 10 minutes, spectroscopic reading was performed at a wavelength of 575 nm using an ultraviolet-visible spectrophotometer (UVmini-1240, Shimadzu Corporation, Kyoto, Japan).

The results of analysis of the reducing sugar content in the hydrolyzate obtained by treating the mildew meat with an ash modulus of 220-260 DEG C and a pressure of 30-70 bar according to the present example are shown in FIG. Reducing sugar content of the mackerel hydrolyzate was the highest among the mackerel hydrolysates. On the other hand, FIG. 5B shows the result of analyzing the reducing sugar content in the hydrolyzate obtained by adding other catalysts to the ashing factor of 220-260 DEG C and 30-70 bar pressure, and treating the deodorized mackerel meat.

The reducing sugar content of the hydrolyzate using sodium hydrogencarbonate catalyst was the highest (24.75 ± 0.38 mg / g) and the reducing sugar content was lowest in the hydrolyzate using acetic acid catalyst (1.97 ± 0.12 mg / g). Therefore, the catalyst, temperature and pressure seem to be important factors in the production of reducing sugars in mackerel hydrolysates. When the temperature and pressure were increased, the amount of reducing sugar decreased in all the deaerated mackerel hydrolysates. Degreasing Reducing sugar obtained from mackerel meat means that it is not stable at high temperatures and pressures.

On the other hand, as shown in Table 1 of Example 1, the moisture contents of non-dried fresh mackerel meat and non-dehydrated dried mackerel meat were 65.77%, 3.45% and 2.35%, respectively. After mass balance, the amino acid and reducing sugar content of all processed mackerel hydrolysates were changed. Table 10 shows the amino acid and reducing sugar content of each mackerel meat. The amino acid content of the hydrolyzate obtained by treating non-dried raw mackerel meat at 260 ° C and 70 bar pressure was the highest.

Analysis of Amino Acid and Reduced Sugar Contents in Meat Hydrolyzate of Mackerel after Substance Balance

Example 6: Antioxidative activity against hydrolysates of mackerel meat

The antioxidant activity of the hydrolyzate was determined by the free radical scavenging activity assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assay, Azo-bis (3-ethylbenzothiazoline-6-sulphonic acid, ABTS) free radical scavenging activity assay and reducing power assay ) Was slightly modified and measured.

(1) DPPH free radical scavenging activity

The scavenging activity of the mackerel hydrolyzate was measured by a slight modification of Yen and Chen 's method. Specifically, 3.95 mL of a 0.1 mM DPPH solution (dissolved in methanol) was slowly added to a test tube containing 0.05 mL of meconium hexhydrolyzate and mixed. The mixture was vortexed for 10 seconds and kept at room temperature for 30 minutes in a dark room. The absorbance of all sample solutions was measured at 517 nm. Percentage of DPPH free radical scavenging performance was calculated using the following equation.

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

(Where As is the absorbance of the hydrolyzate derived from mackerel meat at 517 nm; Ac is the absorbance of the blank at 571 nm). The sample blank and control (0.5 mg / mL trolox dissolved in methanol) was also performed according to the same method.

The results of DPPH free radical scavenging activity analysis according to this example are shown in FIGS. 6A and 6B. All mackerel hydrolyzate was analyzed to be an efficient scavenger for DPPH free radicals. Among the hydrolysates of processed mackerel, high DPPH radical scavenging activity was observed in dehydrated mackerel hydrolyzate.

Compared to the case of using the sublimation factor alone, the highest DPPHH free radical scavenging activity was exhibited in the hydrolyzate using a sodium hydrogencarbonate catalyst at a temperature of 220 ° C and a pressure of 30 bar, and the maximum sago effect was 80.96 ± 2.10%. The DPPH radical scavenging activity was 43.50 ± 0.73% when trolox (0.5 mg / mL) was used as a standard antioxidant. When the temperature and pressure were increased, the effect of erasing was reduced in all hydrolysates. The DPPH radical scavenging activity of the hydrolyzate obtained by using gas, acid, and sodium chloride as the catalyst was decreased compared to the case where only the sublimation coefficient was used alone.

