KR20180055120A - Protein Hydrolysate of larva of Tenebrio molitor, Method for Preparing the Same and Food Comprising the Same - Google Patents

Abstract

The present invention relates to a protein hydrolysate of mealworm (Tenebrio molitor) larvae, a production method thereof, and a functional food composition containing the same. According to the present invention, the protein hydrolysate of mealworm larvae shows excellent antioxidative effects, and particularly enables acquisition of physiologically active peptides exhibiting antioxidative effects at high yield when produced by the production method of the present invention. The functional food composition using the same can derive high antioxidative effects, hepatoprotective functions, and stress relief effects.

Description

TECHNICAL FIELD [0001] The present invention relates to a protein hydrolyzate of Tenebrio molitor (mealworm) larva, a method for preparing the same, and an antioxidant, a composition for protecting the liver and a composition for reducing stress, }

The present invention relates to a protein hydrolyzate of brown larch, a method for producing the protein hydrolyzate, and a functional food composition containing the same.

Recently, the FAO has announced plans to revitalize edible insects as a future food resource as a policy to solve the food shortage problem caused by diseases and environmental pollution, and interest in this is increasing worldwide. In line with this, the Ministry of Food, Agriculture, Forestry and Fisheries has announced the "Five-Year Comprehensive Plan for Insect Industry Development" and has been supporting the insect industry accordingly.

Insects are the most diverse species on the planet, with more than one million species reported, but more than one million species are expected to remain unreported. Insects have been used as medicines for some time, and silkworms, grasshoppers and zines have been reported to be effective in the treatment of chronic diseases such as diabetes, inflammatory diseases, liver diseases and arteriosclerosis.

Brown Tortoise ( Tenebrio molitor , mealworm) is an insect of beetle, and it is called "brown rice duck". It is distributed in Korea and other parts of the world. It has a strong adaptability and it has a short transfiguration period. It is easy. In addition, nutritional components of brown goat have very high nutritional content, such as 2.90% moisture, 50.32% crude protein, 33.70% crude fat, 3.76% inquiry and 4.81% crude fiber. In relation to this, brown dough is a high-protein material with a very high protein content and is registered as a food source for food insecticides in 2016, thereby increasing interest in the use of brown dough. Brown goat larvae contain more essential amino acids than soybeans, have higher unsaturated fatty acid content than meat, and are rich in vitamin A, iron, and dietary fiber. Moreover, unlike livestock breeding, there is no need to improve the land required for insect rearing, and it is possible to easily feed large amounts of insects with only a small amount of feed, which is economical.

Studies on the brown goose larvae in Korea include studies on the cytotoxic effect of the brown goose larvae extract on hepatocarcinoma cells and the inhibition of melanin formation of the brown goose-ethanol extracts. In contrast, brown gooseberry larvae The research on physiologically active peptides is in short supply.

Physiologically active peptides are generally defined as peptides with small molecular weight with physiological activity. They are usually composed of 3-20 amino acids, and their activities vary depending on the composition and sequence of amino acids. In addition, it is small in size and can be easily absorbed into living body, and exhibits various functional properties. The effects of the physiologically active peptides studied so far include improvement of the antioxidant function of the milk protein hydrolyzate, prevention of the oxidation reaction in the processed food, and a new antimicrobial peptide of the egg white hydrolyzate. In addition, antihypertensive, antithrombotic, And the like have been actively studied for the functionalization of physiologically active peptides.

On the other hand, antioxidants are compounds that have the function of preventing or delaying the change of the smell and flavor of food caused by oxidation, the rancidity of the fattening and the discoloration of the food, and also the artificial compounds, Much of the material is known. As the interest in antioxidants that can suppress the oxidation reaction has increased, various synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), butyl hydroquinone and tert-butylhydroquinone (TBHQ).

However, since the safety controversy of these synthetic antioxidants has been constantly raised, legal restrictions on the amount of the antioxidants have been strictly regulated. As a result, studies on natural antioxidants that are relatively safe have been actively conducted to replace them.

Thus, as described above, the development of a technique for obtaining a physiologically active peptide obtained from a brown goat larva, which can be mass-produced and used as a natural antioxidant, is easy to breed and has a great number of nutrients. It is necessary to develop new natural antioxidants.

