KR20220015657A - Functional low molecular peptide from the Mytilus coruscus with enhanced antioxidative activity and method for making the same - Google Patents

Abstract

The present invention relates to an antioxidant functional composition containing a sea mussel protein-derived low molecular weight peptide as an active ingredient, and a production method thereof. More specifically, the present invention relates to the functional composition and a food composition showing antioxidant efficacy containing sea mussel protein-derived low molecular weight peptide that optimizes the sea mussel protein through a hydrolysis process, and obtains by hydrolysis. The present invention comprises the food composition comprising the sea mussel-derived low molecular weight peptide as the active ingredient.

Description

Functional low molecular peptide from the Mytilus coruscus with enhanced antioxidative activity and method for making the same}

The present invention relates to a functional composition with antioxidant effect containing a mussel protein-derived low molecular weight peptide as an active ingredient and a method for preparing the same, and more specifically, to a mussel protein obtained by optimizing and hydrolyzing the mussel protein through a hydrolysis process. It relates to a functional composition and a food composition that contain the derived low-molecular peptide and exhibit antioxidant efficacy.

Mussel (sea mussel) is a kind of bivalve clams classified in the order Mussels and Mussels, and it is widely distributed around the world and is widely spotlighted as a food material. Mussels, meaning red clams, are named 'damchae', meaning light vegetables of the sea in the Gyuhapchongseo (1809) compiled by Mr. Bingheogak. About 20 types of mussels are distributed along the coast of Korea. Traditional wild mussels mean Mytilus coruscus , but domestically farmed mussels refer to Mytilus edulis , a pearl mussel. Therefore, mussels commonly found in the market are pearl mussels and are commonly called mussels because they are confused with wild mussels.

Antioxidant is a substance that inhibits oxidation, and is a generic term for substances that eliminate or attenuate the action of so-called oxidizing substances, including hydroxyl radicals, superoxide anions, and hydrogen peroxide, which are called active oxygen or oxygen free radicals. it means As low molecular weight compounds, glutathione, N-acetylcysteine, ascorbic acid, α-tocopherol, butylated hydroxyanisole (BHA), catechin, quercetin, uric acid, bilirubin, glucose, flavonoids, etc. are known, and recently, red wine extract Natural antioxidant products such as , tomatoes, and blueberries are in the spotlight.

Studies on the oxidation of fats related to free radicals, oxidative stress and antioxidants have been intensively studied in recent years. Synthetic antioxidants such as BHA (butylated hydroxy-anisole) and BHT (butylated hydroxytoluene) have been commercialized and used, but recently, DNA damage and toxicity caused by these derivatives have been revealed. Although physiologically active peptides are in an inactivated state in the protein to which they belong, it has been reported that when they exhibit physiological activity by hydrolysis, they exhibit the same function as hormones. Among various raw materials, hydrolyzates of peptide proteins that exhibit antioxidant, antihypertensive, anticancer, and angiotensin-converting enzyme inhibitory functions have been reported in seafood protein.

Human protein is a major component of connective tissue and is distributed in skin, bones, joints, blood vessels, membranes of organs, and hair, etc., and is a major protein that supports and maintains the human body. However, as we get older, more synthesized protein is destroyed and excreted, and in particular, because it is larger than normal protein, it is known that 90% of it is excreted without being easily digested and absorbed by the body when ingested. Therefore, small molecule peptides are known as the best way to solve this problem.

Therefore, there is an urgent need for the development of ingredients that are safe for the living body, have stable active ingredients, and above all, have excellent effects compared to substances having an existing antioxidant effect.

Patent Document 1: KR 10-2012-0049042 A (2012.05.16) “Antioxidant composition containing hydrolyzate of mussels as an active ingredient”

Therefore, the technical problem to be solved in the present invention is to evaluate the antioxidant activity of hydrolysates according to the type of enzyme of mussel protein, and a method for producing a low molecular weight peptide derived from mussel protein through an optimal production process, and a safe and excellent antioxidant effect as a natural edible material to provide a composition.

In order to solve the above technical problem, the present invention provides an antioxidant composition comprising a mussel protein-derived low molecular weight peptide as an active ingredient.

In addition, the present invention provides a food composition comprising a mussel protein-derived low molecular weight peptide as an active ingredient.

In addition, in the present invention, there is provided a method for producing a low-molecular-weight peptide derived from mussel protein, comprising the steps of (a) pulverizing mature mussels to prepare a slurry, and (b) adding a proteolytic enzyme to the mussel slurry and reacting it. to provide.