(2) ABTS free radical scavenging activity

Subsequently, the method of Zheleva-Dimitorva et al. (2010) was slightly modified in relation to ABTS free radical scavenging analysis of hydrolysates derived from mackerel meat obtained using different temperatures and pressures and different catalysts. ABTS ⁺ was dissolved in water to a concentration of 7 mM / L. 2.45 mM / L potassium sulphate was reacted with an equal amount of ABTS stock solution, and the mixture was allowed to stand in the dark room at room temperature for 16 hours to produce ABTS ⁺ .

For the test of the sample, the ABTS ⁺ stock solution was diluted with 80% methanol so that the absorbance was 0.70 ± 0.02 at 734 nm. After 0.075 mL of each hydrolyzate was diluted with ABTS ⁺ 4.975 mL, the mixture was left in the dark at room temperature for 6 minutes. The absorbance of all sample solutions was measured at 734 nm. ABTS ⁺ free radical scavenging activity was calculated using the following equation.

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

(Where As is the absorbance of the sample and control at 734 nm; Ac is the absorbance of the blank at 734 nm). A sample blank and control (1.0 mg / mL Trolox in 80% methanol) was also performed according to this method.

The results of ABTS free radical scavenging activity analysis according to this example are shown in FIGS. 7A and 7B. ABTS is an excellent method for evaluating lipophilic antioxidants as well as hydrophilic antioxidants. As shown in FIG. 7A, the ABTS radical scavenging activity was higher in the hydrolyzate derived from dehydrated and dried mackerel meat than the hydrolyzate derived from non-dried raw mackerel meat and non-dehydrated mackerel meat.

On the other hand, as shown in FIG. 7B, when catalysts were used at an 260 ° C temperature and 70 bar pressure, except for formic acid and acetic acid (44.29 ± 1.10%, 63.20 ± 1.22% ABTS scavenging activity was higher than 99% in all hydrolysates obtained from dehydrated mackerel meat using different catalysts. When Troxox (1.0 mg / mL) was used as a standard antioxidant, the ABTS radical scavenging activity was 81.95 ± 1.25%. When the temperature and pressure were increased, there was no significant difference in ABTS radical scavenging activity in all hydrolysates.

(3) Analysis of reduction power

The reducing power of the hydrolyzate, a sample derived from mackerel meat, was measured according to Oyaiza method (1986). A hydrolyzate sample (0.5 mL) derived from mackerel meat was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at &lt; RTI ID = 0.0 &gt; 50 C &lt; / RTI &gt;

A 10% trichloroacetic acid fraction (2.5 mL) was added to the mixture and centrifuged at 3000 rpm for 10 minutes. The upper layer (2.5 mL) of the solution was mixed with 2.5 mL of distilled water and 2.5 mL of 0.1% ferric chloride, and the absorbance at 700 nm was measured. An increase in the absorbance of the reaction mixture indicates an increase in reducing power. The ascorbic acid (1.0 mg / mL) in the controller was carried out according to the same method.

The results of the analysis of the reduction power according to the present embodiment are shown in Figs. 8A and 8B. Reducing power can serve as a key indicator of potential antioxidant activity as an indicator of antioxidant capacity and antioxidant effects are known to be associated with the occurrence of reductones. An increase in absorbance in the reaction mixture indicates an increase in reducing power.

As shown in Fig. 8A, the reducing power of the hydrolyzate derived from dehydrated mackerel meat was higher than the hydrolyzate derived from other processed mackerel meat. When the temperature and pressure were increased, the reducing effect was slightly reduced. (Absorbance at 1.28 ± 0.05 and 1.17 ± 0.04 at 700 nm, respectively) at 220 ° C. and 30 bar pressure, 260 ° C. temperature and 70 bar pressure, respectively. The reducing power was greatly increased in the hydrolyzate (absorbance was 1.97 ± 0.07 and 2.10 ± 0.08 at 700 nm, respectively). The effect of reducing power on hydrolyzate using acid was lower than other catalysts.