1. Korean Registered Patent No. 10-1404566 (registered on May 30, 2014).

An object of the present invention is to provide a protein hydrolyzate excellent in antioxidant effect.

Another object of the present invention is to provide a functional food composition which is excellent in antioxidative effect and can be utilized in various ways.

It is still another object of the present invention to provide a method for producing a protein hydrolyzate having an excellent antioxidative effect.

The present invention to achieve the above object, proteolytic enzyme, Alcala dehydratase (Alcalase) the hydrolysis to mealworm (Tenebrio molitor , mealworm) or a protein hydrolyzate of at least one protein selected from the group consisting of Flavourzyme, Neutrase, Pepsin, Subtilisin and Pancreatin. It provides proteolytic hydrolysates of larvae ( Tenebrio molitor, mealworm) hydrolyzed with degrading enzymes and alkalase.

In order to accomplish the above other objects, the present invention provides a functional food composition for antioxidation, a functional food composition for liver protection or a functional food composition for stress reduction, which comprises the protein hydrolyzate of the brown larch larva.

The addition the present invention to another aspect is, (a) the substrate is dried mealworm (Tenebrio molitor, mealworm) and mixed with a powder of the larvae in water or a buffer solution from 1 to 10% (w / v) substrate solution Lt; / RTI > (b) heating the substrate solution to inactivate the self-enzyme contained in the substrate solution; (c) mixing the substrate solution with a protein hydrolyzing enzyme in an amount of 0.1 to 5% (w / v) relative to the brown goat larvae powder to prepare a mixture; (d) preparing a reaction mixture by reacting a protein hydrolyzing enzyme and a brown larvae larva in the mixture; (e) heating the reaction mixture to inactivate the protein hydrolyzing enzyme to obtain a reaction mixture; (f) centrifuging the mixed solution obtained in step (e) to take a supernatant; (g) centrifuging the supernatant using a filtration membrane; And (h) separating a protein hydrolyzate having a weight average molecular weight of 0.01 kDa or more and 3 kDa or less.

Since the brown gill larva protein hydrolyzate of the present invention contains a physiologically active peptide having a bioactive peptide of 3 kDa or less by using a specific enzyme, the functional food composition containing the protein hydrolyzate can reduce stress, It can be usefully used for protecting liver cells from oxygen to prevent or improve diseases associated with active oxygen.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram illustrating a method of preparing a brown gill larva protein hydrolyzate according to one embodiment of the present invention.
FIG. 2 is a result of SDS-PAGE of the brown larvae protein hydrolyzate according to another embodiment of the present invention.
FIG. 3 is a graph showing the yield of the brown gill larva protein hydrolyzate according to the embodiment of FIG. 2. FIG.
FIG. 4 is a graph showing the degree of protein hydrolysis of the brown gill larva protein hydrolyzate according to the embodiment of FIG. 2. FIG.
FIG. 5 is a graph showing DPPH radical scavenging activity of physiologically active peptides in the brown gill larva protein hydrolyzate according to another embodiment of the present invention.
FIG. 6 is a graph showing the results of measuring the ABTS radical scavenging activity of physiologically active peptides in the brown gill larva protein hydrolyzate according to the embodiment of FIG. 5. FIG.
FIG. 7 shows the results of measurement of the hydrogen peroxide scavenging activity of the physiologically active peptides in the hydrolysates of the larvae of the larvae according to the embodiment of FIG.
FIG. 8 shows the results of confirming the effect of inhibiting linoleic acid oxidation of physiologically active peptides in the hydrolysates of the larvae of the larvae according to the embodiment of FIG.
FIG. 9 shows the results of confirming the hepatocyte protective effect of the brown gill larva protein hydrolyzate according to another embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides a protein hydrolyzate of a larva ( Tenebrio molitor, mealworm) hydrolyzed with a protein hydrolyzing enzyme, Alcalase.

In the present invention, "Alcalase" is a protein hydrolyzing enzyme produced from bacteria or fungi, and can exhibit strong protein resolution under the condition of about pH 7-9.