In the present invention, the antioxidant composition and the food composition may contain as an active ingredient a mussel protein-derived low molecular weight peptide, which is prepared by the above method.

The term antioxidant effect used in the present invention refers to a substance that suppresses oxidation reaction, and a substance that eliminates or attenuates the action of so-called oxidizing substances, including hydroxyl radicals, superoxide anions, hydrogen peroxide, etc. called active oxygen or free radicals. As a generic meaning, it is used in the same meaning as terms such as antioxidant composition, antioxidant, and antioxidant composition.

The low molecular weight peptide derived from mussel protein of the present invention can exhibit excellent antioxidant effect by scavenging DPPH radicals.

According to one preferred embodiment of the present invention, there is provided a food composition for antioxidants comprising a mussel protein-derived low molecular weight peptide as an active ingredient.

The food of the present invention may be a health supplement, health functional food, functional food, etc., but is not limited thereto, and it includes adding the compound of the present invention or an acceptable salt thereof to natural food, processed food, general food materials, etc. .

The food composition of the present invention may contain the mussel protein-derived low molecular weight peptide alone or may be used in combination with other foods or food compositions, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (prevention, improvement or therapeutic treatment).

In general, the mussel protein-derived low molecular weight peptide of the present invention may be added in an amount of 0.1 to 70% by weight, preferably 2 to 50% by weight, based on 100% by weight of the raw material of the food or beverage when preparing food or beverage.

The effective dose of the mussel protein-derived low molecular weight peptide of the present invention may be less than the above range in the case of long-term intake for health and hygiene or health control, and the active ingredient has no problem in terms of safety. It can also be used in amounts beyond the range.

There is no particular limitation on the type of the food. Examples of foods to which the mussel protein-derived low molecular weight peptide of the present invention can be added include fish cakes, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, Beverages, teas, drinks, alcoholic beverages and vitamin complexes, and other nutritional supplements may be mentioned, but are not limited to these types of foods.

According to one preferred embodiment of the present invention, there is provided a feed composition for antioxidants comprising a mussel protein-derived low molecular weight peptide as an active ingredient.

The feed composition of the present invention may be distributed together with a conventional feed, and the feed composition of the present invention may be added to a conventional feed composition to prepare and use a functional feed composition. In addition, the feed composition of the present invention may further include a functional ingredient in addition to the mussel protein-derived low molecular weight peptide. When preparing a functional feed composition in which the conventional feed composition and the low molecular weight peptide derived from the mussel protein of the present invention are mixed, the low molecular weight peptide derived from the mussel protein of the present invention is 0.01 to 30.00% by weight, preferably 0.01 to 20.00, based on the total feed composition It may be added in an amount in weight percent. The effective dose of the mussel protein-derived low molecular weight peptide in the feed composition can be used according to the effective dose of the food composition, but in the case of long-term intake for the purpose of continuous health control, it may be less than the above range, and the active ingredient is Since there is no problem, it can be used in an amount beyond the above range as needed.

The feed composition of the present invention is for livestock or poultry. The livestock or poultry is cattle, pigs, chickens, horses, sheep, donkeys, mules, wild boars, rabbits, quails, ducks, roosters, cockfowl, pigeons, turkeys, dogs, cats, monkeys, hamsters, mice, rats, guinea pigs , parrots, parakeets, canaries, etc., but not limited thereto, and any mammals or birds other than humans that can be reared at home will be the subject of the feed composition of the present invention.

The composition comprising the mussel protein-derived low-molecular-weight peptide of the present invention as an active ingredient has excellent DPPH radical scavenging ability, exhibits an antioxidant effect, and has no toxicity to cells, so it is expected to be usefully used as an antioxidant food composition.

1 is a graph showing the results of analyzing the antioxidant capacity of DPPH of low molecular weight peptides produced under different proteases and hydrolysis conditions.
2 is a graph showing the results of analyzing the superanion scavenging antioxidant capacity of mussel protein low molecular weight peptides produced using different proteases.
3 is a graph showing the results of analyzing the hydroxyl radical scavenging antioxidant ability of mussel protein low molecular weight peptides produced using different proteases.
4 is a graph showing the results of analyzing the molecular weight of the hydrolyzate of mussels using bromelamin and flavourzyme according to the present invention.
5 is a graph showing the results of analyzing the distribution of amino acids constituting the mussel small molecule peptide according to the present invention.
Figure 6 shows the surface reaction analysis (RSM) results of the optimal production conditions for mussel small molecule peptides according to the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it is to those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention. it will be self-evident

Frozen mature pearl mussels ( Mytilus edulis ) were purchased. In the present invention, protein and peptide analysis was performed using a BCA protein analysis kit (Thermo scientific, Seoul, Korea). Tri-chloro-acetic (TCA) was used for peptide and protein precipitation (Sigma Company, Seoul, Korea). Proteolytic enzymes alcalase, bromelain, flavourzyme, neutrase, papain and protamex were purchased from Daejong Trading (Seoul, Korea), and Trypsin was purchased from Sigma company (Seoul, Korea).