These results indicate that hydrolysates derived from de-mackerel meat potentially contain substances such as peptides or amino acids. Peptides or amino acids can be used for proton donating, electron donating and chain breaking, , And they terminate the radical chain reaction by reacting with free radicals and converting them into more stable products. Hydrophobic amino acids such as tyrosine, methionine, histidine, lysine, and tryptophan are generally known as antioxidants.

Example 7: Analysis of ACE inhibitory activity against hydrolysates of mackerel meat

The inhibitory activity of angiotensin converting enzyme (ACE) on the hydrolyzate slightly modified Chang's o-phthalaldehyde (OPA) method. Each hydrolyzate sample of mackerel meat was diluted at a ratio of 1: 0.25 (v / v) to Buffer-A containing 20 mM sodium borate and 0.3 M NaCl (pH 8.3 with HCl). 100 [mu] l of the diluted hydrolyzate sample, 100 [mu] l of the ACE solution (40 mU / ml Buffer-A) and the mixture containing the HHL solution (15 mM Buffer-A) The reaction was carried out.

The other mixture contained 100 μl of the diluted sample, 200 μl of Buffer-A (Mix 2), 100 μl of Buffer-A, 100 μl of ACE solution (40 mU / ml Buffer-A) And 100 [mu] l of HHL solution (15 mM in Buffer-A) to obtain data for 100 reactions. 1.5 mL of OPA solution (10 mg / mL dissolved in ethanol) dissolved in 100 mL of Buffer-B (pH 12.0 with NaOH) containing 0.1 M sodium borate and 0.2 M NaOH and 2-mercaptoethanol solution (5 μl / mg dissolved in ethanol) were mixed. To obtain the background absorbance of the OPA reagent prepared at least 1 hour before the experiment, a fourth mixture (Buffer 4) containing 300 μl of Buffer-A was used Respectively. Captoprile (synthetic ACE inhibitor 10 [mu] g / mL) was used as a standard instead of the diluted hydrolyzate sample. 3 mL of basic OPA reagent was added to complete the enzyme reaction.

With respect to the OPA method, the absorbance (A ₁ ) for mixture 1, the absorbance (A ₂ ) for mixture 2, the absorbance (A ₃ ) for mixture 3 and the absorbance (A ₄ ) for mixture 4 are 20 Min and then measured at a wavelength of 390 nm. The inhibition rate I (%) was calculated according to the following equation.

According to this example, the ACE suppression effect on the hydrolyzate obtained by treating the subcodes of high temperature (220-260 DEG C) and high pressure (30-70 bar) on the mackerel meat of other processes and the other catalysts in the subcycle The results of measuring the ACE inhibitory effect on the hydrolyzate obtained by treating deinked mackerel meat are shown in FIGS. 9A and 9B, respectively.

As shown in FIG. 9A, ACE-inhibiting activity exceeding 60% was measured in the hydrolysates derived from mackerel meat of all the processes. Among them, the ACE-inhibiting effect was the highest in the hydrolysates derived from the deodorized mackerel meat. In the case of using the catalyst, the ACE-inhibiting activity of more than 94% was measured in the hydrolyzate derived from all deodorized mackerel meat.

When the synthetic ACE inhibitor captopril was applied at a concentration of 10 μg / mL, the ACE-inhibiting activity was 93 ± 2.82%. The percentages of ACE inhibition in hydrolysates derived from sardine, pork and chicken feathers were 70%, 72.3%, and 49.6%, respectively, which were much lower than those of dehydrated mackerel meat. The hydrolyzate derived from dextrose mackerel contains all the amino acids and peptides at high concentrations (results not shown) and appears to be involved in ACE inhibition.