In one embodiment of the present invention, the protein hydrolyzate of the larvae hydrolyzed with the alkaline protease exhibits a very high antioxidative effect and the effect of protecting the hepatocyte from the active oxygen, and the protein hydrolyzate is useful as various functional foods Can be utilized.

The present invention also relates to a pharmaceutical composition comprising at least one protein hydrolyzing enzyme selected from the group consisting of Flavourzyme, Neutrase, Pepsin, Subtilisin and Pancreatin, A brown goose hydrolyzed with Alcalase ( Tenebrio molitor , and mealworm) protein hydrolysates, wherein the protein hydrolytic enzymes are not limited to flavorzyme, ntrease, pepsin, subtilisin, and pancreatin.

In the present invention, "flavorzyme" is a protein hydrolyzing enzyme obtainable from fungi, and can exhibit protein resolution at a pH of about 6-7.

In the present invention, "Neutrase" is a protein hydrolyzing enzyme obtainable from Bacillus subtilis, and can exhibit proteolytic activity by hydrolyzing a peptide bond inside the protein at a pH of about 5.5 to 7.5.

In the present invention, " pepsin " is a protein hydrolyzing enzyme secreted in the stomach chief cell of an animal. The protein hydrolyzing enzyme can hydrolyze a portion of the protein having an aromatic amino acid such as phenylalanine and tyrosine.

In the present invention, " Subtilisin " is a serine protease, which can be obtained from Bacillus sp., And has an optimal active pH of about neutral to alkaline.

In the present invention, " pancreatin " is a protein hydrolyzing enzyme produced in the pancreas of an animal and can exhibit protein resolution around pH 8.0.

At this time, the protein hydrolyzate may include peptides of 0.01 kDa or more and 3 kDa or less, and the peptides may be also referred to as physiologically active peptides.

Thus, the protein hydrolysates containing the physiologically active peptides can exhibit an antioxidative effect.

Furthermore, the present invention provides a functional food composition for antioxidation or a functional food composition for liver protection or a functional food composition for stress reduction comprising the protein hydrolyzate of brown larch larva.

That is, since the protein hydrolyzate includes a physiologically active peptide, functional food compositions containing it as an active ingredient can exhibit an excellent antioxidative effect, a liver protecting effect from active oxygen, and an effect of reducing cell stress.

The functional food composition may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, A carbonating agent used in organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, antiseptics, glycerin, alcohols and carbonated beverages, and the like. May be further contained. These components may be used independently or in combination.

In addition, the functional food composition may further include a food additive, and the suitability of the food additive as a "food additive" is not limited to the corresponding item in the General Rules and General Test Methods approved by the Food and Drug Administration Shall be determined according to the relevant standards and standards.

Examples of the products that have been used in the above-mentioned "food additives" include natural products such as ketones, chemical products such as glycine, potassium citrate, nicotinic acid and cinnamic acid, sensory coloring matter, licorice extract, crystalline cellulose, high- - Mixed preparations such as a sodium glutamate preparation, a noodle-added alkaline agent, a preservative preparation, and a tar coloring agent.

The functional food may be in the form of any one of meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol and vitamin complex , But is not limited thereto.

At this time, the protein hydrolysates of the larvae of the present invention, which are added to foods in the process of producing the functional food, can be appropriately added or decreased as needed.

In addition, the present invention relates to a process for the preparation of (a) a dried brown gill ( Tenebrio molitor , mealworm) 1 to 10% (w / v) of the larval powder in water or buffer solution to prepare a substrate solution; (b) heating the substrate solution to inactivate the self-enzyme contained in the substrate solution; (c) mixing the substrate solution with a protein hydrolyzing enzyme in an amount of 0.1 to 5% (w / v) relative to the brown goat larvae powder to prepare a mixture; (d) preparing a reaction mixture by reacting a protein hydrolyzing enzyme and a brown larvae larva in the mixture; (e) heating the reaction mixture to inactivate the protein hydrolyzing enzyme to obtain a reaction mixture; (f) centrifuging the mixed solution obtained in step (e) to take a supernatant; (g) centrifuging the supernatant using a filtration membrane; And (h) separating a protein hydrolyzate having a weight average molecular weight of 0.01 kDa or more and 3 kDa or less. When the protein hydrolyzate of brown larvae is prepared by the above-described method, the physiologically active peptide with the highest yield can be obtained.