The enzymatic hydrolyzate was separated using an ultrafilter (Amicon TCF-10; Amicon Co., USA). Amino acid analysis was quantitatively analyzed with an automatic analyzer (S433-H, Sykam GmbH, Germany, Munich). A cation separation column (LCA K06/Na) was used for the amino acid automatic analyzer column, the column size was 4.6 × 150 mm, the column temperature was 57-74°C, and the flow rates of the buffer and OPA reagent were 0.45 mL/min, 0.25, respectively. mL/min.

[Example 1] Preparation of mussel protein enzyme hydrolyzate

Put the raw, semi-dried mussel raw material in a predetermined amount of purified water, administer a commercial protease in the enzyme reactor, hydrolyze it, and then separate it using an 8 mesh screen to discard the residue after the enzyme reaction. The filtrate was put in a concentrator, concentrated to 40 Brix, heated at 95°C for 10 minutes to stop the enzymatic reaction, and then concentrated again to 60 Brix, and a certain amount of the concentrated solution was added at a temperature of 45-60°C and vacuum degree of 720-740mmHg under vacuum. Dry for 5 hours.

[Example 2] Manufacturing process of low molecular weight peptide using mussel protein

Bromelain was added to mature mussels and reacted for 4 hours at an optimum temperature of 53° C., pH 7.2, and an enzyme concentration of 3% (relative to mussel protein). After the reaction, the concentration of the protein hydrolyzate treated with the residue was 12-15 Brix, and after concentration to 30 Brix, salt and an extender were added, and the concentration was continued to 55 Brix. The basic formulation table is shown in [Table 1] below.

In terms of mussels produced in Korea, on average, 30 g per piece, the proportion of edible parts is 50%. The important nutrients of the edible part consisted of 1.5 g of protein, 0.6 g of carbohydrate, and 0.2 g of lipid. The raw mussel used in the present invention contains 9.7 g of protein, 4.0 g of carbohydrate, 1.2 g of lipid, and 82.80 g of moisture per 100 g. The self-dried mussels are 56.10 g of protein, but a prototype was produced using the self-made mussels due to the low processability and high cost.

Experimental Example 1. Yield of mussel small-molecular peptide production by commercial protease

The production yield results of the protein hydrolyzate (low molecular weight peptide) contained in the mussel slurry using the commercial proteases alcalase, bromelain, flavourzyme, neutrase, prozyme, and protamex are shown in FIG. 1 . For the protein hydrolysis reaction, the enzyme reactor (working volume; 800 ml) prepared in the present invention was used. Hydrolysis reaction conditions were carried out according to the instructions (manual) of the company that provided the enzyme, unless otherwise specified. The hydrolysis yield was different depending on the type of enzyme used. This is thought to be because the position of the hydrolyzed peptide bond is different depending on the characteristics of the enzyme and the size of the average molecular weight of the produced peptide is different. The yields of the enzymes alcalase, bromelain, flavourzyme, neutrase, prozyme, and protamex used were 37.36, 67.87, 50.23, 32.27, 58.07 and 54.23%, respectively. The highest yield was obtained with bromelain. On the other hand, the lowest yield was found in neutrase.

Experimental Example 2. Antioxidant properties of mussel protein low molecular weight peptide (in vitro test)

Protein hydrolysates were prepared from mussels using proteases alcalase, bromelain, flavourzyme, neutrase, prozyme, and protamex. Optimal conditions for the enzyme reaction were performed under the conditions provided by the enzyme supplier. After the enzymatic reaction, the unreacted portion of the low molecular weight peptide and the reaction product were separated by centrifugation. The reaction products were pulverized in a vacuum dryer.

1. 1.1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity

1 shows the results of analysis of DPPH antioxidant capacity of small molecule peptides produced under different proteases and hydrolysis conditions. The DPPH antioxidant capacity of hydrolysates of mussels was found to be affected by the type of enzyme used. This is thought to be because the peptide types of hydrolyzates are different because the enzymes used have different action sites for peptide bonds. The DPPH antioxidant capacity of hydrolysates was approximately 25.50 to 68.20% or more on average. The scavenging activity of bromelain, flavourzyme, prozyme, and protamex was similar, but hydrolysates by alcalase and neutrase were relatively low. According to the research results, it is known that the antioxidant capacity of peptides is higher as 1) non-polar and aromatic amino acids among the constituent amino acids, and 2) the molecular weight is small. These study results may explain the reason for the different antioxidant capacity depending on the protease enzymes used.