Example 8: Antimicrobial activity against hydrolysates derived from mackerel meat

The antimicrobial activity of hydrolyzate samples was determined by slightly modifying the paper disc diffusion assay. All microorganisms used in accordance with the present invention ( Escherichia coli KCCM 11234, Bacillus cereus KCCM 40022, S almonella typhimurium KCCM 11862 , Staphylococcus aureus KCCM 40050, Pseudomonas aeruginosa KCCM 11803, Listeria monocytogenes KCCM 40307) Center of Microorganism, KCCM).

Advnatec paper disks (10 mm) containing hydrolyzate samples (80 [mu] l) were placed on Mueller-Hilton agar plate coated with microbial suspension before treatment with sterile cotton wool, incubated at 37 [deg.] C for 24 hours The diameter of each inhibition area (mm) was measured afterwards. The analysis results according to this embodiment are shown in Table 11 below. Antimicrobial activity was not exhibited in hydrolysates derived from all mackerel meat, except hydrolyzate obtained by using sodium hydroxide or sodium hydrogencarbonate as a catalyst in the asymptotic ratio and treating it with deodorized mackerel meat.

As shown in Table 11, the hydrolyzate derived from deodorized mackerel meat showed a inhibition zone (mm) for six microorganism strains using a disk diffusion method. The hydrolyzate derived from mackerel meat obtained by using sodium hydroxide and sodium hydrogencarbonate as catalysts at 220-260 ℃ temperature and 30 - 70 bar pressure sublimation suppressed the growth of six microbial strains. In particular, the hydrolyzate obtained using sodium hydrogencarbonate as a catalyst at 260 DEG C temperature and 70 bar pressure sublimation showed the most effective antimicrobial effect against E. coli (inhibition zone diameter 13.25 mm).

The hydrolyzate obtained by using sodium hydroxide or sodium bicarbonate as a catalyst at 260 DEG C and 70 bar pressure sublimation showed the most effective antimicrobial effect against S. typhimurium (inhibition zone diameter 13.00 mm). This may be due to the presence of antimicrobial peptides or amino acids in these hydrolysates.

Antimicrobial Activity of Hydrolyzate Derived from Mackerel Meat Obtained by Catalyzed Asymmetry

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: gas cylinder 2: gas regulator
4: Stirrer 5: Electric heater
8: Reactor

Claims (6)

Drying the mackerel muscle;
Crushing the dried mackerel meat;
Removing oil from the crushed mackerel meat to obtain oat mackerel meat; And
Reacting the de-mackerel mackerel meat with an iron having a temperature of 150 to 350 DEG C and a pressure of 4 to 400 bar to hydrolyze the de-mackerel mackerel meat to recover the physiologically active ingredient;
The step of obtaining the deacidized mackerel meat comprises treating the ground mackerel meat with supercritical carbon dioxide having a temperature of 40 to 60 DEG C and a pressure of 300 to 500 bar, and in the step of hydrolyzing the de- A catalyst selected from the group consisting of acetic acid, sodium hydroxide, sodium chloride, sodium bicarbonate, carbon dioxide gas, nitrogen gas and a combination thereof is used, and the step of recovering the physiologically active ingredient comprises: Is mixed at a ratio of 1:10 to 1: 30 w / v to obtain a physiologically active ingredient from the mackerel meat. delete The method according to claim 1,
Wherein the physiologically active component obtained by hydrolysis of the mackerel meat is obtained from mackerel meat containing amino acid and reducing sugar. The method according to claim 1,
Wherein the physiologically active ingredient obtained by hydrolysis of the mackerel meat has any one of an antioxidant activity, an inhibitory activity of angiotensin converting enzyme (ACE) and an antimicrobial activity effect, A method for obtaining a physiologically active ingredient.
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