At this time, the substrate solution may be prepared by mixing the brown germ larva powder dried in step (a) with water or a buffer solution at 3 to 7% (w / v), more preferably 4% (w / v) to prepare a substrate solution.

The buffer solution may preferably be a sodium phosphate buffer.

Preferably, in step (c), the protein hydrolyzing enzyme may be mixed with the brown goat larva powder at a concentration of 0.5 to 3% (w / v), more preferably 1% (w / v) to prepare a mixed solution.

Further, in the step (b) or (e), the reaction mixture may be heated at 60 ° C to 100 ° C to inactivate the self-enzyme or the protein hydrolase, preferably 75 ° C to 95 ° C, more preferably , It is possible to inactivate the self-enzyme or the protein hydrolase by heating at 90 ° C.

The protein hydrolyzing enzyme may be an Alcalase.

Alternatively, the protein hydrolase is selected from the group consisting of Alcalase and Flavourzyme, Neutrase, Pepsin, Subtilisin and Pancreatin. But the present invention is not limited thereto.

In other words, the protein hydrolyzate of brown larch larva according to the present invention, the method for producing the protein hydrolyzate, and the functional food composition containing the protein hydrolyzate according to the present invention can increase the yield of the physiologically active peptide showing an antioxidative effect by using a specific type of protein hydrolyzing enzyme, Can be utilized.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. It is to be understood that both the foregoing description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. no.

<Preparation Example 1> Preparation of sample

For the experiment, it was produced and sold on March 18, 2016 by MG Natural Co., Ltd. (Mudung-myeon, Damyang-gun, Jeollanam-do). The larvae of vacuum-dried brown duck were purchased and stored in frozen ware .

In addition, all the experimental data were expressed as mean and standard error (mean ± standard error) at least 3 times and SPSS Statistics 23 (Version 23.0, Inc, Chicago, IL, USA) was used for statistical analysis. One-way ANOVA was used to verify the significance of differences between groups, and post-test was performed using Scheffe's multiple range test. If the significance (P-value) was less than 0.05, it was judged to be statistically significant.

Example 1 Preparation of Protein Hydrolyzate of Brown Goat Protein

As shown in FIG. 1, the lyophilized brown gill larva powder prepared in Preparation Example 1 was dissolved in 10 mM sodium phosphate buffer (pH 7.0) to prepare a 4% (w / v) substrate solution. Lt; / RTI &gt; for 20 minutes. 1% (w / v) of protein hydrolytic enzymes (alcalase, flavourzyme, neutrase, bromelain, papain) Each enzyme was treated and reacted at 55 ℃ for 24 hours. At this time, each of the peptides was sampled by 5 mL of reaction mixture every hour to perform TNBS and SDS-electrophoresis. After 24 hours, the final reaction mixture was heated at 90 DEG C for 20 minutes to inactivate the enzyme. Then, the reaction solution was centrifuged at 13,000 × g for 20 minutes to obtain a supernatant. The supernatant was further centrifuged (5,000 × g) for 2 hours using an ultrafiltration membrane to obtain a protein hydrolyzate having a molecular weight of 3 kDa or less. The protein hydrolysates were lyophilized and stored at 20 ° C.

Example 2: Identification of physical properties of the larval protein hydrolyzate

(1) Sodium Dodecyl Sulfonate Polyacrylamide  Gel electrophoresis dodecylsulfate 폴리acrylamide  gel electrophoresis, SDS-PAGE)

In order to identify peptides of brown larvae according to protein hydrolytic enzymes, SDS-PAGE patterns of proteins were measured and the degradation patterns of the proteins in the respective hydrolysates were examined. SDS-PAGE was carried out using Laemmli (Laemmli, UK, 1970. Cleavage of structural proteins during the assembly of the bacteriophage T4, nature 227: 680-685), 15% , Hydrolyzed proteins were centrifuged at 12,000 rpm at 4 ° C for 10 minutes, and 20 μl of the supernatant was loaded on the gel and electrophoresed at 80 V for about 2 hours. The bands were then stained using coomassie brilliant blue and decolorized with 30% methanol containing 10% acetic acid. As a control, an enzyme-free substrate solution was used. Protein pattern analysis was performed by scanning the polyacrylamide gel using Gel Logic 2200 PRO and imaging the image. At this time, a molecular weight marker was obtained from BIO-RAD (Bio-Rad Laboratories, CA, USA).