2. Superoxide radical scavenging ability

Figure 2 shows the results of analysis of superanion scavenging antioxidant capacity of mussel protein low molecular weight peptides produced using different proteases. It was found that the superanion scavenging antioxidant ability of mussel small-molecular peptides was affected by the type of enzyme used. The superanion-scavenging antioxidant capacity of hydrolysates was about 16.73 to 69.16% or more, and unlike the DPPH-scavenging antioxidant capacity, alcalase and bromelain showed a similar distribution. This result is thought to be due to the different peptide types of hydrolyzates because the enzymes used have different action sites for peptide bonds. Also, it is considered that the types of radicals to be removed are different depending on the hydrolyzate.

3. Hydroxy radical scavenging ability

Figure 3 shows the difference in the results of analysis of the hydroxyl radical scavenging antioxidant capacity of mussel protein low molecular weight peptides produced using different proteases. The hydroxyl radical scavenging antioxidant ability of hydrolysates of mussels was found to be affected by the type of enzyme used. The antioxidant capacity of hydrolysates was distributed from about 10.0 to 70.0% or more. Unlike superoxide radical scavenging antioxidant capacity, alcalase had the lowest antioxidant capacity, and the remaining enzymes showed about 40% or more, showing a similar trend to that of DPPH radical scavenging activity. . This result is thought to be due to the different peptide types of hydrolyzates because the enzymes used have different action sites for peptide bonds. According to the research results, it is known that the smaller the molecular weight of the peptides, the higher the antioxidant capacity. Also, according to the research results of other researchers, the increase in the hydroxyl radical scavenging power decreased with the size of the peptide, and it was reported that a peptide with a low molecular weight is more effective as a strong hydroxyl radical scavenger than a peptide with a higher molecular weight. This is consistent with the fact that the antioxidant power is different depending on the proportion of the hydroxyl radical scavenging peptide and the non-removing peptide in the hydrolyzate.

4. Antioxidant inhibitory concentration of the produced low molecular weight peptide

The mussel protein hydrolysates showed different DPPH scavenging ability, superoxide radical scavenging ability, and hydroxy radical scavenging ability depending on the type of enzyme used. Although the DPPH scavenging ability and the hydroxy radical scavenging ability were different for the hydrolyzate, the trends were similar. These reasons can be attributed to the difference in molecular weight of the hydrolyzate produced for each enzyme used and the difference in the type of peptide. To compare the antioxidant inhibitory ability of hydrolysates, each hydrolyzate was evaluated for IC50. As shown in [Table 2], overall, the antioxidant inhibitory ability was significantly different depending on the enzyme used and the antioxidant inhibition target.

The hydrolyzate produced by Alcalase showed high inhibitory concentrations in the order of hydroxy radical scavenging ability, superoxide radical scavenging ability, and DPPH scavenging activity. Superoxide radical scavenging activity, hydroxy radical scavenging activity and DPPH scavenging activity inhibitory concentrations were 1.2, 5.80, 12.40 and 44.7 mg/ml, respectively. The hydrolyzate produced by bromelain showed high inhibitory concentrations in the order of metal chelation ability, superoxide radical scavenging ability, DPPH scavenging ability and hydroxy radical scavenging ability. The metal chelation ability, superoxide radical scavenging ability, hydroxy radical scavenging ability and DPPH scavenging activity inhibitory concentrations were 3.08, 6.8, 16.32 and 6.10 mg/ml, respectively. The hydrolyzate produced by Flavorzyme showed high inhibitory concentrations in the order of metal chelation ability, superoxide radical scavenging ability, DPPH scavenging ability and hydroxyl radical scavenging ability. The metal chelation ability, superoxide radical scavenging ability, hydroxy radical scavenging ability and DPPH scavenging activity inhibitory concentrations were 2.05, 8.20, 8.14 and 10.1 mg/ml, respectively. The hydrolyzate produced by neutrase showed a high inhibitory concentration in the order of metal chelation ability, superoxide radical scavenging ability, DPPH scavenging ability and hydroxy radical scavenging ability. The metal chelation ability, superoxide radical scavenging ability, hydroxy radical scavenging ability and DPPH scavenging activity inhibitory concentrations were 0.83, 7.20, 10.97 and 23.9 mg/ml, respectively. The hydrolyzate produced by the prozyme showed high inhibitory concentrations in the order of metal chelation ability, superoxide radical scavenging ability, DPPH scavenging ability and hydroxy radical scavenging ability. The metal chelation ability, superoxide radical scavenging ability, hydroxy radical scavenging ability and DPPH scavenging activity inhibitory concentrations were 2.19, 11.2, 13.12 and 17.6 mg/ml, respectively. The hydrolyzate produced by Protamex showed high inhibitory concentration in the order of metal chelation ability, superoxide radical scavenging ability, DPPH scavenging ability and hydroxy radical scavenging ability. The metal chelation ability, superoxide radical scavenging ability, hydroxy radical scavenging ability and DPPH scavenging activity inhibitory concentrations were 2.63, 11.2, 13.12 and 17.6 mg/ml, respectively.

Experimental Example 3. Measurement of molecular weight of mussel protein low molecular weight peptide

1. MALDI-TOF Chromatogram

To determine the molecular weight of the hydrolyzed mussel hydrolyzate using Bromelamin, Flavorzyme, Protamex, and Prozyme, Matrix Assisted Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry (MALDI-TOF-MS) analysis was performed.

2 μL of the mussel hydrolyzate sample was attached to a polished steel 384 target plate. 10mg/mL of α-Cyano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA and ACN 1:1 mixed solution was mixed and dried. Each sample was analyzed using MALDI TOF MS of a flex Control 3.0 mass spectrometer (Bruker Daltonics, Germany). Data were acquired by accumulating data from 300 laser shots in cationization and reflection modes at 100-4,000 m/z.

As shown in FIG. 4, the molecular weight of the hydrolyzate obtained by hydrolyzing mussels using bromelamin and flavorzyme was analyzed using a MALDI-TOF mass spectrometer. was in the range of about 500 to 600 Da. In addition, the molecular weight range of the peptide hydrolyzed using the flavourzyme was about 350 to 730 Da. When hydrolyzed using Protamex, the molecular weight range of the peptide was about 490 to 1,020 Da, and when treated with a prozyme enzyme under the same conditions, the molecular weight range of the peptide was about 500 to 730 Da.

As a result, most of the low molecular weight peptides have a total average molecular weight of 128 Da. The minimum number of peptide bonds is 4 or less in the case of bromelain and 2-5 in the case of flavorzyme. Protamex was distributed in the range of 500-1,000, 4-7, and prozyme was also distributed in the range of 2-5.

The distribution of the average molecular weight of the produced low molecular weight peptides shows that the hydrolyzate of bromelain consists of 2 peptides, 5 flavors, 5 types of protamex, and 5 types of prozymes.

2. Bromelain and flavourzyme Amino acids composing mussel-derived peptides

Fig. 5 shows the distribution of constituent amino acids when the mussel small-molecular peptide was hydrolyzed with bromelain enzyme. The distribution of constituent amino acids showed the highest content in the order of glutamic acid (6.08%), aspartic acid (10.07%), and arginine (8.72%), among which essential amino acids (threonine, valine, methionine, isoleucine, leucine, phenylalanine, histidine, lysine) was found to be 40.55%. It is known that the amino acid composition of proteins and protein hydrolysates greatly affects the functionality [19]. The hydrophobicity of the peptide is known to have high functionality because it is easy to reach the target point by passing through the phospholipid layer of the cell membrane. Hydrophobic amino acids in the mussel protein hydrolyzate accounted for 42.54% of the total, which seems to be related to the antioxidant function. Lysine and tyrosine, which exhibit antioxidant functions, donate electrons, whereas histidine has an imidazole structure and has been reported to have a strong radical scavenging ability.

The mussel protein hydrolyzate showed different amino acid distribution according to the type of enzyme used. The distribution of amino acids hydrolyzed by bromelamin, flavorzyme, and enzymes is shown in FIG. 5 . A shows the distribution of amino acids hydrolyzed using Bromelamin enzyme, and B shows the distribution of amino acids hydrolyzed using Flavorzyme enzyme. C shows the distribution of amino acids hydrolyzed using the Protamex enzyme, and D shows the distribution of amino acids hydrolyzed using the Prozyme enzyme.

5A shows the distribution of constituent amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine, threonine) when hydrolyzed with Bromelamin enzyme. Among the distribution of constituent amino acids, glutamic acid showed the highest content at 12.679%, and among the distribution of essential amino acids, leucine was found to occupy the largest content at 10.309%.