As a result, as shown in Fig. 2, in the case of the hydrolyzate of the alkaline protease ([1] in Fig. 2) compared with the control group not treated with the enzyme The bands of this size did not appear to be degraded. From this, it was confirmed that the solution treated with the alkalase had many low molecular peptides, and the hydrolysis degree of the alkalase was excellent. Nutraaze ([2] in Fig. 2) showed a band of more than 10 kDa than protein hydrolyzate using alkalase. Plaborozymes ([3] in Fig. 2) showed peptides with large molecular weights due to an increase in the number of bands as compared with hydrolysates of alkalase and Ntrease. On the other hand, protein hydrolysates of bromelain ([2] in Fig. 2) and papain (in Fig. 2 [5]) show clear protein bands of 10 kDa to 25 kDa and confirm that they contain a large number of peptides of this molecular size I could.

When SDS-PAGE results are summarized, it was found that the hydrolysates of the enzyme hydrolyzate contain a large amount of low molecular weight peptides in the order of alkalase, ntrease, flavorzyme, bromelain and papain.

(2) Measurement of yield of hydrolyzate

The enzymatic degradation was carried out for 24 hours under optimal decomposition conditions in order to prepare the protein hydrolyzate of brown larvae using the enzyme in Example 1 above. Namely, a brown goat larva sample mixed with a buffer solution of pH 7.0, and alkalase, flavozyme, ntrease, bromelain and papain were added for 24 hours to inactivate the enzyme. To inactivate the enzyme, . The supernatant was collected by centrifugation, and peptides of 3 kDa or less were isolated using an ultrafiltration membrane at 5,000 xg for 2 hours at 8 ° C. Thereafter, the hydrolyzate of the brown larch larvae protein was prepared by lyophilization. As shown in the following equation 1, considering the salt by 10 mM sodium phosphate buffer, the hydrolyzate of the brown larch larva protein was measured as a ratio The yield of the hydrolyzate was calculated.

[Formula 1]

As a result, as shown in Fig. 3, the yield of protein hydrolyzate with alkalase was the highest at 44.71%, and that of Nutrase was 34.33%, flavorzyme 23.17%, bromelain 15.75%, papain 11.82% Respectively. This is in agreement with the result of SDS-PAGE analysis of the yield of the 3 kDa peptide of the protein hydrolyzate treated with the alkalase. Therefore, protein hydrolysates obtained by treating enzymes of higher yields such as alkalase, flavorzyme and Ntrease can be used economically and productively.

(3) Measurement of hydrolysis degree of protein

In order to confirm how much of the protein is hydrolyzed by the enzymes, the protein was hydrolyzed with a peptide of Tricholobenzenesulfonic acid (TNBS) method (G-Biosciences Co.) Was measured. When the protein is hydrolyzed, the concentration of the effective amino group increases in proportion to the degree of hydrolysis.

Specifically, the hydrolysis degree of the larval larvae protein over time at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after preparing three kinds of enzyme and substrate mixtures as in Example 1 Respectively. 0.01% TNBS was prepared by mixing 990 μl of 10 mM sodium bicarbonate (pH 8.5) and 10 μl of 1% TNBS, and 50 μl of TNBS and 100 μl of the reaction mixture taken over time were mixed and incubated at 37 ° C After the reaction for 2 hours, 10% sodium dodecylsulfate (10% SDS) and 1N hydrogen chloride (1N HCl) were added to stop the reaction. The absorbance of each mixture was measured at 335 nm using a microplate spectrophotometer. The results were then converted to tyrosine content.

As a result, as shown in Fig. 4, flavorzyme 5429.35 / / mL, alcalase 4781.38 / / mL, neutraze 3155.55 / / mL, bromelain 1800.00 / / mL and papain 1782.61 / / mL. This is similar to the result of SDS-PAGE and yield measurement.

Then, the antioxidative activities were investigated using the hydrolysates of alcalase, flavozyme and ntrease, which had excellent hydrolysis degree.