FIG. 5B shows the distribution of constituent amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine, and threonine) when hydrolyzed with a Flavorzyme enzyme. Among the distribution of constituent amino acids, valine had the highest content at 13.137%, and it was found that it occupies the highest content among the distribution of essential amino acids.

5C shows the distribution of constituent amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine, threonine) when hydrolyzed with Protamex enzyme. Among the distribution of constituent amino acids, valine had the highest content at 17.338%, and it was found that it occupies the highest content among the distribution of essential amino acids.

5D shows the distribution of constituent amino acids and essential amino acids (valine, methionine, isoleucine, leucine, phenylalanine, lysine, threonine) when hydrolyzed with a prozyme enzyme. Among the distribution of constituent amino acids, valine occupied the largest content at 19.359%, and it was found that it occupies the most content among the distribution of essential amino acids.

In addition, cystine, tyrosine, and arginine were hardly found in the amino acid distribution of Bromelamin, Flavorzyme, Protamex, and Prozyme enzymes. The highest total amino acid content was the flavourzyme enzyme, and the essential amino acid content was found to be 52.355% among these enzymes.

The amino acids constituting the hydrolyzate of mussels hydrolyzed with bromelamin, flavourzyme, protamex, and prozyme enzymes were classified into non-polar amino acids, polar amino acids, cationic amino acids, and anionic amino acids and shown in [Table 3]. As for the content of non-polar amino acids, valine showed the highest content when hydrolyzed with prozyme enzyme, leucine showed the highest content when hydrolyzed with bromelamin enzyme, and valine showed the highest content when hydrolyzed with flavorzyme and protamex enzymes content was shown. The content of polar amino acids showed the highest glycine content when hydrolyzed by the flavourzyme enzyme, the highest glycine content when hydrolyzed by the bromelamin enzyme, and the highest in threonine when hydrolyzed by the protamex and prozyme enzymes. showed As for the content of cationic amino acids, lysine showed the highest content when hydrolyzed with flavorzyme and bromelamin enzymes, and histidine showed the highest content when hydrolyzed with protamex and prozyme enzymes. When hydrolyzed with prozyme enzyme, aspartic acid showed the highest content, and when hydrolyzed with bromelamin enzyme, glutamic acid showed the highest content.

Experimental Example 4. Optimization of production of mussel small-molecular peptides by enzymes by surface reaction analysis (RSM)

The degree of hydrolysis was measured by designing an experiment using the Box-Behnken design for the three experimental variables of temperature, pH, and amount of enzyme, which are the process condition factors for enzymatic hydrolysis of mussel protein. As a result of the hydrolysis test of mussel protein, measured values were obtained in the range of minimum 21.2 - maximum 72.5% of the hydrolysis yield under the conditions of a reaction temperature of 40-60 ° C, a reaction pH of 6-8, and an enzyme concentration of 1-3%. In the enzymatic reaction conditions, the yield at the center of the reaction ranged from 51.2 to 61.2%, with an average of 55.16%. The highest yield was 72.5% at a reaction temperature of 60°C, pH 7, and an enzyme concentration of 3%.

1. Optimal production conditions according to temperature and pH

As shown in Figure 6, the concentration of the input enzyme was fixed at 2%, and as a result of the analysis of the surface reaction at the reaction temperature range of 40-60 °C and the reaction pH range of 6-8, the reaction temperature and pH affect protein hydrolysis. has been shown to give The hydrolysis according to the reaction temperature gradually increased from 40°C to 50°C and then gradually decreased after passing the normal point around 53.5°C. decreased gradually. Therefore, when the concentration of the input enzyme was 2%, the maximum value of the hydrolysis degree was determined as a stationary point at a reaction temperature of about 53.5° C. and a reaction pH of about 7.2, and the degree of hydrolysis at this time was about 55.9%.

2. Optimal production conditions according to temperature and amount of enzyme

As shown in FIG. 6 , the reaction temperature and enzyme concentration affect protein hydrolysis as a result of analysis of the surface reaction at the reaction temperature range of 40-60° C. and the enzyme concentration range of 1-3% with the enzyme reaction pH fixed at 7.0. has been shown to give Hydrolysis according to the reaction temperature gradually increased from 40°C to 50°C, and showed a tendency to gradually decrease after passing around 51°C. showed a steadily increasing trend. Therefore, when the reaction pH was fixed at 7, the maximum value of the degree of hydrolysis did not appear in the experimental range, but as a result of ridge analysis, it was determined at a reaction temperature of about 51° C. and an enzyme concentration of 3%, and the degree of hydrolysis at this time was about 68.2%.