<Example 3> Measurement of antioxidative activity of the obtained peptides

1) Measurement of DPPH radical scavenging activity

The DPPH radical is a substance used to evaluate the ability of an antioxidant to donate a hydrogen atom or electron, and is converted to a stable form of the compound by receiving hydrogen or electrons, and the radical-specific violet turns into a pale yellow. Using this principle, the DPPH free radical scavenging activity was confirmed by measuring the reducing power of the protein hydrolysates against α, α-diphenyl-β-picryl hydrazine (α, diphenyl-ß-picryhydrazyl, DPPH) .

Specifically, the protein hydrolyzate samples obtained in Example 1 were diluted to 0, 100, 250, 500 or 1000 μg / mL using 99% methanol, and 160 μl of the diluted samples were dispensed into 96-well plates . Subsequently, 40 μl of a 0.2 mM DPPH solution dissolved in methanol was dispensed into wells in which the sample was placed, and left at room temperature for 30 minutes. Then, the absorbance was measured at 517 nm using a microplate spectrophotometer. And the RC ₅₀ value, which is the concentration of the sample required to reduce the absorbance of the gelatinous larval protein hydrolyzate of each enzyme by ½ by using [Equation 2] below. Trolox was used as a positive control for activity comparison.

[Formula 2]

As a result, as shown in FIG. 5, the RC ₅₀ value of the alkaline protease hydrolyzate was 339.34 / / mL, the flavorzyme 379.35 / / mL, and the neutraceutical 398.93 / / mL, DPPH radical scavenging activity was the highest.

Compared with the results of the study of the antioxidant activity and the physiological function of the Anomala albopilosa extracts, Journal of Food Science and Nutrition 36 (6): 670-677), the ethanol extract of bronze beetle larvae The RC ₅₀ value was 483.39 ㎍ / mL, indicating that the DPPH radical scavenging activity of the brown duck protein hydrolyzate was much better.

In addition, Yu et al. (2008. ACE inhibitory activity and antioxidative activity of hydrolyzate of pupa), Korean Journal of Food Science and Nutrition 37 (2): 136-140.) This nutrient pupa was treated with Ntase, Alcalase, Trypsin and pepsin checking the DPPH radical scavenging activity of the protein hydrolysates result, the RC ₅₀ value of Alcala kinase protein hydrolyzate with 396.09 ㎍ / mL, Nutra azepin the 352.75 ㎍ / mL, trypsin is 677.44 ㎍ / mL, pepsin is 626.09 ㎍ / mL appear. In other words, the results of alkalase and ntrease showed similar tendency to that of brown duck larvae protein hydrolyzate, and trypsin and pepsin showed slightly lower scavenging activity.

2) Measurement of ABTS radical scavenging activity

The ABTS radical scavenging activity assay can be compared with the value of Trolox, which is a standard substance, and it is widely used as a method for measuring antioxidant activity in vitro as well as measuring antioxidant activity in vivo. When ABTS and potassium persulfate are placed in a dark place, ABTS radicals are produced. When the radicals are cleared by antioxidants in the sample, dark cyan ABTS radicals are discolored, which is measured by absorbance.

Specifically, ABTS, which was 7 mM in final concentration, 2.45 mM and potassium sulfide were mixed, respectively, and then left in a dark room for 24 hours to form radicals. Then, the concentration of the ABTS solution was diluted with phosphate buffered saline (PBS, pH 7.4) such that the absorbance value was 0.70 ± 0.02 at 734 nm. 20 μl of each protein hydrolyzate was added to 180 μl of the diluted ABTS solution, and the mixture was allowed to stand at 30 ° C for 1 minute and absorbance was measured at 734 nm. And the RC ₅₀ value, which is the concentration of the sample required to reduce the absorbance of the hydrolyzate of the larvae of the larvae of each of the enzymes, measured in accordance with the following formula 3, to 1/2. At this time, Trolox was used as a positive control for the activity comparison.

[Formula 3]

As a result, as shown in FIG. 6, the protein hydrolyzate obtained through the treatment with Ntrease was found to have an RC ₅₀ of 29.84 / / mL, an alkalase of 31.71 / / mL and a flavozyme of 56.03 / / It was confirmed that the radical scavenging activity of the hydrolyzate was the highest.

The results of this study are summarized as follows. Lee et al. (2008. Apoptosis induction and antioxidative activity of avocado extract, Korean Journal of Food Science and Nutrition 37 (3): 269-275) As a result of comparison, the flesh of avocado was 38.4% at 200 ㎍ / mL, and the hydrolysates of brown algae protein in this study were 69.80% at 50 ㎍ / mL, 49.38% for flavozyme, Was 71.41%, indicating that the ABTS radical scavenging performance was much better.

3) Hydrogen peroxide, H ₂ O ₂ ) Measurement of scavenging activity

Hydrogen peroxide (H ₂ O ₂ ), a reducing metabolite of oxygen, is formed in the mitochondria and peroxisomes of normal cells stimulated by various external factors. It induces DNA and protein damage or is a component of biomembranes such as unsaturated fatty acids To produce lipid peroxides, which are known to induce hypogonadism, aging and adult diseases. In the present invention, the H ₂ O ₂ scavenging activity of the brownish larva larvae hydrolyzate was measured using ABTS, a substrate of peroxidase. Specifically, such as HE Muller (1985. Detection of hydrogen peroxide produced by microorganisms on an ABTS peroxidase medium Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene Series A:.. Medical Microbiology, Infectious Diseases, Virology, Parasitology 259 (2): 151-154 ), 20 μl of each protein hydrolyzate diluted by concentration in distilled water, 100 μl of PBS, and 20 μl of 1 mM H ₂ O ₂ were added to a 96-well plate, followed by reaction at 37 ° C for 5 minutes in an incubator. Subsequently, 30 μl of 1.25 mM ABTS and 30 μl of 1 U / ml peroxidase were added, reacted at 37 ° C. for 10 minutes, and the absorbance was measured at 405 nm using a microplate spectrophotometer. At this time, Trolox was used as a positive control for the activity comparison.

As a result, as shown in Fig. 7, the RC ₅₀ value of the hydrolyzed alkaline protease was 65.94 / / mL, 121.67 / / mL of Ntase and 70.38 / / mL of flavozyme. Comparing these results with studies carried out so far, Hwang et al. (Chokeberry (Aronia melancocarpa) antioxidant content and antioxidant activity of hot-water extract of the Journal of the Korea Food and 2014. drying 46 (3): 0.303 to 308 ) Is the H ₂ O ₂ The results shown in the scavenging RC ₅₀ value, which is interposed in 172.05 ㎍ / mL bar, showed excellent natural product oh H ₂ O ₂ scavenging of mealworm larvae protein hydrolyzate than Catalonia extract.

4) Confirmation of antioxidative effect on oxidation of linoleic acid

The peroxide generated in the early stage of lipid oxidation reacts with ferrous chloride to produce a red ferric chloride dye. When the oxidation of lipid continues, a low-molecular compound such as malonaldehyde Which again binds with thiobarbituric acid (TBA) to form a red compound. Therefore, in this experiment, the antioxidant effect of hydrolysates of brown larch larvae was evaluated using linoleic acid, an unsaturated fatty acid, as a substrate.

Specifically, among the three antioxidative tests described above, an alkalase hydrolyzate having the highest antioxidant activity was used. First, 2 mL of 50 mM sodium phosphate buffer (pH 7.0), 1 mL of 2.52% linoleic acid, 1 mL of absolute alcohol, and 1 mL of alkaline protease hydrolyzate at each concentration were mixed to prepare a linoleic acid reaction substrate solution And then shaken at 100 rpm at 40 ° C. Then, 100 μl of 20% trichloroacetic acid (TCA; w / v), 100 μl of 0.8% TBA and 50 μl of a linolenic acid reaction substrate solution were mixed well and reacted at 95 ° C. or higher for 20 minutes. Cooled and centrifuged at 3000 rpm for 15 minutes to measure 180 μl of supernatant at 532 nm absorbance. This procedure was performed three times a day at intervals, and Trolox was used as a positive control for activity comparison.

As a result, as shown in FIG. 8, the lipid peroxides of the negative control group (no protein hydrolysates were not added) increased with time, and when the absorbance value at the third day of the negative control group was taken as 100% Oxidation of lipid peroxide was also significantly inhibited by about 27, 33, 40 and 68%, respectively, as the concentration of the degraded product increased to 100, 200, 400 and 800 ㎍ / mL.