3. Optimal production conditions according to pH and enzyme amount

As shown in FIG. 6 , the reaction pH and enzyme concentration affect protein hydrolysis as a result of analysis of the surface reaction at the reaction pH range of 6-8 and the enzyme concentration range of 1-3% with the enzyme reaction temperature fixed at 50° C. has been shown to give The hydrolysis according to the reaction pH showed a tendency to gradually increase at pH 6 and then gradually decrease after passing around pH 7.3. showed a trend. Therefore, when the temperature was fixed at 50°C, the maximum value of the degree of hydrolysis did not appear in the experimental range, but as a result of ridge analysis, it was determined at a reaction temperature of about pH 7.3 and an enzyme concentration of 3%, and the degree of hydrolysis at this time was about 71.60%. From these results, the optimal process obtained in the surface reaction analysis was determined at an enzyme reaction temperature of 55° C., a reaction pH of 6.5, and the amount of enzyme 3% (w/v). Under optimal conditions, the degree of protein hydrolysis reached 67.5%.

4. Optimal conditions according to temperature, pH and amount of enzyme

In summary, as a result of the analysis of the surface reaction, the maximum value was not found within the experimental section, but as a result of the analysis of the ridgeline, temperature has the most influence on the hydrolysis of mussel protein. As the temperature increases, it can be seen that the degree of hydrolysis increases rapidly and then decreases rapidly. On the other hand, the degree of hydrolysis showed a tendency to steadily increase as the concentration of the enzyme increased regardless of the reaction pH and temperature. Therefore, it can be seen that the effect on hydrolysis is large in the order of the concentration of the enzyme, the reaction temperature, and the reaction pH.

Under the optimal reaction conditions, the hydrolysis reaction conditions for mussel protein are determined as a temperature of 53°C, a reaction pH of 7.2, and an enzyme concentration of 3% based on the hydrolysis yield, the appropriateness (cost) of the use of the enzyme, and the efficiency of the process, and the predicted yield is 70- 73%.

Experimental Example 5. Mussel Small Molecular Peptide Efficacy Experiment

As a result of evaluating the production yield of mussel protein hydrolyzate, establishment of an optimal production process and processing characteristics, and antioxidant DPPH, in vivo and in vivo efficacy experiments on bromelain and flavourzyme enzyme hydrolysates were conducted at Daejeon University College of Oriental Medicine, which operates an efficacy analysis laboratory. was selected as a consignment institution.

1. Antioxidant properties of mussel protein hydrolyzate (in vitro, cellular level test)

(1) Cell viability

As shown in [Table 4], as a result of measuring the cell viability, Bromelain and Flavorzyme did not show toxicity to cells at a concentration of 5% or less.

(2) Intracellular superoxide dismutase (SOD) activity

As shown in [Table 5], as a result of measuring the intracellular SOD activity, Bromelain and Flavorzyme showed a significant (**: p<0.01, ***: p<0.001) increase compared to the control at 5% concentration.

(3) intracellular catalase activity

As shown in [Table 6], as a result of measuring intracellular catalase activity, Bromelain and Flavorzyme were significantly more significant than the control group at all concentrations (*: p<0.05, **: p<0.01, ***: p< 0.001) increased.

(4) Intracellular GSH peroxidase (GPx) activity

As shown in [Table 7], as a result of measuring intracellular Gpx activity, Bromelain and Flavorzyme were significantly more significant than the control group at a concentration of 1% or more (*: p <0.05, **: p <0.01, ***: p<0.001) increased.

(5) Intracellular GSH reductase (GR) activity

As shown in [Table 8], as a result of measuring intracellular GR activity, Bromelain and Flavorzyme were significantly higher than the control group at all concentrations (* : p <0.05, ** : p < 0.01, *** : p < 0.001) increased.

(6) Intracellular glutathione (GSH) content

As shown in [Table 9], as a result of measuring the intracellular GSH content, Bromelain and Flavorzyme showed a significant (**: p<0.01, ***: p<0.001) increase compared to the control at a concentration of 1% or more. appear.

(7) Free fatty acid content in cells

As shown in [Table 10], as a result of measuring the intracellular free fatty acid content, Bromelain was significantly more than 2% concentration and Flavourzyme compared to the control group at all concentrations (*: p <0.05, **: p < 0.01 , *** : p<0.001 ) decreased.

(8) Intracellular lipid peroxidation (MDA) content

As shown in [Table 11], as a result of measuring the intracellular MDA content, Bromelain was significantly higher than the control at a concentration of 2% or more and Flavorzyme at a concentration of 1% or more (*: p<0.05, **: p<0.01) , *** : p<0.001 ) decreased.