Example 4 [0064] The active oxygen (ROS, H ₂ O ₂ ) &Lt; / RTI &gt;

(1) Cell culture

AML-12 cells, which were normal mouse hepatocytes, were purchased from the American Type Culture Collection (Manassas, Va., USA) and were inoculated with 10% Fetal Bovine Serum (FBS), 1% insulin- selenium (insulin-transferrin-selenium; ITS ), 1% antibiotics (penicillin / streptomycin), and dexamethasone in 5% CO _2, 37 ℃ conditions using DMEM / F-12 medium containing (40 ng / mL) Lt; / RTI &gt;

(2) Measurement of cell viability

AML-12 cells were plated at a density of 2 × 10 ⁴ cells / well in a 96-well plate and incubated for 24 hours. After incubation for 24 hours, The hydrolysates of carrageenan, flavozyme, and ntrease were treated for 24 hours at each concentration. Then, 7 mM H ₂ O ₂ was treated for 1 hour, and 20 μL of MTT reagent (2.5 mg / mL) was added to each well for 3 hours. Then, the supernatant was removed, and the formazan formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and absorbance was measured at 550 nm. The hepatocyte protective effect was calculated by evaluating the relative cell viability of each treatment group based on the survival rate of the H ₂ O ₂ -free group (control group).

As a result, as shown in FIG. 9, the survival rate of AML-12 cells was reduced to about 21% by 7 mM H ₂ O ₂ , but in the case of cells treated with each protein hydrolyzate, The survival rate was increased. Among them, hydrolysates of alkaline protease hydrolysates showed cell survival rates of 53, 68 and 75% at concentrations of 1, 2.5, and 5 mg / mL, respectively, showing the greatest effect of protecting hepatocytes from reactive oxygen species cytotoxicity .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. Do. That is, the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

The protein hydrolyzate, Alcalase, was hydrolyzed with a brown goat ( Tenebrio molitor, mealworm) protein hydrolyzate of larvae. (Alcalase), which is selected from the group consisting of Flavorzyme, Neutrase, Pepsin, Subtilisin and Pancreatin. Proteolytic hydrolyzate of hydrolyzed brown duck ( Tenebrio molitor, mealworm) larvae. [3] The protein hydrolyzate according to claim 1 or 2, wherein the protein hydrolyzate comprises peptides of 0.01 kDa or more and 3 kDa or less. [3] The protein hydrolyzate according to claim 1 or 2, wherein the protein hydrolyzate exhibits an antioxidative effect. A functional food composition for antioxidation comprising a protein hydrolyzate of a larva of the brown gourd according to claim 1 or 2. A functional food composition for liver protection comprising the protein hydrolyzate of a larva of the brown gurrant according to claim 1 or 2. A functional food composition for stress reduction comprising a protein hydrolyzate of a brown larch larval according to claim 1 or 2. (a) a dry brown duck ( Tenebrio molitor , mealworm) 1 to 10% (w / v) of the larval powder in water or buffer solution to prepare a substrate solution; (b) heating the substrate solution to inactivate the self-enzyme contained in the substrate solution; (c) mixing the substrate solution with a protein hydrolyzing enzyme in an amount of 0.1 to 5% (w / v) relative to the brown goat larvae powder to prepare a mixture; (d) preparing a reaction mixture by reacting a protein hydrolyzing enzyme and a brown larvae larva in the mixture; (e) heating the reaction mixture to inactivate the protein hydrolyzing enzyme to obtain a reaction mixture; (f) centrifuging the mixed solution obtained in step (e) to take a supernatant; (g) centrifuging the supernatant using a filtration membrane; And (h) separating a protein hydrolyzate having a weight average molecular weight of 0.01 kDa or more and 3 kDa or less. 9. The method according to claim 8, wherein the protein hydrolyzing enzyme is Alcalase. The method according to claim 8, wherein the proteinase is selected from the group consisting of Alcalase, Flavourzyme, Neutrase, Pepsin, Subtilisin and Pancreatin. Wherein the protein hydrolyzate is at least one selected from the group consisting of:

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