As a result of the antioxidant activity test, fermented mussels (FTP) showed a concentration-dependent effect (*: p＜0.05, **: p<0.01, ***: p<0.001).

Experimental Example 6. Antioxidant properties of mussel protein hydrolyzate (in vitro, animal test)

1. Gene expression level

(1) iNOS

As shown in [Table 12], as a result of measuring the iNOS gene expression level in the spleen tissue, a significant (***: p<0.001) decrease was observed in all groups administered with FTP compared to the control group.

(2) IL-6

As shown in [Table 13], as a result of measuring the IL-6 gene expression level in the spleen tissue, a significant (**: p<0.01) decrease was observed in all administration groups of 400 mg/kg FTP compared to the control group.

(3) IL-1β

As shown in [Table 14], as a result of measuring the IL-1β gene expression level in the spleen tissue, there was a significant (*: p<0.05, ***: p<0.001) decrease in all administration groups of FTP compared to the control group. appear.

(4) TNF-α

As shown in [Table 15], as a result of measuring the TNF-α gene expression level in the spleen tissue, a significant (**: p<0.01, ***: p<0.001) decrease in all administration groups of FTP compared to the control group has appeared

(5) COX-2

As shown in [Table 16], as a result of measuring the expression level of the COX-2 gene in the spleen tissue, there was a significant (*: p<0.05, ***: p<0.001) decrease in all administration groups of FTP compared to the control group. appear.

2. Biomarker production amount

(1) IL-6

As shown in [Table 17], as a result of measuring the amount of IL-6 production in the serum, all of the FTP administration groups decreased compared to the control group, but no significance was observed.

(2) IL-1β

As shown in [Table 18], as a result of measuring the amount of IL-1β production in serum, a significant (***: p<0.001) decrease was observed in the FTP 400 mg/kg administration group compared to the control group.

(3) TNF-α

As shown in [Table 19], as a result of measuring the amount of TNF-α production in the serum, a significant (***: p<0.001) decrease was observed in all administration groups of FTP compared to the control group.

As a result of the anti-inflammatory activity test, the expression levels of iNOS, IL-6, IL-1β, COX-2, and TNF-α genes in the spleen of fermented mussel (FTP) were concentration-dependent (* : p <0.05, ** : p < 0.01, ***: p<0.001).

3. Antioxidant Enzyme Activity

(1) SOD

As shown in Table 20, as a result of measuring the SOD activity in the liver tissue, a significant (***: p<0.001) increase was observed in the 400 mg/kg administration group of FTP compared to the control group.

(2) Catalase

As shown in [Table 21], as a result of measuring catalase activity in liver tissue, a significant (***: p<0.001) increase was observed in the 400 mg/kg administration group of FTP compared to the control group.

(3) GPx

As shown in [Table 22], as a result of measuring GPx activity in liver tissue, a significant (**: p<0.01) increase was observed in the 400 mg/kg administration group of FTP compared to the control group.

(4) GR

As shown in [Table 23], as a result of measuring GR activity in the liver tissue, a significant (***: p<0.001) increase was observed in the 400 mg/kg administration group of FTP compared to the control group.

(5) GSH

As shown in [Table 24], as a result of measuring the GSH level in the liver tissue, a significant (***: p<0.001) increase was observed in the 400 mg/kg administration group of FFTP compared to the control group.

(6) Free fatty acid

As shown in [Table 25], as a result of measuring the level of free fatty acid in the liver tissue, a significant (***: p <0.001) decrease was observed in all administration groups of FTP compared to the control group.

(7) MDA

As shown in [Table 26], as a result of measuring the MDA level in the liver tissue, a significant (***: p <0.001) decrease was observed in all administration groups of FTP compared to the control group.

As a result of the antioxidant activity test, fermented mussels (FTP) showed a concentration-dependent (**: p＜0.01, ** *: p<0.001) was increased.

Claims (4)

A food composition comprising a mussel-derived low molecular weight peptide as an active ingredient.
The method of claim 1,
The low molecular weight peptide is a food composition, characterized in that it is prepared by hydrolyzing the mussel protein with a protease enzyme.
The method of claim 1,
The mussel-derived low molecular weight peptide is a food composition, characterized in that it exhibits an antioxidant effect.
A method for preparing a composition comprising a mussel-derived low molecular weight peptide as an active ingredient, comprising the steps of:
(a) grinding the mussels to prepare a slurry; and
(b) reacting by adding a proteolytic enzyme to the mussel slurry

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